Ismail Iraqui

Yasmina Chekkal

Nada Jmari

Violena Pietrobon

Karine Fre ´on

Audrey Costes

Sarah A E Lambert
email: sarah.lambert@curie.fr.

Recovery of Arrested Replication Forks by Homologous Recombination Is Error-Prone

Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrestbased assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.

I would like to thank …

• Professor Rossignol Jean-Michel, who accepted to be the President of the jury • Drs Arcangioli B. and Pasero P. for the reading, corrections and critics they have made on the manuscript

• Drs Lopes M. and Quivy J.P. who accepted to be examiners of my PhD thesis

• Dr Sarah Lambert for the possibility she gave me to do my PhD in her lab and for her help and advices, in these three years and half.

• Pasts and actual members of the lab, who provided me a lot of aid during this period: Karine Fréon for first, Audrey Costes, Ismail Iraqui, Joël Blaisonneau and Martine Heude.

• I would like to thank also Mounira Amor-Guéret to have hosted me in her Unit at the Institut Curie and all members of UMR 3348 for the help and the good moments we passed together, especially Dodo, Pierre-Marie, Sylvain, Ludo and Yulia. Thanks also to the personal of the "milieux", always kind and hard-worker.

• Grazie a mia mamma e mio papà che mi hanno aiutato nei momenti di difficoltà e a Bilal che mi é stato vicino specialmente in questi ultimi mesi. Vorrei ringraziare i miei amici italiani: la mitica Angela che mi ha salvato con la bibliogafia e con un mucchio di altre cose; Simonetta e i nostri film del sabato sera che non sono mai riuscita a vedere perché mi addormentavo sempre prima della fine; Valeria perché é una sciroccata che mette tutti di buon umore; Giuseppe per il suo senso pratico nei momenti davvero duri..., Alessandro e Giusy anche se sono rimasti a Parigi troppo poco.

• This work was supported by funds from Marie Curie Actions (European Initial training network), Institut Curie, CNRS (Centre National de la Recherche Scientifique), from "Fondation ARC pour la recherche sur le cancer" and "Agence Nationale de la Recherche"

PREAMBLE

DNA replication and subsequent segregation of chromosomes requires the coordinated action of a complex network of pathways, which ensure the accurate transmission of the genetic material. DNA replication is a fundamental process for every life form that needs to be orchestrated with chromatin disassembly/assembly, maintenance of epigenetic marks, and DNA repair pathway in order to maintain genome stability and inheritance of epigenetic information (for example to label different chromatin structure heterochromatin versus euchromatin). Defects in the coordination of DNA metabolism pathways during chromosomes replication can result in genomic and epi-genomic instability and ultimately trigger cancer development. Indeed, genome instability is not only a prominent common feature for most, if not all, types of cancer but importantly, it is strongly associated with cancer predisposition in many human syndromes.

Chromosomes replication can be challenged by various endogenous and environmental factors, interfering with the progression of replication fork. Therefore, cells have also to coordinate DNA synthesis with mechanisms ensuring the stability and the recovery of halted forks. Among these mechanisms are included the "checkpoints" and the "homologous recombination" pathways. The DNA damage response promotes cell survival by activating cell cycle checkpoints, allowing time for DNA repair prior to resumption of cell cycle progression, or DNA synthesis resumption before entering into mitosis. Homologous recombination is a universal mechanism that supports DNA repair and robust DNA replication. However, due to its ability to promote genetic exchanges between repeated sequences dispersed through the genome, recombination also contributes to chromosomal rearrangements (mainly by Non Allelic Homologous Recombination, NAHR). Therefore, homologous recombination needs to be accurately regulated to promote efficient DNA repair and robust DNA replication while limiting aberrant recombination outcomes, contributing to genome instability. Nevertheless, mechanisms regulating allelic versus non allelic recombination remain poorly understood.

Replication-coupled chromatin assembly is a vital process that ensures correct wrapping of newly replicated DNA around nucleosomes and proper inheritance of chromatin marks. The Chromatin Assembly Factor 1 (CAF-1) is a histone H3/H4 chaperone playing a major role in replication-coupled nucleosomal deposition. Homologous recombination includes a DNA synthesis step and little is known on the associated chromatin assembly during homologous recombination. During my PhD, I investigated the role of CAF-1 during the mechanisms of homologous recombination. The data obtained support a model according to which CAF-1 promotes histones deposition during homologous recombination. By doing so, CAF-1 allows the stabilization of early recombination intermediates (D-loop), by preventing their dissociations by DNA helicases. Thus, CAF-1 appears to be part of an equilibrium that regulates stability/dissociation of early recombination events. Importantly, I found that the role of CAF-1 in this equilibrium is of particular importance during non-allelic recombination, revealing a novel regulation level of homologous recombination mechanisms and outcomes by chromatin assembly.

Note to the readers:

The introduction covers several aspects of the maintenance of genome stability, including DNA repair, Homologous Recombination, and chromatin assembly. Our knowledge on these concepts came from studies undertaken in several organisms such as yeast models (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and metazoans. Because our main understanding of homologous recombination results from studies using the yeast S. cerevisiae, the mechanisms of recombination are mainly presented in the context of budding yeast. In contrast, our main understanding of chromatin assembly during DNA replication came from studies undertaken in metazoans, especially in mammals; thus the mechanism of chromatin assembly is mainly presented in the context of human cells. Tables provided correspondences between yeast and human homologues are included.

DNA REPAIR PATHWAYS

DNA lesions arise from three main causes: environmental agents, products of cellular metabolism and the natural instability of some chemical bonds in DNA. DNA repair mechanisms are evolutionary conserved from prokaryotic to eukaryotic cells, thus revealing the importance of maintaining genomic integrity. Mutations in genes involved in DNA repair are often associated with human diseases with a broad range of clinical features, included cancer susceptibility. The association between cancer predisposition and mutations in DNA repair genes illustrates the importance of DNA repair pathways in maintaining genome stability. During the evolution, cells have developed multiple DNA repair pathways according to the nature of the DNA lesion (Figure n.1).

In this chapter, I will present a brief description of the DNA repair pathways and then I will focus on homologous recombination, which is the predominant repair pathway that was investigated during my PhD.

Base Excision Repair (BER)

DNA lesions that do not significantly distort the DNA backbone are corrected by the base excision repair (BER) pathway (Figure 1). A wide variety of damaged DNA bases are recognized by BER: DNA bases chemically modified by oxidation or alkylation, abasic sites resulting from base loss, uracil resulting from deamination of cytosine or from misincorporation during DNA replication, fragmented pyrimidines [START_REF] Beranek | Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents[END_REF][START_REF] Christmann | Mechanisms of human DNA repair: an update[END_REF]; [START_REF] Barnes | Repair and genetic consequences of endogenous DNA base damage in mammalian cells[END_REF][START_REF] Dizdaroglu | Formamidopyrimidines in DNA: mechanisms of formation, repair, and biological effects[END_REF][START_REF] Hegde | Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells[END_REF][START_REF] Svilar | Base excision repair and lesion-dependent subpathways for repair of oxidative DNA damage[END_REF]. Reactive oxygen species cause DNA oxidative lesions and among them, the most mutagenic one is the 7,8-dihydro-8-oxoguanine (8-oxogunanine). Indeed, if not repaired, 8-oxoguanine can form a base pair with adenine during DNA synthesis ultimately leading to GC to TA transversions [START_REF] Kasai | Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair[END_REF][START_REF] Hazra | Multiple DNA glycosylases for repair of 8-oxoguanine and their potential in vivo functions[END_REF][START_REF] David | Base-excision repair of oxidative DNA damage[END_REF]). This pathway is initiated by a specific DNA N-glycosylase that recognizes the damaged base and cleaves it, thus producing an apurinic/apyrimidinic site. Other enzymes are then recruited to excise the remaining sugar fragment. A DNA polymerase inserts the correct base and a DNA ligase seals the nick, thereby repairing the damage [START_REF] Lindahl | An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues[END_REF][START_REF] Haring | Recognition of oxidized abasic sites by repair endonucleases[END_REF][START_REF] Kubota | Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein[END_REF][START_REF] Aburatani | Cloning and characterization of mammalian 8hydroxyguanine-specific DNA glycosylase/apurinic, apyrimidinic lyase, a functional mutM homologue[END_REF][START_REF] Morland | Human DNA glycosylases of the bacterial Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA[END_REF].

There are two distinct repair pathways: the short-patch pathway involves a single nucleotide replacement followed by ligation (implication of polymerase  and ligase III) and the longpatch pathway involves DNA synthesis of 2-6 nucleotides (by polymerase ), removal of displaced nucleotide overhang by a specific Flap endonuclease and sealing of the nick (ligase I) [START_REF] Frosina | Two pathways for base excision repair in mammalian cells[END_REF][START_REF] Klungland | Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1)[END_REF].

Nucleotide Excision Repair (NER)

Lesions that significantly distort the DNA helix are excised by the nucleotide excision repair (NER) pathway. NER is a highly conserved DNA repair pathway which removes lesions such as those caused by UV light photoproducts and cyclobutane pyrimidine dimers) [START_REF] Setlow | The Disappearance of Thymine Dimers from DNA: An Error-Correcting Mechanism[END_REF][START_REF] Setlow | Cyclobutane-type pyrimidine dimers in polynucleotides[END_REF][START_REF] Ford | Expression of wild-type p53 is required for efficient global genomic nucleotide excision repair in UV-irradiated human fibroblasts[END_REF][START_REF] Dip | Mechanisms of DNA damage recognition and strand discrimination in human nucleotide excision repair[END_REF]). NER covers two pathways: the transcription-coupled repair (TCR) and the global genome repair (GGR) pathways. TCR occurs when the lesion is located in the transcribed strand of active genes and thus disturbs transcription elongation, while GCR occurs when the damage is in the non-transcribed strand of active genes or untranscribed regions of the genome [START_REF] Tornaletti | Structural characterization of RNA polymerase II complexes arrested by a cyclobutane pyrimidine dimer in the transcribed strand of template DNA[END_REF][START_REF] Li | Dissecting transcription-coupled and global genomic repair in the chromatin of yeast GAL1-10 genes[END_REF]. Whereas the same core set of enzymes is used by TCR and GGR, these two pathways rely on different proteins for the initial recognition of the DNA lesion. An intriguing hypothesis is that detection of lesions during TCR (stalling of the transcription machinery) could trigger global relaxation of chromatin structure throughout the genome, thus enhancing access of repair factors for GGR [START_REF] Rubbi | p53 is a chromatin accessibility factor for nucleotide excision repair of DNA damage[END_REF]. The excision of the DNA lesion involves four key events: the recognition of the lesion, the excision of the damaged nucleotide and of flanking regions, the filling of the gap by DNA synthesis and finally ligation of the nick [START_REF] Huang | Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5' and the 6th phosphodiester bond 3' to the photodimer[END_REF][START_REF] Lin | Active site of (A)BC excinuclease. I. Evidence for 5' incision by UvrC through a catalytic site involving Asp399, Asp438, Asp466, and His538 residues[END_REF][START_REF] Mu | Reconstitution of human DNA repair excision nuclease in a highly defined system[END_REF][START_REF] Araujo | Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK[END_REF][START_REF] Hoeijmakers | Genome maintenance mechanisms for preventing cancer[END_REF][START_REF] Guo | SnapShot: nucleotide excision repair[END_REF].

The most notorious disease, resulting from defects in NER pathway, is the Xeroderma Pigmentosum (XP) disease. XP patients present a high risk of skin cancer and eye damage predisposition due to an extreme sun sensitivity (Setlow, Regan et al. 1969;[START_REF] Kraemer | New areas of focus at workshop on human diseases involving DNA repair deficiency and premature aging[END_REF]).

Mismatch Repair (MMR)

The DNA mismatch repair (MMR) pathway is responsible for the removal of mispaired bases or small DNA loop (insertion-deletion loops or IDLs). These errors are caused by DNA polymerases during DNA replication [START_REF] Iyer | DNA mismatch repair: functions and mechanisms[END_REF]). Wagner and Meselson postulated that mismatch repair contributes to replication fidelity in Escherichia coli. This hypothesis was based on the fact that MMR is preferentially directed to the newly synthesized DNA strand [START_REF] Wagner | Repair tracts in mismatched DNA heteroduplexes[END_REF]. In prokaryotes, transient hemimethylation during DNA replication distinguishes the two strands: the parental but not the newly synthetized is methylated [START_REF] Pukkila | Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coli[END_REF][START_REF] Lyons | Kinetics of methylation in Escherichia coli K-12[END_REF]. In eukaryotes, the MMR pathway is temporally coupled to DNA replication, and MMR components have been recently identified as associated with replication centres [START_REF] Hombauer | Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates[END_REF][START_REF] Hombauer | Mismatch repair, but not heteroduplex rejection, is temporally coupled to DNA replication[END_REF]. However, the exact mechanism allowing discriminating the parental strand from the newly synthesized one by MMR remains unclear. The replication factor PCNA, which associates with DNA polymerases, might provide a physical link between repair and replication by acting as a strand signal at the fork [START_REF] Umar | Requirement for PCNA in DNA mismatch repair at a step preceding DNA resynthesis[END_REF]. Another hypothesis, involving incorporation of rNTPs into newly synthetized strands, has recently emerged. Indeed, during normal DNA replication, DNA polymerases have the ability to incorporate rNTPs, instead of dNTPs, at a frequency of one rNTP every 1.250 nucleotides [START_REF] Mcelhinny | Differential correction of lagging-strand replication errors made by DNA polymerases {alpha} and {delta}[END_REF]. The surprising high level of rNTPs incorporation into DNA molecules during DNA replication might serve as a signal to recognize the parental strand from newly synthetized one by eukaryotic MMR [START_REF] Lazzaro | RNase H and postreplication repair protect cells from ribonucleotides incorporated in DNA[END_REF].

Subsequent steps to the recognition of base-base mismatches are: a) recognition of the nascent strand and generation of a nick, b) unwinding of the 3' end of the nicked strand, c) exonucleolytic degradation of error-containing section of the newly synthesized strand, d) filling of the resulting gap by DNA synthesis and sealing of the repair nick by DNA ligase [START_REF] Teixeira | Single-nucleotide polymorphisms of the Trypanosoma cruzi MSH2 gene support the existence of three phylogenetic lineages presenting differences in mismatch-repair efficiency[END_REF][START_REF] Guo | Differential requirement for proliferating cell nuclear antigen in 5' and 3' nick-directed excision in human mismatch repair[END_REF][START_REF] Constantin | Human mismatch repair: reconstitution of a nickdirected bidirectional reaction[END_REF][START_REF] Jiricny | The multifaceted mismatch-repair system[END_REF][START_REF] Geng | In vitro studies of DNA mismatch repair proteins[END_REF][START_REF] Pena-Diaz | Noncanonical mismatch repair as a source of genomic instability in human cells[END_REF].

In humans, the mutator phenotype conferred by defects in MMR contributes to the initiation and promotion of multistage carcinogenesis [START_REF] Loeb | Multiple mutations and cancer[END_REF]. Loss of MMR efficiency leads to cancer predisposition syndromes such as the hereditary non-polyposis colon cancer syndrome (HNPCC) [START_REF] Liu | Analysis of mismatch repair genes in hereditary nonpolyposis colorectal cancer patients[END_REF]. Loss of MMR efficiency has also been implicated in the development of a subset of sporadic tumors that occur in a variety of tissues [START_REF] Eshleman | Microsatellite instability in inherited and sporadic neoplasms[END_REF][START_REF] Peltomaki | Genotype and phenotype in hereditary nonpolyposis colon cancer: a study of families with different vs. shared predisposing mutations[END_REF][START_REF] Jiricny | The multifaceted mismatch-repair system[END_REF][START_REF] Jun | DNA mismatch repair system. Classical and fresh roles[END_REF]).

Non Homologous End-Joining (NHEJ)

Among all DNA lesions, double-strand breaks (DSBs) are considered as the most toxic lesions, if not repaired. They are caused by genotoxic agents such as ionizing radiation, but they are also programmed to orchestrate physiological processes, like in V(D)J recombination (somatic recombination). V(D)J recombination is necessary for the development of the vertebrate immune system [START_REF] Leder | Recombination events that activate, diversify, and delete immunoglobulin genes[END_REF][START_REF] Yaneva | Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic-force microscopy studies[END_REF][START_REF] Coster | A Dual Interaction Between the DNA Damage Response Protein MDC1 and the RAG1 Subunit of the V(D)J Recombinase[END_REF]. Another example is the programmed DSB introduced by the HO endonuclease to promote mating type switching in the yeast Saccharomyces cerevisiae [START_REF] Haber | Transpositions and translocations induced by site-specific double-strand breaks in budding yeast[END_REF].

DSBs can also jeopardize genome stability by generating genome rearrangements such as translocations, and thus trigger carcinogenesis by inactivation of tumor suppressor genes or activation of oncogenes. Homologous Recombination (HR) and Non Homologous End Joining (NHEJ) are the two pathways involved in DSB repair. The predominance of one pathway over the other varies according to the organism, but also according to the stage of the cell cycle and the nature of DNA ends at the DSB site [START_REF] Takata | Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells[END_REF]. For example, in human cells, in which DSBs are mainly repaired by NHEJ, the maximal use of HR occurs during DNA replication [START_REF] Karanam | Quantitative live cell imaging reveals a gradual shift between DNA repair mechanisms and a maximal use of HR in mid S phase[END_REF]. In contrast, DSBs are mainly repaired by HR in yeast models, but NHEJ occurs also in G1 cells [START_REF] Roth | Relative rates of homologous and nonhomologous recombination in transfected DNA[END_REF][START_REF] Godwin | Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells[END_REF][START_REF] Kanaar | Molecular mechanisms of DNA double strand break repair[END_REF]. Moreover, there is also a possible competition between two key enzymes of NHEJ and HR (Ku 70/80 and Rad52, respectively) for binding of DNA ends [START_REF] Van Dyck | Binding of double-strand breaks in DNA by human Rad52 protein[END_REF][START_REF] Pierce | Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells[END_REF].

Non-Homologous End Joining pathway is highly conserved among organisms. NHEJ was firstly discovered in mammalian cells, but NHEJ is present in all eukaryotes and bacteria.

NHEJ consists of the direct joining of two DNA ends and requires at least four steps: a) detection of the DSB b) formation of a molecular bridge that holds the DNA ends together c) a processing mechanism that modifies non-matching and/or damaged nucleotide at DNA ends into compatible ends and d) final ligation step. In vertebrates, these steps require an intimate interplay among the core NHEJ machinery, composed of XRCC4 (for X-ray repair Cross Complementing group 4), DNA ligase IV, the Ku70/80 heterodimer and the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) [START_REF] Roth | Mechanisms of nonhomologous recombination in mammalian cells[END_REF][START_REF] Yaneva | Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic-force microscopy studies[END_REF][START_REF] Walker | Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair[END_REF][START_REF] Mahajan | Association of DNA polymerase mu (pol mu) with Ku and ligase IV: role for pol mu in end-joining double-strand break repair[END_REF][START_REF] Mari | Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4[END_REF][START_REF] Spagnolo | Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair[END_REF][START_REF] Weterings | The endless tale of non-homologous end-joining[END_REF] When DNA ends are blunt, NHEJ is a an error-free mechanism (van Heemst, [START_REF] Van Heemst | End-joining of blunt DNA double-strand breaks in mammalian fibroblasts is precise and requires DNA-PK and XRCC4[END_REF]) while in presence of complex lesions due to multiple damages, the ligation process leads often to deletions [START_REF] Roth | Mechanisms of nonhomologous recombination in mammalian cells[END_REF][START_REF] Moore | Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae[END_REF][START_REF] Wilson | Yeast DNA ligase IV mediates non-homologous DNA end joining[END_REF][START_REF] Pardo | Mismatch tolerance by DNA polymerase Pol4 in the course of nonhomologous end joining in Saccharomyces cerevisiae[END_REF]. Defects in NHEJ components lead to a severe immunodeficiency disease termed SCID (Severe Combined Immune Deficiency). In this pathology, B cells and T cells of the adaptive immune system are impaired leading to an increase susceptibility to infectious diseases [START_REF] Bosma | A severe combined immunodeficiency mutation in the mouse[END_REF][START_REF] Perryman | Molecular pathology of severe combined immunodeficiency in mice, horses, and dogs[END_REF] O 'Driscoll and Jeggo 2006;[START_REF] Van Der Burg | DNA-PKcs deficiency in human: long predicted, finally found[END_REF]. Furthermore, cells derived from patients with a defect in NHEJ are highly radiosensitive [START_REF] Riballo | Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient[END_REF][START_REF] Buck | Severe combined immunodeficiency and microcephaly in siblings with hypomorphic mutations in DNA ligase IV[END_REF]).

HOMOLOGOUS RECOMBINATION

Homologous recombination (HR) is a universally conserved mechanism that promotes DNA repair and supports DNA replication. The fundamental function of HR is to promote strand exchange reactions, leading to genetic exchanges between homologous DNA sequences [START_REF] Paques | Multiple pathways of recombination induced by doublestrand breaks in Saccharomyces cerevisiae[END_REF][START_REF] Mimitou | Nucleases and helicases take center stage in homologous recombination[END_REF]. The basic substrate of HR is a single stranded DNA (ssDNA) on which the recombinase Rad51 forms a nucleoprotein filament. This Rad51-coated ssDNA is then competent for the search of homologous sequences in the genome. Beyond its function in the repair of DSBs and single stranded gaps (ssDNA gaps), HR has multiple functions in eukaryotes such as support of DNA replication, alternative mechanism for telomere maintenance, diversification of immunoglobulin chains, programmed genetic diversity and chromosomes segregation in meiosis.

Alongside its potential benefits, HR is also considered as a source of genome instability.

Indeed, the ability of HR to recombine repeated sequences dispersed throughout the genome can lead to genome rearrangements. Knowing that the human genome contains up to 10 % of repeated sequences, HR has to be accurately regulated to promote efficient DNA repair and to support DNA replication, while limiting aberrant outcomes contributing to cancer development or to genomic disorders. So, HR can be considered a "double-edged sword" for genome stability maintenance (Lambert and Carr 2005;[START_REF] Stankiewicz | The genomic basis of disease, mechanisms and assays for genomic disorders[END_REF][START_REF] Lee | A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders[END_REF]Aguilera and Gomez-Gonzalez 2008;[START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF][START_REF] Simmons | What have studies of genomic disorders taught us about our genome?[END_REF].

Thanks to genetic, biochemical and molecular studies, the different steps of HR are well characterized during the repair of DSBs. Therefore, the molecular mechanism of HR will be first described in the context of DSB repair, and then in the context of DNA replication. Moreover, a particular attention will be given to the process of early joint-molecules (D-loop).

Early steps of homologous recombination

In response to a DSB, DNA ends are resected in 5' to 3' orientation to create a 3' singlestranded DNA. The Mre11-Rad50-Xrs2 (MRX) complex participates in early steps of end resection, via its DNA end-processing activities [START_REF] Nelms | In situ visualization of DNA double-strand break repair in human fibroblasts[END_REF]Lisby, Barlow et al. 2004;[START_REF] Mimitou | Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing[END_REF]. The exonuclease activity of Mre11 in vitro operates in the opposite polarity to that required for DSB resection in vivo, so there are additional factors that cooperate with MRX to facilitate DSB processing, such as Sae2 [START_REF] Rattray | Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: a role for SAE2/COM1[END_REF][START_REF] Clerici | The Saccharomyces cerevisiae Sae2 protein promotes resection and bridging of double strand break ends[END_REF][START_REF] Limbo | Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control double-strand break repair by homologous recombination[END_REF][START_REF] Sartori | Human CtIP promotes DNA end resection[END_REF]). Sae2 in collaboration with MRX creates a minimally resected DNA intermediate. This intermediate is further processed by the 5'-3' exonuclease Exo1 or the exonuclease Dna2 in conjunction with the helicase Sgs1 (Figure n.2). The resection can result into kilobase-sized 3' ssDNA overhangs [START_REF] Lee | Complementation between N-terminal Saccharomyces cerevisiae mre11 alleles in DNA repair and telomere length maintenance[END_REF][START_REF] Tran | EXO1-A multi-tasking eukaryotic nuclease[END_REF][START_REF] Limbo | Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control double-strand break repair by homologous recombination[END_REF][START_REF] Sartori | Human CtIP promotes DNA end resection[END_REF][START_REF] Gravel | DNA helicases Sgs1 and BLM promote DNA double-strand break resection[END_REF][START_REF] Mimitou | Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing[END_REF][START_REF] Zhu | Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends[END_REF][START_REF] Bolderson | Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks[END_REF][START_REF] Cejka | DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2[END_REF][START_REF] Niu | Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae[END_REF]. The resulting ssDNA is initially stabilized through the binding of RPA (Replication protein A), the eukaryotic DNA single stranded binding protein (Figure n.2) [START_REF] Alani | Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding protein[END_REF].

With the assistance of recombination mediator proteins (RMPs), the recombinase Rad51 nucleates onto ssDNA to form a nucleoprotein filament (pre-synaptic step). This step is mediated by Rad52 the main RMP, which displaces RPA from ssDNA to facilitate Rad51 nucleation onto ssDNA. The Rad51 nucleoprotein filament is then the competent intermediate to initiate the search of homology and the genetic exchange between homologous DNA molecules (Figure n.2). The nucleoprotein filament is able to invade a homologous DNA duplex, to pair the invading ssDNA with the complementary strand of the DNA duplex and to displace the non-complementary strand (synaptic step). The resulted three-stranded intermediate referred to as a joint-molecule, is termed displacement loop (D-loop) [START_REF] New | Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A[END_REF]Shinohara, Shinohara et al. 1998). Once the D-loop has been formed, DNA polymerases extend the 3' single stranded DNA invading end, using the donor duplex as a template to restore the lost information (post-synaptic step) (Figure n.2).

Different models have been proposed to explain the step of homology search. The first model proposes that Rad51-filament binds nonspecifically to a dsDNA and then linearly diffuses or slides along the dsDNA, searching for homology [START_REF] Gonda | By searching processively RecA protein pairs DNA molecules that share a limited stretch of homology[END_REF]. A second model proposes random three-dimensional collisions between the invading strand and the genomic DNA [START_REF] Adzuma | No sliding during homology search by RecA protein[END_REF][START_REF] Gupta | Human Rad51 protein can form homologous joints in the absence of net strand exchange[END_REF][START_REF] Folta-Stogniew | Exchange of DNA base pairs that coincides with recognition of homology promoted by E. coli RecA protein[END_REF]). The search for homology takes place in a chromatin environment and it is logical to speculate about the effect of nucleosomes on the search of homology or on the DNA invasion step. Nonetheless, nucleosomes do not impose a barrier for successful search of homology and strand invasion. Indeed Rad51-filaments are sufficient to locate the homologous sequence and to form initial joints molecules, even on the surface of a nucleosome in vitro [START_REF] Sinha | A Rad51 presynaptic filament is sufficient to capture nucleosomal homology during recombinational repair of a DNA double-strand break[END_REF].

Figure n.2: Mechanism of DSB repair by homologous recombination

The initial step of HR consists in the 5'-3' end resection, generating a 3' overhang ssDNA coated by RPA. Then Rad51 nucleates on RPA-coated ssDNA to form a nucleofilament which triggers strand invasion to form a Dloop. In the classical DSBR model, the second end is captured, leading to the formation of double Holliday junctions (HJs) that are resolved by cleavage, giving rise to crossing-over (CO) or non CO products, or resolved by dissolution, giving rise only to non CO products. In the SDSA pathway, the invaded strand is displaced after elongation and result only in non CO products [START_REF] Mimitou | Nucleases and helicases take center stage in homologous recombination[END_REF].

Models of homologous recombination

Over the last 30 years, several models of recombination have been proposed to report the type of genetic exchanges observed during mitotic or meiotic recombination. The main models are described in details below.

Double Strand Break Repair (DSBR)

The Double Strand Break Repair model was proposed by Szostak and collaborators in 1983 [START_REF] Szostak | The double-strand-break repair model for recombination[END_REF] (Figure n.2). In this model, both ends of the DSB are resected to generate 3' ssDNA overhang ends. One of the two ends invades a homologue duplex to form a D-loop structure. This process is catalyzed by the recombinase Rad51 and the main recombination mediator protein Rad52. The invading 3' end primes DNA synthesis using the complementary strand as a donor template. The 3' overhang of the second end is captured by the displaced strand and then elongated by DNA synthesis. The second endcapture is thought to be facilitated by the single strand annealing activity of Rad52 (see below) [START_REF] Sugiyama | Sequence-specific recognition of double-stranded DNA by cooperative strand invasion[END_REF][START_REF] Mcilwraith | DNA repair synthesis facilitates RAD52-mediated second-end capture during DSB repair[END_REF][START_REF] Nimonkar | Rad52 promotes second-end DNA capture in double-stranded break repair to form complement-stabilized joint molecules[END_REF]. A double Holliday Junction (HJs) is then formed, which is resolved either by cleavage through the activities of HJ resolvases (Mus81/Mms4, Slx4, Yen1), or by dissolution through the helicase activity of the Sgs1/Rmi1/Top3 complex [START_REF] Holliday | The Induction of Mitotic Recombination by Mitomycin C in Ustilago and Saccharomyces[END_REF][START_REF] Boddy | Mus81-Eme1 are essential components of a Holliday junction resolvase[END_REF][START_REF] Ira | Srs2 and Sgs1-Top3 suppress crossovers during doublestrand break repair in yeast[END_REF][START_REF] Osman | Generating crossovers by resolution of nicked Holliday junctions: a role for Mus81-Eme1 in meiosis[END_REF][START_REF] Wu | The Bloom's syndrome helicase suppresses crossing over during homologous recombination[END_REF][START_REF] Hollingsworth | The Mus81 solution to resolution: generating meiotic crossovers without Holliday junctions[END_REF][START_REF] Ip | Identification of Holliday junction resolvases from humans and yeast[END_REF][START_REF] Singh | Roles of RECQ helicases in recombination based DNA repair, genomic stability and aging[END_REF] (Figure n.2).

According to the strands cleaved by HJs resolvases, the genetic exchange results in either gene conversion associated with a cross-over (CO, i.e. the reciprocal exchange of flanking markers), or to gene conversion not associated with CO (i.e. no reciprocal exchange of flanking markers, see bottom panel of Figure n.2). In contrast, resolution of HJs by dissolution gives rise only to non CO products [START_REF] Elborough | Resolution of synthetic Holliday junctions in DNA by an endonuclease activity from calf thymus[END_REF][START_REF] Hyde | Resolution of recombination intermediates by a mammalian activity functionally analogous to Escherichia coli RuvC resolvase[END_REF][START_REF] Paques | Multiple pathways of recombination induced by doublestrand breaks in Saccharomyces cerevisiae[END_REF][START_REF] Taylor | Cleavage mechanism of human Mus81-Eme1 acting on Holliday-junction structures[END_REF]. Mitotic cross-overs can result in chromosomal rearrangement such as translocations and thus can be potential threats for genome stability (see below).

Initially, the model of Szostak hypothesized that either the 3'-or the 5'-end of the DSB could be degraded [START_REF] Szostak | The double-strand-break repair model for recombination[END_REF]. Nonetheless, the results obtained by Sun et al. have demonstrated that resection occurs only in the 5'-3' direction [START_REF] Sun | Genetic and physical analyses of sister chromatid exchange in yeast meiosis[END_REF].

The model of Szostak well describes meiotic recombination events that are mainly associated with COs. However, mitotic recombination is less often associated with COs, thus leading to the emergence of an alternative recombination model for the repair of DSB: the synthesisdependent strand annealing (SDSA) pathway.

Synthesis Dependent Strand Annealing (SDSA)

In the Synthesis Dependent strand Annealing model, the invaded strand is elongated, displaced from the D-loop, and anneals back with the 3'ssDNA overhang of the second end (Figure n.2). The remaining gap is then filled by DNA synthesis and the nicks are ligated. The SDSA model forms only non COs products with no alteration of the genetic information of the donor duplex (Figure n.2, right panel) [START_REF] Nassif | Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair[END_REF][START_REF] Paques | Multiple pathways of recombination induced by doublestrand breaks in Saccharomyces cerevisiae[END_REF][START_REF] Mcmahill | Synthesis-dependent strand annealing in meiosis[END_REF][START_REF] Mimitou | Nucleases and helicases take center stage in homologous recombination[END_REF]). It appears that the SDSA is the predominant pathway of DSB repair in vegetative cells [START_REF] Elliott | Double-strand breaks and translocations in cancer[END_REF][START_REF] Maloisel | A role for DNA polymerase delta in gene conversion and crossing over during meiosis in Saccharomyces cerevisiae[END_REF]).

Break Induced Replication (BIR)

Break Induced Replication is an alternative model of DSB repair and it occurs when only one end of a double-strand break shares homology with a template, thus preventing the capture of the second DSB-end (Figure n.3). BIR has the particularity to promote efficient DNA synthesis over hundreds of kilobases, through the assembly of a single replisome. Therefore, BIR is thought to play a key role in the repair of broken replication forks and in the maintenance of eroded telomeres in cells lacking telomerase, as DNA synthesis can progress until the rebuilt-fork meets a converging fork, or progress from the break to the template chromosomes end (Bosco and Haber 1998;[START_REF] Cullen | Break-induced loss of heterozygosity in fission yeast: dual roles for homologous recombination in promoting translocations and preventing de novo telomere addition[END_REF][START_REF] Lydeard | Break-induced replication and telomerase-independent telomere maintenance require Pol32[END_REF]Lydeard, Lipkin-Moore et al. 2010;Moriel-Carretero and Aguilera 2010).

The first steps of BIR are similar to the ones of the DSBR model, resulting in a D-loop structure and the 3' end of the invading strand is used to prime DNA synthesis [START_REF] Davis | RAD51-dependent break-induced replication in yeast[END_REF][START_REF] Malkova | RAD51-dependent break-induced replication differs in kinetics and checkpoint responses from RAD51-mediated gene conversion[END_REF]. BIR requires all essential replication proteins, with the exception of those needed for pre-RC assembly. Thus, both leading and lagging strand DNA synthesis are involved in BIR, in contrast to SDSA that requires only leading strand DNA synthesis [START_REF] Lydeard | Break-induced replication and telomerase-independent telomere maintenance require Pol32[END_REF]Lydeard, Lipkin-Moore et al. 2010). In S. cerevisiae, the requirement of a non-essential polymerase δ subunit (Pol32) distinguishes the DNA synthesis associated to BIR from DNA synthesis associated with canonical replication [START_REF] Smith | Template switching during break-induced replication[END_REF]. Moreover, BIR is prone to template exchange and is hyper-mutagenic, displaying increased template switching plus frameshift mutagenesis [START_REF] Gerik | Characterization of the two small subunits of Saccharomyces cerevisiae DNA polymerase delta[END_REF][START_REF] Smith | Template switching during break-induced replication[END_REF][START_REF] Deem | Break-induced replication is highly inaccurate[END_REF].

How BIR allows extensive DNA replication and rebuilding of a replication fork remains unclear. One possible scenario is that the invading strand in the D-loop is unstable and undergoes multiple dissociation/invasion cycles. In this instance, the DNA would be conservatively replicated because all newly synthesized DNA would be associated with the sequences extending from the repair broken end (Figure n.3A) [START_REF] Formosa | DNA synthesis dependent on genetic recombination: characterization of a reaction catalyzed by purified bacteriophage T4 proteins[END_REF].

Alternatively, the D-loop might be converted into a complete unidirectional replication fork, progressing along the donor chromosome. The fact that all replication factors necessary for the elongation step of canonical replication forks are also required for BIR supports this model (Lydeard, Lipkin-Moore et al. 2010). This results in two semi-conservatively replicated molecules and a single Holliday Junction that needs to be resolved (Figure n.3B and C). BIR is known to drive chromosome rearrangements, such as long tracts of gene conversion, nonreciprocal translocations and multiple templates switching (Bosco and Haber 1998;[START_REF] Ruiz | Chromosomal translocations caused by either pol32-dependent or pol32-independent triparental break-induced replication[END_REF]). This takes place when multiple rounds of invasion/DNA synthesis/dissociation occur within dispersed repeated sequences (McEachern and Haber 2006;[START_REF] Smith | Template switching during break-induced replication[END_REF]).

Single Strand Annealing (SSA)

When DSB occurs between two direct repeats, the repair of the broken chromosome can result in the deletion of the region between the two direct repeats, leaving a single copy of the repeat. This repair event refers to the Single Strand Annealing (SSA) pathway that was discovered in mammals, and then largely studied in the yeast S. cerevisiae [START_REF] Lin | Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process[END_REF][START_REF] Ivanov | RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae[END_REF][START_REF] Paques | Multiple pathways of recombination induced by doublestrand breaks in Saccharomyces cerevisiae[END_REF].

The first step of SSA is an extensive resection of the 5'-3' DNA end until the homology between direct repeat is exposed as ssDNA (Figure n.4A). The long 3' end is bound by RMPs such as Rad52 and Rad59 that promote the annealing of complementary ssDNA strands [START_REF] Smith | A mutation in the gene encoding the Saccharomyces cerevisiae single-stranded DNA-binding protein Rfa1 stimulates a RAD52independent pathway for direct-repeat recombination[END_REF][START_REF] Smith | An allele of RFA1 suppresses RAD52-dependent doublestrand break repair in Saccharomyces cerevisiae[END_REF][START_REF] Sugawara | DNA length dependence of the single-strand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair[END_REF] [START_REF] Ivanov | RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae[END_REF]. The final step of SSA is the filling of ss-gaps by DNA synthesis and ligation [START_REF] Thacker | A mechanism for deletion formation in DNA by human cell extracts: the involvement of short sequence repeats[END_REF]). As SSA results in deletion of genetic material, it is considered as a non-conservative mechanism [START_REF] Sugawara | DNA length dependence of the single-strand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair[END_REF].

C)

Non-homologous 3'flap are cleaved by the Rad1/Rad10 nuclease complex.

Positive and negative regulators of homologous recombination

The different steps of HR (pre-synaptic, synaptic and post-synaptic) are tightly regulated by positive and negative regulators. Positive regulators are mainly Recombination Mediators Proteins (RMPs) that facilitate Rad51 nucleation onto ssDNA, stabilize the Rad51-filament or favor the strand invasion step. In contrast, negative regulators destabilize the nucleofilament and favor D-loop dissociation. Both negative and positive regulators of HR influence the mode of DSB repair and thus recombination outcome (CO versus non CO products).

Recombination Mediators Proteins (RMPs)

Rad51 nucleates onto a RPA-coated ssDNA. RPA plays a dual role in the formation of Rad51 filament. In one hand, RPA has a positive action by preventing the formation of secondary structures in ssDNA [START_REF] Kowalczykowski | Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein. Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA[END_REF]. But, on the other hand, Rad51 nucleation is prevented when ssDNA is covered by RPA, as RPA as a greater affinity for ssDNA than Rad51. Therefore, proteins that can either overwhelm the inhibitory effect of RPA on Rad51-binding to ssDNA or promote Rad51 nucleofilament formation are defined as Recombination Mediators Proteins (RMPs) (Table n.1). RMPs can facilitate Rad51 loading onto resected ssDNA, increasing the stability of the nucleofilament. One way to stabilize the nucleofilament is to regulate the ATP-hydrolysis activity (ATPase) of Rad51. The recombinase Rad51 binds ssDNA in an ATP-dependent manner. ATP-binding by Rad51 promotes a structural alteration of the protein that is necessary for efficient and cooperative nucleation of Rad51 onto ssDNA [START_REF] Namsaraev | Binding of Rad51p to DNA. Interaction of Rad51p with single-and double-stranded DNA[END_REF]. The ssDNA within the filament is then stretched [START_REF] Ogawa | RecA-like recombination proteins in eukaryotes: functions and structures of RAD51 genes[END_REF]) and this stretching is essential for fast and efficient homology search [START_REF] Klapstein | Physics of RecA-mediated homologous recognition[END_REF]Chen and Ferec 2008). ATP hydrolysis leads to a loss of Rad51 affinity for ssDNA and a subsequent dissociation of the nucleofilament. Therefore, RMPs regulate the stability of the filament by regulating the ATPase activity of Rad51. Alternatively, RMPs can protect the Rad51 filament from dissociation by negative regulators.

Table n.1: Recombination mediator proteins

As mentioned above, the main positive regulator of HR is the Rad52 protein that interacts with RPA and ssDNA, to form a ring-like structure. Rad52 facilitates the pre-synaptic step: it binds to ssDNA, displaces RPA from ssDNA, and thus facilitates the binding of Rad51 onto ssDNA [START_REF] Resnick | The repair of double-strand breaks in chromosomal DNA of yeast[END_REF][START_REF] Ogawa | A species-specific interaction of rad51 and rad52 proteins in eukaryotes[END_REF]Sung 1997;[START_REF] Benson | Synergistic actions of Rad51 and Rad52 in recombination and DNA repair[END_REF][START_REF] New | Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A[END_REF]Shinohara and Ogawa 1998) (Figure n.5). Genetic and physical interactions have been reported between the N-terminus of Rad52 and RPA [START_REF] Hays | Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins[END_REF][START_REF] Hays | Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA[END_REF] while the C-terminus of Rad52 interacts with Rad51 protein (Shinohara and Ogawa 1998;[START_REF] Song | Functional interactions among yeast Rad51 recombinase, Rad52 mediator, and replication protein A in DNA strand exchange[END_REF][START_REF] Sugawara | In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination[END_REF]. Rad52 displays also ssDNA annealing activity which functions independently of Rad51, for example to capture the second DNA end during the repair of DSB or to promote SSA [START_REF] Mortensen | DNA strand annealing is promoted by the yeast Rad52 protein[END_REF][START_REF] Van Dyck | Binding of double-strand breaks in DNA by human Rad52 protein[END_REF][START_REF] Mcilwraith | DNA repair synthesis facilitates RAD52-mediated second-end capture during DSB repair[END_REF][START_REF] Nimonkar | Rad52 promotes second-end DNA capture in double-stranded break repair to form complement-stabilized joint molecules[END_REF]). Rad59 is considered as a Rad52 paralog, sharing significant sequence homology with the Nterminal domain of S. cerevisiae Rad52 but lacking the C-terminal domain involved in Rad51 interaction [START_REF] Davis | The yeast recombinational repair protein Rad59 interacts with Rad52 and stimulates single-strand annealing[END_REF][START_REF] Wu | DNA annealing mediated by Rad52 and Rad59 proteins[END_REF]). Rad52 via its N-terminal region interacts with Rad59 in vivo and in vitro, thus forming heteromeric rings [START_REF] Davis | The Rad52-Rad59 complex interacts with Rad51 and replication protein A[END_REF]. Rad59 is involved in Rad51-independent recombination, suggesting that Rad59 could stimulate Rad52 single strand annealing activities to promote SSA and/or the second end capture during DSBR. The cooperation between Rad59 and Rad52 is particularly important when the length of direct repeats is reduced [START_REF] Bai | A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae[END_REF][START_REF] Sugawara | DNA length dependence of the single-strand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair[END_REF][START_REF] Davis | The yeast recombinational repair protein Rad59 interacts with Rad52 and stimulates single-strand annealing[END_REF][START_REF] Wu | DNA annealing mediated by Rad52 and Rad59 proteins[END_REF]).

Rad55 and Rad57 proteins are Rad51 paralogs, forming a heterodimer to promote the formation and stabilization of Rad51 nucleoprotein filament. Firstly, Rad55-Rad57 heterodimer mediates Rad51 binding to the ssDNA [START_REF] Heyer | The search for the right partner: homologous pairing and DNA strand exchange proteins in eukaryotes[END_REF]Sung 1997;[START_REF] Sugawara | In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination[END_REF]. Secondly, the Rad55-Rad57 heterodimer forms a co-filament with Rad51 which is more resistant to dissociation by the DNA helicase Srs2, than the Rad51-filament alone. Moreover, the heterodimer stabilizes the Rad51-nucleofilament, increasing the possibility of pairing of Rad51-nucleoprotein filament with a homologue sequence (Sung 1997;[START_REF] Fortin | Mutations in yeast Rad51 that partially bypass the requirement for Rad55 and Rad57 in DNA repair by increasing the stability of Rad51-DNA complexes[END_REF][START_REF] Liu | Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation[END_REF].

The Shu complex (Shu1-Shu2-Psy3-Csm2) has been shown genetically to promote DNA repair through HR [START_REF] Huang | A genomewide screen in Saccharomyces cerevisiae for genes that suppress the accumulation of mutations[END_REF][START_REF] Shor | A genetic screen for top3 suppressors in Saccharomyces cerevisiae identifies SHU1, SHU2, PSY3 and CSM2: four genes involved in error-free DNA repair[END_REF][START_REF] Mankouri | Shu proteins promote the formation of homologous recombination intermediates that are processed by Sgs1-Rmi1-Top3[END_REF]). The Shu complex contains Rad51 paralogs (Shu1 and Psy3) that stabilize the Rad51 nucleofilament by inhibiting its disassembly by the DNA helicase Srs2, notably through Shu1 that modulates Srs2 recruitment, [START_REF] Shor | A genetic screen for top3 suppressors in Saccharomyces cerevisiae identifies SHU1, SHU2, PSY3 and CSM2: four genes involved in error-free DNA repair[END_REF][START_REF] Mankouri | Shu proteins promote the formation of homologous recombination intermediates that are processed by Sgs1-Rmi1-Top3[END_REF][START_REF] Ball | The yeast Shu complex couples error-free postreplication repair to homologous recombination[END_REF][START_REF] Choi | The Smc5/6 complex and Esc2 influence multiple replication-associated recombination processes in Saccharomyces cerevisiae[END_REF][START_REF] Bernstein | The Shu complex, which contains Rad51 paralogues, promotes DNA repair through inhibition of the Srs2 anti-recombinase[END_REF].

Both RMPs Rad55/Rad57 and the Shu complex illustrate how the assembly and disassembly of Rad51 presynaptic filament is governed by the balance between positive and negative regulators of HR [START_REF] Liu | Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation[END_REF].

Rad54 is another positive regulator of recombination acting at the presynaptic, synaptic and post-synaptic steps of HR. Rad54 is a member of the Snf2-family of SF2 helicases, whose components are involved in chromatin remodeling during transcription and DNA repair [START_REF] Flaus | Identification of multiple distinct Snf2 subfamilies with conserved structural motifs[END_REF][START_REF] Heyer | Rad54: the Swiss Army knife of homologous recombination?[END_REF]. Rather than operating as DNA helicases, the Snf2-related proteins are viewed as motor proteins using the energy of ATP hydrolysis to translocate on dsDNA, and thus inducing topological changes [START_REF] Pazin | SWI2/SNF2 and related proteins: ATP-driven motors that disrupt protein-DNA interactions?[END_REF][START_REF] Tan | Mouse Rad54 affects DNA conformation and DNAdamage-induced Rad51 foci formation[END_REF][START_REF] Mazin | Rad54 protein is targeted to pairing loci by the Rad51 nucleoprotein filament[END_REF][START_REF] Van Komen | Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54[END_REF].

Moving along the DNA filament, Rad54 creates positive and negative supercoils. This negative supercoil could favor the unwinding of the donor duplex and thus facilitates the strand invasion step [START_REF] Van Komen | Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54[END_REF].

Rad54 stabilizes Rad51 nucleoprotein filaments in vitro [START_REF] Mazin | A novel function of Rad54 protein. Stabilization of the Rad51 nucleoprotein filament[END_REF] but the main function of Rad54 in vivo is post-synaptic to promote the extension of the invading 3'end by DNA synthesis, and thus stabilizing the D-loop structure [START_REF] Sugawara | In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination[END_REF][START_REF] Li | Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics[END_REF]. First, Rad54 increases the rate of branch migration in an ATPdependent manner [START_REF] Solinger | Rad54 protein stimulates the postsynaptic phase of Rad51 protein-mediated DNA strand exchange[END_REF]. Second, Rad54 promotes Rad51 dissociation from the DNA duplex in the D-loop structure (Figure n.5). Indeed, even upon ATP hydrolysis Rad51 has a slow turnover and releases slowly from the DNA duplex. Rad54 facilitates Rad51 dissociation from the DNA duplex, in an ATP-dependent manner, thus allowing the extension of the 3'invading end [START_REF] Sung | Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein[END_REF][START_REF] Zaitseva | The DNA binding properties of Saccharomyces cerevisiae Rad51 protein[END_REF][START_REF] Kiianitsa | Rad54 protein exerts diverse modes of ATPase activity on duplex DNA partially and fully covered with Rad51 protein[END_REF][START_REF] Solinger | Rad54, a Swi2/Snf2-like recombinational repair protein, disassembles Rad51:dsDNA filaments[END_REF].

Regulation of early steps of homologous recombination

Investigations of the dynamics of the early steps of HR during the repair of a site-specific DSB have provided further knowledge on the regulation of HR [START_REF] Sugawara | In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination[END_REF][START_REF] Wolner | Recruitment of the recombinational repair machinery to a DNA double-strand break in yeast[END_REF][START_REF] Wang | Role of DNA replication proteins in double-strand breakinduced recombination in Saccharomyces cerevisiae[END_REF][START_REF] Hicks | Real-time analysis of double-strand DNA break repair by homologous recombination[END_REF]. The 5'-3' end-resection appears to be a fast process as the recruitment of RPA at the site of DSB coincides with DSB induction. In contrast, Rad51 nucleation onto ssDNA occurs 30 minutes after DSB induction, suggesting that the formation of Rad51 nucleoprotein filament is a slow process. The search of homology and the strand invasion step are rather fast processes as Rad51 associates with the donor template only 10 minutes after Rad51 filament formation.

Finally, the initiation of DNA synthesis to elongate the 3' invading end occurs 30-40 minutes after the initial association of Rad51 with the donor template. This delay may reflect the reversible nature of the initial Rad51-filament in association with the donor DNA molecule (subsequent cycles of pairing of the ssDNA with the homologue sequence). In support of this, molecular studies have revealed that stable and complete strand exchange is a complex mechanism.

Fluorescence resonance energy transfer (FRET) analyses indicate a rapid annealing exchange of A:T base pairs between the invading nucleoprotein filament and the duplex donor. This A:T exchange is a key step to allow the search of homology [START_REF] Gupta | Human Rad51 protein can form homologous joints in the absence of net strand exchange[END_REF]. The initial interactions between nucleoprotein filament and homologue sequence are promoted by the extended structure of ssDNA within the Rad51 filament [START_REF] Klapstein | Physics of RecA-mediated homologous recognition[END_REF]) and stabilized by Rad51 itself, therefore implicating a protein-protein interaction [START_REF] Bianco | DNA strand exchange proteins: a biochemical and physical comparison[END_REF][START_REF] Sung | Rad51 recombinase and recombination mediators[END_REF][START_REF] Sinha | A Rad51 presynaptic filament is sufficient to capture nucleosomal homology during recombinational repair of a DNA double-strand break[END_REF]. These early interactions lead to a metastable DNA joint (paranemic joints), in which the 3' end of the Rad51-filament is not engaged in extended base-pairing interactions. The paranemic joint is then converted in a plectonemic joint in which the ssDNA bases pairs with its complementary strand in the D-loop structure [START_REF] Riddles | The formation of plectonemic joints by the recA protein of Escherichia coli. Requirement for ATP hydrolysis[END_REF]. The ATPase activity of Rad54 plays a crucial role in the conversion of the paranemic joint into a protein-independent joint.

Recent data demonstrated that within 10 seconds of Rad54 addition, 20% of the maximum number of stable joints was formed, in vitro. In contrast, this value is reached in about 2 minutes in the absence of Rad54 [START_REF] Sinha | A Rad51 presynaptic filament is sufficient to capture nucleosomal homology during recombinational repair of a DNA double-strand break[END_REF]. Therefore Rad54 converts initial metastable products into a stable joint molecule termed the D-loop.

The recombinase Rad51 itself plays a critical role in the further stabilization of the nascent Dloop. Indeed, Rad51 not only promotes the formation of branched structure (D-loop) but also promotes branch migration in the 3'-5' direction relative to the invading single stranded DNA (Figure n.6) [START_REF] Murayama | Formation and branch migration of Holliday junctions mediated by eukaryotic recombinases[END_REF]. In vitro, Rad51-dependent branch migration occurs probably by stabilizing base pairing between heteroduplexes and allows a complete 4strand exchange reaction [START_REF] Hall | Homologous pairing and strand exchange promoted by the Escherichia coli RecT protein[END_REF][START_REF] Namsaraev | Rad51 uses one mechanism to drive DNA strand exchange in both directions[END_REF]. Branch migration by Rad51 results in the migration of the first HJ behind the initial point of strand invasion and in the increase of the length of the heteroduplex, thus stabilizing the D-loop. As mentioned above, a delay of 40 minutes has been detected between initial recognition of Rad51-mediated strand exchange and observable primer extension initiation in budding yeast [START_REF] Hicks | Real-time analysis of double-strand DNA break repair by homologous recombination[END_REF]. A similar delay of 30 minutes to initiate 3' end polymerization has been observed in mammalian cells [START_REF] Si | A strand invasion 3' polymerization intermediate of mammalian homologous recombination[END_REF]. This might also suggest that the assembly of a DNA synthesis machinery takes time. Extension of the invading 3'end has been proposed to be mediated by the DNA polymerase eta (pol ɳ). Indeed, in vitro studies have shown that only pol ɳ is able to extend 3'end within a D-loop structure, but no other polymerases such as pol delta (pol δ), pol iota (pol ι), polymerases T4 and T7 [START_REF] Mcilwraith | Human DNA polymerase eta promotes DNA synthesis from strand invasion intermediates of homologous recombination[END_REF]. In support of this, genetic data in DT40 cells showed that defect in pol ɳ results in impaired DSB repair by HR [START_REF] Kawamoto | Dual roles for DNA polymerase eta in homologous DNA recombination and translesion DNA synthesis[END_REF].

Moreover, pol ɳ was found to be recruited during the repair of broken replication forks in xenopus [START_REF] Hashimoto | RAD51-and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks[END_REF]). Nonetheless, further biochemical studies have established conditions in which pol δ is able to prime DNA synthesis on a D-loop structure [START_REF] Sebesta | Reconstitution of DNA repair synthesis in vitro and the role of polymerase and helicase activities[END_REF]. The interaction between pol δ and the replication factor PCNA is crucial for D-loop extension by pol δ. Several genetic evidences support a role for pol δ in mitotic gene conversion and extension of DNA heteroduplex. Indeed, DNA synthesis during gene conversion is mutagenic and pol δ contributes to it; pol δ and pol ε are also required to support extensive DNA synthesis during BIR [START_REF] Lydeard | Break-induced replication and telomerase-independent telomere maintenance require Pol32[END_REF][START_REF] Maloisel | DNA polymerase delta is preferentially recruited during homologous recombination to promote heteroduplex DNA extension[END_REF][START_REF] Hicks | Differential roles for DNA polymerases eta, zeta, and REV1 in lesion bypass of intrastrand versus interstrand DNA cross-links[END_REF]. Therefore, the exact nature of the DNA synthesis machinery involved in the extension of the heteroduplex within the D-loop remains unclear (Lydeard, Lipkin-Moore et al. 2010;[START_REF] Hashimoto | RAD51-and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks[END_REF]).

In competition with the D-loop extension by DNA polymerases, phenomena of D-loop dissociation by several DNA helicases also influence the mode of DSB repair and recombination outcomes. These DNA helicases acting during the early steps of recombination are considered as negative regulators of HR (Table n.2 and Figure n.5).

The DNA helicase/translocase Srs2 is a 3'-5' DNA helicase that suppresses crossover during mitotic recombination by dissociating joint molecules [START_REF] Fabre | Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication[END_REF][START_REF] Aylon | Molecular dissection of mitotic recombination in the yeast Saccharomyces cerevisiae[END_REF][START_REF] Ira | Srs2 and Sgs1-Top3 suppress crossovers during doublestrand break repair in yeast[END_REF][START_REF] Robert | Mrc1 and Srs2 are major actors in the regulation of spontaneous crossover[END_REF]). Srs2 is defined as an anti-recombinase because the deletion of Srs2 leads to a hyper-recombination phenotype, especially an increase in CO-products, which is rescued by deleting Rad51 [START_REF] Chanet | Semidominant mutations in the yeast Rad51 protein and their relationships with the Srs2 helicase[END_REF][START_REF] Gangloff | Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases[END_REF]. In vitro studies have established a role for Srs2 in the disassembly of Rad51 nucleoprotein filament [START_REF] Krejci | DNA helicase Srs2 disrupts the Rad51 presynaptic filament[END_REF][START_REF] Veaute | The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments[END_REF]. This activity requires the interaction between Srs2 and Rad51 with subsequent ATP hydrolysis to weaken the Rad51-DNA interaction [START_REF] Antony | Srs2 disassembles Rad51 filaments by a proteinprotein interaction triggering ATP turnover and dissociation of Rad51 from DNA[END_REF][START_REF] Colavito | Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption[END_REF]. Moreover, the helicase activity of Srs2 is stimulated by the presence of Rad51 filament, resulting in the ability of Srs2 to unwind D-loop structure [START_REF] Dupaigne | The Srs2 helicase activity is stimulated by Rad51 filaments on dsDNA: implications for crossover incidence during mitotic recombination[END_REF]. Therefore, it is thought that Srs2 promotes SDSA pathway, thus preventing mitotic COs.

Table n.2: Negative regulators of homologous recombination

The DNA helicase Sgs1 belongs to the 3'-5' RecQ helicase family. Sgs1 acts at different steps during the repair of DSB by HR. During late steps, the complex Sgs1/Rmi1/Topoisomerase III promotes the dissolution of double HJs, thus preventing mitotic Cos [START_REF] Ira | Srs2 and Sgs1-Top3 suppress crossovers during doublestrand break repair in yeast[END_REF][START_REF] Wu | The Bloom's syndrome helicase suppresses crossing over during homologous recombination[END_REF][START_REF] Ahmad | The N-terminal region of the Schizosaccharomyces pombe RecQ helicase, Rqh1p, physically interacts with Topoisomerase III and is required for Rqh1p function[END_REF][START_REF] Chang | RMI1/NCE4, a suppressor of genome instability, encodes a member of the RecQ helicase/Topo III complex[END_REF][START_REF] Cejka | DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2[END_REF]. Sgs1 also promotes end-resection to generate long 3'ssDNA end. In addition, Sgs1 regulates the Rad51-nucleoprotein filament by promoting the disassembly of Rad51 from ssDNA. It is proposed that acting at the postsynaptic step, Sgs1 prevents aberrant multiple strand invasion events [START_REF] Oh | BLM ortholog, Sgs1, prevents aberrant crossing-over by suppressing formation of multichromatid joint molecules[END_REF][START_REF] Oh | RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, resolve aberrant joint molecules during meiotic recombination[END_REF]. The helicase BLM, the human homologue of Sgs1, is able to disrupt the binding of Rad51 to ssDNA and thus to promote DSB repair by the SDSA pathway [START_REF] Bugreev | Novel pro-and anti-recombination activities of the Bloom's syndrome helicase[END_REF]. Finally, experimental evidences in Drosophila melanogaster show that BLM can unwind ssDNA tail from D-loops thus providing a mechanism of channeling the D-loop towards the SDSA pathway [START_REF] Adams | Drosophila BLM in double-strand break repair by synthesis-dependent strand annealing[END_REF][START_REF] Mcvey | Formation of deletions during double-strand break repair in Drosophila DmBlm mutants occurs after strand invasion[END_REF]).

In budding yeast, cells depleted for Sgs1 and Srs2 are either unviable or present a very slow growth phenotype. These phenotypes are suppressed by inactivation of recombination genes involved in the presynaptic step, such as RAD51, RAD55 and RAD57 [START_REF] Gangloff | Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases[END_REF]. These data suggest that accumulation of unresolved Joint-Molecules, or Rad51 bound to ssDNA are lethal recombination intermediates. Similar genetic interactions were uncovered in fission yeast [START_REF] Doe | The involvement of Srs2 in post-replication repair and homologous recombination in fission yeast[END_REF].

The helicase Mph1 is an ATP-dependent helicase with 3'-5' polarity that works independently of Srs2 and Sgs1 [START_REF] Prakash | Saccharomyces cerevisiae MPH1 gene, required for homologous recombination-mediated mutation avoidance, encodes a 3' to 5' DNA helicase[END_REF]. Deletion of Mph1 results in an increased rate of mitotic COs. Purified Mph1 binds and unwinds D-loops, by dissociating the invading DNA strand from the template donor. Thus, Mph1 might promote repair of DSB by promoting the SDSA pathway [START_REF] Entian | Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach[END_REF][START_REF] Schurer | Yeast MPH1 gene functions in an error-free DNA damage bypass pathway that requires genes from Homologous recombination, but not from postreplicative repair[END_REF][START_REF] Gari | Remodeling of DNA replication structures by the branch point translocase FANCM[END_REF][START_REF] Sun | The FANCM ortholog Fml1 promotes recombination at stalled replication forks and limits crossing over during DNA double-strand break repair[END_REF][START_REF] Prakash | Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination[END_REF][START_REF] Zheng | Processing of DNA structures via DNA unwinding and branch migration by the S. cerevisiae Mph1 protein[END_REF]. Similarly, Fml1, the S. pombe homologue of Mph1, also prevents mitotic COs by disrupting D-loop intermediates [START_REF] Sun | The FANCM ortholog Fml1 promotes recombination at stalled replication forks and limits crossing over during DNA double-strand break repair[END_REF].

Homologous Recombination: a support for DNA replication

HR plays an important role during DNA replication, notably by ensuring the repair or restart of halted replication forks. In eukaryotes, DNA replication is initiated at multiple replication origins that are activated according to a spatio-temporal program (Huberman and Riggs 1966;Wu and Gilbert 1996;[START_REF] Kelly | Regulation of chromosome replication[END_REF][START_REF] Raghuraman | Replication dynamics of the yeast genome[END_REF][START_REF] Mechali | Eukaryotic DNA replication origins: many choices for appropriate answers[END_REF]. Once activated, a replication origin gives birth to two replication forks that progress bidirectionally until they merge with a converging fork. Once established, a single replication fork will replicate several tens of thousands of bases before meeting a converging fork.

During DNA synthesis, fork progression can be impeded by many obstacles that refer to Replication Fork Barriers (RFBs), including DNA damages, structure-forming sequences (inverted repeats, G-quadruplexes, hairpins), collisions between advancing forks and transcription machineries, and non-histone proteins bound to DNA (Lambert and Carr 2005;Mirkin and Mirkin 2007) (Figure n.7). In many organisms, natural RFBs results in replication forks pausing during each S-phase at specific loci such as centromeres, telomeres, replication slow zones, tRNA, ribosomal DNA and repeated sequences. Moreover, impediments to fork progression can destabilize the replication machinery and RFBs are known hot spots of recombination, chromosome breakages and rearrangements in yeast models. Thus, replication fork progression requires a well-orchestrated machinery to ensure forks movement, stability, reactivation and fusion with a converging fork. A single halted fork does not necessarily prevent the completion of DNA replication because the incoming of an opposite fork initiated from an adjacent origin will replicate up to the site of the arrested fork. Thus, in contrast to the bacterial chromosome replication, the rescue of impeded forks does not rely exclusively on fork-restart mechanisms, but also on activation of dormant origins (Figure n.7). Indeed, in response to replication stress, activation of dormant origins is recognized as an efficient mechanism ensuring complete chromosomal replication [START_REF] Woodward | Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress[END_REF][START_REF] Ge | Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress[END_REF][START_REF] Petermann | Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair[END_REF][START_REF] Kawabata | Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression[END_REF]. However, at unidirectional replication regions (as the ribosomal DNA locus), or when two converging forks are impeded (Figure n.7) or in regions of paucity in origins as human fragile sites, long travelling forks might have also more risk of accident and fork-restart mechanisms might then become essential to complete DNA replication (Le Tallec, Dutrillaux et al. 2011;[START_REF] Letessier | Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site[END_REF]).

Among fork-restart pathways, both the DNA replication checkpoint and HR are pivotal mechanisms in escorting the progression of replication forks. The integrity of replication forks is guaranteed by the DNA replication checkpoint that maintains the replisome in a replicationcompetent state to keep DNA polymerases associated with the site of nucleotide incorporation [START_REF] Katou | S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex[END_REF]Branzei and Foiani 2010;De Piccoli, Katou et al. 2012). How the DNA replication checkpoint modulates replisome activities to maintain it functional at the site of halted fork remains a current debate in the field. In addition, the DNA replication checkpoint also regulates nucleases activities (as Exo1 or Mus81) to preserve the integrity of forked structures [START_REF] Cotta-Ramusino | Exo1 processes stalled replication forks and counteracts fork reversal in checkpoint-defective cells[END_REF][START_REF] Kai | Replication checkpoint kinase Cds1 regulates Mus81 to preserve genome integrity during replication stress[END_REF][START_REF] Froget | Cleavage of stalled forks by fission yeast Mus81/Eme1 in absence of DNA replication checkpoint[END_REF]. In case of replisome malfunctioning (that refers to as collapsed forks in the literature) or loss of components of the replisome at broken forks, the resumption of DNA synthesis needs the replisome to be rebuilt. In eukaryotes, several evidences indicate that HR is able to promote resumption of DNA synthesis and to rebuild replication machinery at halted replication forks. In mammals, defects in HR result in a decreased fork velocity and, in budding yeast, in the accumulation of unreplicated gaps in response to replication-blocking agents [START_REF] Daboussi | A homologous recombination defect affects replication-fork progression in mammalian cells[END_REF][START_REF] Alabert | Differential regulation of homologous recombination at DNA breaks and replication forks by the Mrc1 branch of the S-phase checkpoint[END_REF]). These data further support the view that HR acts as fork-escort mechanism to support robust DNA replication, thanks to the ability of HR to repair ssDNA gaps left in the rear of moving forks, the ability of HR to restart or repair halted fork, and the ability of HR to protect stalled forks.

Post-replication repair by homologous recombination

Homologous recombination is an efficient mechanism to seal ssDNA gaps in the rear of the moving fork that has encountered a DNA lesion (Figure n.8,middle panel). Several evidences show that HR is required to complete DNA replication when the parental DNA is damaged.

For example, analysis of in vivo purified damaged replication forks by electronic microscopy have shown that ssDNA gaps accumulate both just at, and behind, the moving fork on both sister-chromatids, especially in the absence of HR [START_REF] Lopes | Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions[END_REF][START_REF] Hashimoto | Rad51 protects nascent DNA from Mre11dependent degradation and promotes continuous DNA synthesis[END_REF]. The formation of ssDNA gaps in the rear of the progressing fork implies that a re-priming event occurs when the synthesis of the leading strand is blocked.

While in vitro studies pointed out that the bacterial replisome is inherently tolerant to DNA damage, the ability of eukaryotic replisome to re-prime DNA synthesis on the leading strand remains to establish [START_REF] Yeeles | The Escherichia coli replisome is inherently DNA damage tolerant[END_REF]. To seal ssDNA gaps, homologous recombination copy the information from the undamaged sister-chromatids. This mechanism, which refers as gap filling or template switch in the literature, is expected to result in recombination intermediates between sister-chromatids. By analyzing replication intermediates, joints-molecules formed between sister-chromatids were identified in S-phase cells replicating parental DNA damaged by MMS-treatment (a basealkylating agent) [START_REF] Liberi | Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase[END_REF][START_REF] Branzei | Template switching: from replication fork repair to genome rearrangements[END_REF]. Although the exact nature of these joints-molecules are not clearly established, their genetic dependencies support the view that they correspond to recombination intermediates resulting from a strand exchange reaction by Rad51 between an intact and a gapped sister-chromatid in the rear of replication forks. Sister-chromatid joint-molecules would then be resolved by the helicase Sgs1 [START_REF] Branzei | Ubc9-and mms21-mediated sumoylation counteracts recombinogenic events at damaged replication forks[END_REF][START_REF] Mankouri | Holliday junction-containing DNA structures persist in cells lacking Sgs1 or Top3 following exposure to DNA damage[END_REF]. In budding yeast, RPA, the RMPs Rad52 and Rad55/Rad57, the 5'-3' nuclease Exo1 and the replicative DNA polymerase delta, but not epsilon, are all required to promote sister-chromatid junctions during replication of damaged template [START_REF] Vanoli | Replication and recombination factors contributing to recombination-dependent bypass of DNA lesions by template switch[END_REF]). Thus, it is possible that ssDNA gaps need to be first resected to recruit RMPs and for Rad51 nucleation on ssDNA gaps. Following strand invasion (either by the Rad51-coated ssDNA gap itself or by the 3' end of the newly synthetized strand of the gapped sister-chromatid), the DNA polymerase delta and its accessory subunit Pol32 would then seal the gap by extending the 3'end.

Replication fork restart and repair by homologous recombination

Both in yeast and mammalian cells, HR is able to restart halted replication forks. Fork passage through ssDNA nick or gap in the parental DNA results in a broken fork with a sisterchromatid being physically detached from the fork (Figure n.8 left panel). Such broken forks are efficiently repaired by homologous recombination through BIR mechanism [START_REF] Roseaulin | Mus81 is essential for sister chromatid recombination at broken replication forks[END_REF][START_REF] Clemente-Ruiz | Chromatin assembly controls replication fork stability[END_REF]Moriel-Carretero and Aguilera 2010;[START_REF] Hashimoto | RAD51-and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks[END_REF]. In this case, BIR allows the rebuilding of a replisome able to synthetize hundreds of kilobases. All components of DNA replication are required for BIR, except those involved in the assembly of pre-replication complexes (Lydeard, Lipkin-Moore et al. 2010). The DNA polymerase α and δ are necessary for the initial extension step while the DNA pol ε has a later requirement (only after 30 Kb of DNA synthesis). In addition, Pol32, the accessory pol  subunit, is required for BIR but dispensable for conventional DNA replication [START_REF] Lydeard | Break-induced replication and telomerase-independent telomere maintenance require Pol32[END_REF]Moriel-Carretero and Aguilera 2010). Thus, the establishment of a fully functional replication apparatus by HR requires time and maturation of the initial steps. Nonetheless, DNA synthesis associated with BIR is highly inaccurate [START_REF] Deem | Break-induced replication is highly inaccurate[END_REF].

Importantly, DSBs at replication forks are not a pre-requirement for recruitment of recombination factors. Indeed, efficient recruitment of recombination proteins was observed at replication forks impeded by DNA-bound proteins. In this case, fork recovery by HR was proposed to be DSB-independent [START_REF] Lambert | Gross chromosomal rearrangements and elevated recombination at an inducible site-specific replication fork barrier[END_REF][START_REF] Mizuno | Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism[END_REF][START_REF] Hashimoto | Rad51 protects nascent DNA from Mre11dependent degradation and promotes continuous DNA synthesis[END_REF][START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]. Moreover, similarly to BIR, halted forks recovered by HR are prone to replication slippage (Iraqui, Chekkal et al. 2012 in press). Thus, it appears that recombination-dependent replisomes are error-prone in contrast to origin-dependent replisomes.

A function of "fork-protection" for homologous recombination

Recent investigations have enlightened a novel function for HR as "fork-stabilizer" and how this function serves the robustness of replication forks progression. The first report on the role of BRCA2 (the main RMP in mammals) in stabilizing halted replication forks came from the lab of Venkitaraman [START_REF] Lomonosov | Stabilization of stalled DNA replication forks by the BRCA2 breast cancer susceptibility protein[END_REF]. In this study, the stability of unidirectional replication forks in the region of the ribosomal DNA (rDNA) was investigated by analysing replication intermediates in murine embryonic fibroblasts. Upon inhibition of DNA replication by HU treatment (an inhibitor of the ribonucleotide reductase leading to depletion of dNTP pool during S-phase), forks were unstable and were liable to breakages in the absence of BRCA2. Recent studies have provided molecular insights in the understanding of the "fork-stabilizer" function of HR. Schlacher and co-authors used DNA fibres techniques to analyse the dynamic of DNA synthesis upon inhibition and resumption of the elongation step. Newly replicated DNA at the fork, synthesized before inhibition of DNA synthesis, was found to be extensively resected in the absence of BRCA2 or Rad51 [START_REF] Schlacher | Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11[END_REF]. Resection of nascent strands is dependent on Mre11, and it was proposed that BRCA2 ensures fork protection by stabilizing the Rad51 filament at stalled fork, notably by regulating its ATPase activity [START_REF] Schlacher | A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2[END_REF]. These data establish that HR, in addition to repairing replicative DNA lesions, has a specific function during DNA replication by protecting the integrity of elongated strands at the replication fork.

Interestingly, Rad51 was previously proposed to protect nascent strands in xenopus and budding yeast [START_REF] Hashimoto | Rad51 protects nascent DNA from Mre11dependent degradation and promotes continuous DNA synthesis[END_REF]. Direct visualisation of in vivo purified forked structures by electronic microscopy has shown that ssDNA gaps accumulate at replication forks upon Rad51 depletion: gaps located behind the moving fork and gaps located at the three-way branched structure of the fork. ssDNA gaps in the rear of the fork result from the activity of the MRN complex, and are likely to reflect defects in post-replication repair by HR. More surprising are ssDNA gaps at the fork that are not dependent on MRN complex, and might reflect a role of HR in preventing uncoupling between leading and lagging strand synthesis. Even in the absence of exogenous DNA damages, 50% of replication forks contain ssDNA of at least 200 nucleotides at the forked structures when Rad51 is depleted. How homologous recombination maintains the coupling between leading and lagging strand synthesis is unclear, but these data support the view that HR escorts the robustness of DNA replication, and provide also molecular basis for a decreased fork speed in the absence of HR [START_REF] Daboussi | A homologous recombination defect affects replication-fork progression in mammalian cells[END_REF]).

How important is the "fork-stabilizer" function of HR? Replication-associated DSBs have been often observed in cells defective for HR. The initial view was that HR is required to repair spontaneous DSB associated with the process of DNA replication [START_REF] Sonoda | Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death[END_REF]). However, replicative DSBs might also be the consequence of defect in stabilizing halted forks that would then create secondary DNA damages such as breaks or incomplete replication [START_REF] Venkitaraman | Functions of BRCA1 and BRCA2 in the biological response to DNA damage[END_REF]). Thus, HR might have a role in preventing breaks by stabilizing replication forks, rather than exclusively by repairing breaks themselves.

Homologous recombination mediates chromosomal rearrangements

Due to its ability to promote genetic exchange between repeated sequences dispersed throughout the genome, HR can be also a source of chromosomal rearrangements. Eukaryotic genomes contain around 10% of repeated sequences, including Alu sequences, a variety of transposons, pseudo-genes and multigenic families sharing sequence homology. Thus, HR is also viewed as a "double-edged sword" for genome stability maintenance. Genome instability is a hallmark of cancers cells and inherited genome modifications are responsible for human genomic disorders (human diseases that result from genomic rearrangements) [START_REF] Lupski | Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits[END_REF]Aguilera and Gomez-Gonzalez 2008;[START_REF] Gu | Mechanisms for human genomic rearrangements[END_REF][START_REF] Lupski | Genomic disorders ten years on[END_REF]. Nonetheless, genetic variation is also the driving force of evolution and genomes must therefore display adequate plasticity to evolve while remaining sufficiently stable to prevent accumulation of mutations and chromosome rearrangements causing fitness disadvantage.

Non Allelic Homologous Recombination (NAHR)

Homologous recombination between dispersed repeated sequences is defined as Non-Allelic Homologous Recombination (NAHR) or ectopic recombination. NAHR outcomes consist of deletions, duplication, inversions, translocations, acentric and dicentric chromosomes (Figure n.9). NAHR accounts for most of the recurrent rearrangements observed in genomic disorders and some of chromosomal rearrangements occurring in cancer cells. 3) Sequence inversion after CO between inversed repeated sequences (NAHR). 4) Interchromosomal CO between repeated sequences leads to reciprocal translocation (NHAR). 5) Acentric and dicentric chromosomes formation after CO between interchromosomal inverted repeats [START_REF] Grabarz | Initiation of DNA double strand break repair: signaling and single-stranded resection dictate the choice between homologous recombination, non-homologous end-joining and alternative end-joining[END_REF].

NAHR is thought to be induced by DSB, resulting in genetic exchange between repeated sequences, associated or not with CO [START_REF] Lupski | Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits[END_REF]. While COs are responsible for major chromosomal rearrangements, gene conversion mediated by NAHR also results in genome modifications [START_REF] Grabarz | Initiation of DNA double strand break repair: signaling and single-stranded resection dictate the choice between homologous recombination, non-homologous end-joining and alternative end-joining[END_REF]. NAHR is also responsible for rearrangements leading to copy number variation of repeated sequences (Figure n. 9), during mitosis and meiosis [START_REF] Shaw | Non-recurrent 17p11.2 deletions are generated by homologous and non-homologous mechanisms[END_REF][START_REF] Liu | Mechanisms for recurrent and complex human genomic rearrangements[END_REF].

Crossing-over between repeated Alu sequences can lead to duplications/deletions, such as in Duchenne or Becker muscular dystrophy and in familiar hypercholesterolemia [START_REF] Lehrman | Duplication of seven exons in LDL receptor gene caused by Alu-Alu recombination in a subject with familial hypercholesterolemia[END_REF][START_REF] Goldmann | Genomic characterization of large rearrangements of the LDLR gene in Czech patients with familial hypercholesterolemia[END_REF][START_REF] Takeshima | Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center[END_REF][START_REF] Wang | Duchenne or Becker muscular dystrophy: a clinical, genetic and immunohistochemical study in China[END_REF][START_REF] Jelassi | Genomic characterization of two deletions in the LDLR gene in Tunisian patients with familial hypercholesterolemia[END_REF]. NAHR between repeated sequences on different chromosomes results in translocations and acentric/dicentric chromosome formation, with subsequent activation of oncogenes or inactivation of oncosuppressors as in the Philadelphia translocation (also known as Philidelphia chromosome) [START_REF] Onno | Rearrangement of the human tre oncogene by homologous recombination between Alu repeats of nucleotide sequences from two different chromosomes[END_REF][START_REF] Eckardt | Coexistence of translocation(1,19) and the Philadelphia chromosome in a child's first presentation of chronic myeloid leukemia in blast crisis treated with dasatinib[END_REF][START_REF] Gahrton | Historical note on the discovery of the Philadelphia chromosome[END_REF]. Moreover, crossover events can also lead to sequence inversions, responsible for mucopolysaccharidose II [START_REF] Bondeson | Inversion of the IDS gene resulting from recombination with IDS-related sequences is a common cause of the Hunter syndrome[END_REF][START_REF] Rathmann | Mucopolysaccharidosis type II (Hunter syndrome): mutation "hot spots" in the iduronate-2-sulfatase gene[END_REF][START_REF] Bunge | Homologous nonallelic recombinations between the iduronate-sulfatase gene and pseudogene cause various intragenic deletions and inversions in patients with mucopolysaccharidosis type II[END_REF].

NAHR can also result in Loss of Heterozygosity (LOH) when recombination occurs between a gene and a pseudogene (Figure n.10). The sequence of the functional gene is replaced by a homologous pseudogene, leading to a loss of function. For example, LOH is responsible for tumorigenesis like for restinoblastoma and squamous cell carcinoma [START_REF] Cavenee | Expression of recessive alleles by chromosomal mechanisms in retinoblastoma[END_REF][START_REF] De Andrade | A molecular study of first and second RB1 mutational hits in retinoblastoma patients[END_REF][START_REF] Lee | Loss of heterozygosity of tumor suppressor genes (p16, Rb, E-cadherin, p53) in hypopharynx squamous cell carcinoma[END_REF]. 2) A gene conversion event between a pseudo-gene and a functional gene can result in LOH and the subsequent inactivation of the functional gene [START_REF] Grabarz | Initiation of DNA double strand break repair: signaling and single-stranded resection dictate the choice between homologous recombination, non-homologous end-joining and alternative end-joining[END_REF].

NHAR requires a minimal length of homology between repeated sequences, as well as a relatively low level of heterology. The minimal length of homology is defined as the minimal efficient processing segment (MEPS) [START_REF] Rubnitz | The minimum amount of homology required for homologous recombination in mammalian cells[END_REF][START_REF] Waldman | Dependence of intrachromosomal recombination in mammalian cells on uninterrupted homology[END_REF]. Above a certain number of base pair homology corresponding to the MEPS, a direct correlation between the length of the sequence homology and HR efficiency has been established. When recombinant sequences are under the MEPS, HR efficiency drops dramatically. In mammalian cells, MEPS is found to be about 200-300 bp [START_REF] Ayares | Sequence homology requirements for intermolecular recombination in mammalian cells[END_REF][START_REF] Liskay | Homology requirement for efficient gene conversion between duplicated chromosomal sequences in mammalian cells[END_REF][START_REF] Lopez | Structural effect of donor DNA on the initiation of recombination for double strand break repair in human nuclear extracts[END_REF]) while in S.

cerevisiae the minimal size is between 26 and 250 bp [START_REF] Ahn | Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system[END_REF][START_REF] Jinks-Robertson | Substrate length requirements for efficient mitotic recombination in Saccharomyces cerevisiae[END_REF][START_REF] Sugawara | DNA length dependence of the single-strand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair[END_REF].

Chromosomal rearrangements by Template Exchange/Switch

Alternatively to canonical NAHR, other models responsible for chromosomal rearrangements mediated by micro-homology have been suggested. In these models, the initial event triggering rearrangement is thought to be linked to halted replication forks, and refers to Fork Stalling and Template Switching (FoSTeS). FoSTeS was initially proposed to describe nonrecurrent rearrangements observed in human genomic disorders: the replication forks stall, nascent strands switch template on the basis of micro-homology, initiate DNA synthesis on a non-contiguous template, and then anneal back to the initial template (Figure n.11) [START_REF] Slack | On the mechanism of gene amplification induced under stress in Escherichia coli[END_REF][START_REF] Lee | A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders[END_REF]. Because similar template switches of nascent strands were reported during BIR, it was then proposed that FoSTeS results from a BIRrelated mechanism mediated by micro-homology and in which HR would not be involved (MMBIR; for Micro-homology Mediated BIR, [START_REF] Lee | A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders[END_REF][START_REF] Zhang | The DNA replication FoSTeS/MMBIR mechanism can generate genomic, genic and exonic complex rearrangements in humans[END_REF]. Genetic and molecular studies have reported the existence of a similar mechanism in budding yeast: rearrangements resulting in segmental duplication are either mediated by homology and are then dependent on HR, or mediated by micro-homology and are independent of all known DNA repair pathways [START_REF] Payen | Segmental duplications arise from Pol32-dependent repair of broken forks through two alternative replication-based mechanisms[END_REF]. Similarly, the group of Ted Weinert reported chromosomal rearrangements induced by replication defects and independent of HR and canonical DSB repair pathways [START_REF] Paek | Fusion of nearby inverted repeats by a replicationbased mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast[END_REF]).

Thus, the FoSTeS or MMBIR model, initially proposed in human cells, was further supported by genetic data in yeast models. 2) DNA synthesis occurs on a non-contiguous template. 3) Several template exchange reactions can occur between multiple stalled forks. 4) then the resumption of replication occurs on the initial template [START_REF] Gu | Mechanisms for human genomic rearrangements[END_REF] The use of programmed Replication Fork Barrier (RFB) in fission yeast has been informative regarding the molecular mechanisms by which HR induces genome rearrangements during DNA replication. Ectopically placed RFBs behave as hot spot of recombination and genome rearrangements [START_REF] Ahn | Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast[END_REF][START_REF] Lambert | Gross chromosomal rearrangements and elevated recombination at an inducible site-specific replication fork barrier[END_REF]. Replication forks halted by the RFB are restarted by HR via a DSB-independent mechanism. During the restart event, nascent strands are proposed to be unwound and can exchange template with a nearby homologous sequence. This homology-driven template exchange of stalled nascent strands occurs as the fork restart and drives NAHR-type. Moreover, our lab recently established that inactivated forks rescued by HR are error-prone and liable to generate small deletions or duplications with micro-homology at the junctions (Iraqui, Chekkal et al. 2012 in press).

Altogether, these data establish that inactivated forks can result in both homology and microhomology-driven rearrangements in a recombination-dependent manner, pointing out a potential role for HR in FoSTeS in human cells (Figure n.12).

CHROMATIN ASSEMBLY DURING DNA REPLICATION

The structural organization of DNA into chromatin is of key importance to regulate genome expression, organization and stability. The basic unit of the chromatin is conserved in all eukaryotes and it is termed the "nucleosome" [START_REF] Kornberg | Chromatin structure: a repeating unit of histones and DNA[END_REF][START_REF] Bradbury | Nucleosomes, histone interactions, and the role of histones H3 and H4[END_REF]).

The first level of chromatin organization is the wrapping of the DNA around the nucleosomes, these latter being successively enveloped in a 30 nm chromatin fiber [START_REF] Kornberg | Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome[END_REF][START_REF] Hayes | Nucleosomes and the chromatin fiber[END_REF]. During meiosis and mitosis, the 30 nm nucleosome array is further packed in metaphase chromosomes (Figure n.13). The chromatin fiber is organized in either euchromatic or heterochromatic domains. Heterochromatin is defined as regions of chromatin that remain cytologically condensed and densely stained throughout the cell cycle, whereas euchromatin is de-condensed during interphase [START_REF] Heitz | Das Heterochromatin der Moose" Jahrbuck wiss[END_REF].

DNA replication occurs in a chromatin context, therefore major changes in chromatin structure are necessary for the replication fork to progress [START_REF] Almouzni | Assembly of spaced chromatin promoted by DNA synthesis in extracts from Xenopus eggs[END_REF][START_REF] Almouzni | Chromatin assembly on replicating DNA in vitro[END_REF]). The nucleosome flow from ahead to behind the replication fork has to be orchestrated with the maintenance of epigenetic information, through the introduction of specific epigenetic marks [START_REF] Sogo | Structure of replicating simian virus 40 minichromosomes. The replication fork, core histone segregation and terminal structures[END_REF][START_REF] Jackson | Deposition of newly synthesized histones: hybrid nucleosomes are not tandemly arranged on daughter DNA strands[END_REF][START_REF] Ransom | Chaperoning histones during DNA replication and repair[END_REF]. Moreover, DNA replication of heterochromatin domains requires additional replication factors, due to the tightly compaction of the chromatin fiber [START_REF] Taddei | Duplication and maintenance of heterochromatin domains[END_REF][START_REF] Ridgway | Chromatin assembly and organization[END_REF][START_REF] Nakatani | Two distinct nucleosome assembly pathways: dependent or independent of DNA synthesis promoted by histone H3.1 and H3.3 complexes[END_REF]. In this chapter, the mechanisms of nucleosome assembly and disassembly during DNA replication are presented with a focus on the histone chaperone CAF-1.

Nucleosome structures

The name "chromatin" was given by Flemming in 1882, based on the property to retain basic dyes, and it derives from the Greek khroma (coloured). The nucleosome contains about 146 DNA base pairs, wrapped around an octamer of positively charged proteins termed "histones" [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF]. Eukaryotes present five conserved families of histones: H1/H5, H2A, H2B, H3, and H4. Histones H2A, H2B, H3 and H4 have been identified as "core histones", while histones H1 and H5 as "linker histones".

The nucleosome is constituted of a core of tetrameric (H3-H4) 2 , associated with two dimers H2A-H2B (Figure n.14). The molecular weight of each core histone is comprised between 11 and 16 kDa, with more than 20% of their amino acid composition being lysines and arginines [START_REF] Kornberg | Chromatin structure: a repeating unit of histones and DNA[END_REF][START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF]). H3 and H4 histones are among the most conserved proteins in all organisms, while H2B and H2A display an appreciable sequence variation among different species. In most cells, a further level of chromatin compaction is present due to the association of nucleosome cores with linker histone H1/H5. In addition to binding to nucleosomes, the H1 or H5 histones bind to the "linker DNA" (DNA region between nucleosomes), facilitating the stabilization of the 30 nm chromatin fiber (Crane-Robinson, [START_REF] Crane-Robinson | Structural studies of chicken erythrocyte histone H5[END_REF][START_REF] Aviles | The conformation of histone H5. Isolation and characterisation of the globular segment[END_REF].

In mammalian cells, different histone variants are present [START_REF] Zweidler | Resolution of histones by polyacrylamide gel electrophoresis in presence of nonionic detergents[END_REF]). These minor forms of "replacement histones" are non-allelic isoforms of major type histones. Histones variants affect the structure and stability of nucleosome and chromatin, and regulate the access of DNA metabolism enzymes to DNA (transcription machinery, DNA damage repair proteins, etc.) [START_REF] Cohen | Stage-specific switches in histone synthesis during embryogenesis of the sea urchin[END_REF][START_REF] Franklin | Non-allelic variants of histones 2a, 2b and 3 in mammals[END_REF]. A classic example is the histone variant H3.1, which is loaded onto replicating DNA, while H3.3 replaces H3.1 in transcribed regions of the genome [START_REF] Ahmad | Histone H3 variants specify modes of chromatin assembly[END_REF][START_REF] Tagami | Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis[END_REF][START_REF] Orsi | Epigenetic and replacement roles of histone variant H3.3 in reproduction and development[END_REF][START_REF] Ray-Gallet | Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity[END_REF]. Another example is the histone H3-like CENP-A, which appears restricted to centromeric chromatin [START_REF] Black | Structural determinants for generating centromeric chromatin[END_REF][START_REF] Dunleavy | H3.3 is deposited at centromeres in S phase as a placeholder for newly assembled CENP-A in G(1) phase[END_REF]) and which could be responsible for the maintenance of centromere identity in eukaryotes [START_REF] Sullivan | Human CENP-A contains a histone H3 related histone fold domain that is required for targeting to the centromere[END_REF]). The histone variant H2A.Z is associated with promoters of actively transcribed genes and is involved in heterochromatin metabolism [START_REF] Guillemette | Variant histone H2A.Z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning[END_REF][START_REF] Billon | Precise deposition of histone H2A.Z in chromatin for genome expression and maintenance[END_REF], while the histone variant H2A.X, which is a mark of DNA double strand breaks, is phosphorylated by checkpoint kinases in response to replication stress and DNA damages, leading to γH2AX [START_REF] Rogakou | DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139[END_REF][START_REF] Rogakou | Megabase chromatin domains involved in DNA double-strand breaks in vivo[END_REF][START_REF] Paull | A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage[END_REF][START_REF] Van Attikum | ATP-dependent chromatin remodeling and DNA double-strand break repair[END_REF]. However, in fission yeast such discrimination among different variants does not exist. Histone H3 and H4 are encoded by three identical genes and the only variant is CenpA, a centromere specific histone H3 variant [START_REF] Lando | Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast[END_REF].

The histone fold domain is involved in histone/histone and histone/DNA interactions and is formed by three α-helixes, connected by two loops. The N-terminal domain is composed of about 15-30 highly positively charged amino acids, which protrude out of the nucleosome surface [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF]). The N-terminal tails are the main sites of several covalent post-translational modifications (PTMs), including acetylation, methylation and phosphorylation, which are involved in the regulation of chromatin dynamics, structure and expression [START_REF] Brownell | Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation[END_REF][START_REF] Hassig | A role for histone deacetylase activity in HDAC1mediated transcriptional repression[END_REF][START_REF] Loyola | Histone chaperones, a supporting role in the limelight[END_REF][START_REF] Park | Histone chaperones in nucleosome eviction and histone exchange[END_REF]. These modifications could participate in a sort of "histone code", in which histone modifications define different chromatin state through the recruitment of specific proteins [START_REF] Strahl | The language of covalent histone modifications[END_REF].

Chromatin assembly and disassembly at replication forks

During DNA synthesis, a transient disruption of nucleosomes occurs ahead of the replication fork. Nucleosomes are then successively transferred onto the nascent strands, behind the moving fork. At the same time, the deposition of newly synthesized histones is essential to restore the correct nucleosome density along the daughter strands (de novo assembly) (Figure n.15) [START_REF] Sogo | Structure of replicating simian virus 40 minichromosomes. The replication fork, core histone segregation and terminal structures[END_REF][START_REF] Jackson | Deposition of newly synthesized histones: hybrid nucleosomes are not tandemly arranged on daughter DNA strands[END_REF][START_REF] Nakatani | Two distinct nucleosome assembly pathways: dependent or independent of DNA synthesis promoted by histone H3.1 and H3.3 complexes[END_REF][START_REF] Ransom | Chaperoning histones during DNA replication and repair[END_REF]).

Different models have been proposed to explain the transfer of parental nucleosomes onto newly replicated DNA. A first model suggests that parental nucleosomes are disrupted into two H2A-H2B dimers and one (H3-H4) 2 tetramer. The entire (H3-H4) 2 tetramer is transferred onto the nascent DNA strand to form a subnucleosome structure. Thereafter, either old or newly synthesized H2A-H2B dimers are added to the subnucleosome structure [START_REF] Jackson | Deposition of newly synthesized histones: hybrid nucleosomes are not tandemly arranged on daughter DNA strands[END_REF]). However, available data do not exclude that a different segregation mechanism leads to a further dissociation of parental (H3-H4) 2 tetramers into two H3-H4 dimers. Therefore, both parental and newly synthesized H3-H4 could be concomitantly present in the same nucleosome deposited onto newly replicated DNA. This could facilitate the incorporation of pre-existing modified histones to newly synthesized histones (Tagami, Ray-Gallet et al. Figure n. 14: (H3-H4) 2 tetramer is held together via a tight network of hydrogen bonds and salt bridges that are confined to a small region defined by a four-helix bundle structure. DNA is shown in gray, H2A in yellow, H2B in red, H3 in blue and H4 in green. Solid ovals represent the regions of interaction between H2A-H2B and (H3-H4) 2 . The dashed line represents the molecular two-fold axis dissecting the H3 four-helix bundle. H2A-H2B dimer makes intimate contacts with both arms of the W-shaped (H3-H4) 2 tetramer. These contacts involve the docking domain of H2A that interacts with the C-terminal β strand in H4 and parts of H3 on one side, and a four helix bundle between the histone fold regions of H4 and H2B. A minor contact made by the H2A L1 loops exists between the two H2A-H2B dimers. The interactions linking H2A-H2B dimers to (H3-H4) 2 tetramer cannot persist in the absence of DNA and thus the histone octamer is not stable under physiological conditions [START_REF] Park | Histone chaperones in nucleosome eviction and histone exchange[END_REF]. 48 2004). These two mechanisms of parental histone segregation could also be differently used during DNA replication or during chromatin remodeling after DNA repair [START_REF] Groth | Regulation of replication fork progression through histone supply and demand[END_REF][START_REF] Groth | Replicating chromatin: a tale of histones[END_REF].

Histones are positively charged proteins and have a general affinity for the negatively charged DNA. Therefore, during DNA replication, histones chaperones are necessary to avoid an indiscriminate bound of histones to DNA. Histone chaperones represent a class of highly conserved eukaryotic proteins that bind single or complexes histones, and release them in a controlled way onto DNA, without being part of the final product [START_REF] Laskey | Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA[END_REF][START_REF] Ray-Gallet | HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis[END_REF][START_REF] Polo | Chromatin assembly: a basic recipe with various flavours[END_REF]. Histone chaperones count many members (Table n.3) that play a key role in the maintenance of genome stability by ensuring proper chromatin structure throughout the cell cycle. Histone chaperones have different relative affinities for histone and histone variants and this might trigger the efficiency by which histones are incorporated into or removed from chromatin [START_REF] Polo | Chromatin assembly: a basic recipe with various flavours[END_REF][START_REF] Eitoku | Histone chaperones: 30 years from isolation to elucidation of the mechanisms of nucleosome assembly and disassembly[END_REF][START_REF] Park | Histone chaperones in nucleosome eviction and histone exchange[END_REF]). Among the H3-H4 histone chaperones, the Chromatin Assembly Factor-1 (CAF-1) appears to have a major role, together with Anti Silencing Factor 1 (Asf1), in assembling nucleosomes onto newly synthesized DNA during replication and repair [START_REF] Krude | Chromatin assembly during DNA replication in somatic cells[END_REF].

Figure n.15:

Nucleosomes metabolism during DNA replication. Parental histones are evicted ahead of the moving fork, and then transferred onto the daughter strands, in a manner that allows histone variants and histone modifications to be maintained. This segregation mechanism operates together with de novo nucleosome assembly to duplicate nucleosomal density and epigenetic information on both daughter strands [START_REF] Groth | Regulation of replication fork progression through histone supply and demand[END_REF].

The role of histone chaperones during DNA replication

During eukaryotic DNA replication, nucleosomes are transiently disrupted ahead of the fork, allowing the replication machinery to progress along the parental DNA. The replicative helicase MCM2-7, together with Cdc45 and the GINS complex (responsible for binding to both chromatin and DNA replication-associated proteins), mediate DNA unwinding, while the FACT (Facilitates Chromatin Transcription complex) complex removes H2A-H2B dimers (Figure n. 16 left). Asf1 interacts with MCM2-7 and triggers the disruption of the remaining parental (H3-H4) 2 tetramer [START_REF] Groth | Regulation of replication fork progression through histone supply and demand[END_REF]. Released histones held in close proximity to the fork, and then segregate onto daughter strands. In this way, parental histones act as a fingerprint for specific post-translational modifications for the newly synthesized histones [START_REF] Natsume | Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4[END_REF][START_REF] Groth | Replicating chromatin: a tale of histones[END_REF][START_REF] Clemente-Ruiz | Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks[END_REF][START_REF] Tanae | Histone chaperone Asf1 plays an essential role in maintaining genomic stability in fission yeast[END_REF]). [START_REF] Das | The histone shuffle: histone chaperones in an energetic dance[END_REF].

Table n.3: Classification of H3-H4 histone chaperones in mammalian cells (adapted from

To maintain correct nucleosome density along the DNA fiber, newly synthesized histones have to be deposited together with parental histones. An important conceptual point is that both evicted and newly synthesized H3-H4 dimers do not associate as tetramers prior to their deposition onto DNA [START_REF] Polo | Chromatin assembly: a basic recipe with various flavours[END_REF]. Indeed, H3 and H4 are firstly loaded onto the daughter strands and then, H2A and H2B are added by the histone chaperone NAP1, to form a mature nucleosome (Figure n. 16 top). Nucleosome assembly onto newly replicated DNA is achieved by the concerted action of several histones H3-H4 chaperones such as CAF-1, Asf1 and Rtt106. However, it is not clear how the actions of these three histone chaperones are coordinated to achieve rapid deposition of histones onto nascent DNA. Asf1 and CAF-1 are known to interact with components of the replication machinery but how Rtt106 is recruited at the replication fork is unknown [START_REF] Li | Acetylation of histone H3 lysine 56 regulates replicationcoupled nucleosome assembly[END_REF]). Asf1 itself does not deposit histones onto replicating DNA, at least in vitro, but synergizes with CAF-1 in replicationcoupled nucleosome assembly [START_REF] Tyler | Interaction between the Drosophila CAF-1 and ASF1 chromatin assembly factors[END_REF][START_REF] Mello | Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway[END_REF]. The interaction between CAF-1 and Asf1 allows the transfer of H3-H4 dimer from Asf1 to CAF-1.

CAF-1 assembles preferentially newly synthesized H3-H4 dimers during DNA replication both on leading and lagging strands (Figure n. 16 right). Rtt106 might physically interact with CAF-1 but it can also function independently of CAF-1 during DNA replication, RNA transcription and heterochromatin assembly [START_REF] Huang | Rtt106p is a histone chaperone involved in heterochromatin-mediated silencing[END_REF][START_REF] Zunder | Two surfaces on the histone chaperone Rtt106 mediate histone binding, replication, and silencing[END_REF].

Importantly, newly synthetized histones harbors specific post-translational modifications including the acetylation of the lysine K56 on histone H3 (H3K56Ac), at least in yeast models. The histone mark H3K56Ac is promoted by the acetyl transferase Rtt109 that exhibits closed interactions with Asf1 and CAF-1 [START_REF] Jackson | Modifications to histones immediately after synthesis[END_REF][START_REF] Sobel | Conservation of deposition-related acetylation sites in newly synthesized histones H3 and H4[END_REF][START_REF] Verreault | Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4[END_REF][START_REF] Verreault | Nucleosomal DNA regulates the core-histonebinding subunit of the human Hat1 acetyltransferase[END_REF][START_REF] Imhof | Purification and properties of the Xenopus Hat1 acetyltransferase: association with the 14-3-3 proteins in the oocyte nucleus[END_REF][START_REF] Kolonko | Catalytic activation of histone acetyltransferase Rtt109 by a histone chaperone[END_REF][START_REF] Clemente-Ruiz | Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks[END_REF][START_REF] Su | Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106[END_REF]. Asf1 transfer H3-H4 dimers to CAF-1. CAF-1 deposits H3-H4 dimers on leading and lagging strand, via its interaction with PCNA. Histone chaperone NAP1 loads H2A-H2B dimers on the (H3-H4) 2 tetramer, to form a complete nucleosome [START_REF] Groth | Replicating chromatin: a tale of histones[END_REF].

Chromatin Assembly Factor 1

In 1986, Stillman identified an activity that preferentially assembles nucleosomes onto replicating DNA [START_REF] Stillman | Chromatin assembly during SV40 DNA replication in vitro[END_REF]). In 1989, Smith and Stillman purified and characterized a replication-dependent chromatin assembly factor: "Chromatin Assembly Factor 1" (CAF-1) [START_REF] Smith | Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro[END_REF]. This study was performed in a cell-free system, in which purified proteins can assemble chromatin during DNA replication and this provided an experimental system to investigate chromatin assembly in vitro. This system was based on the papovavirus SV40, which replicates in the nucleus of the host cell as a circular chromosome. Moreover, SV40 nucleosomes structure and histones composition are identical to those of the host [START_REF] Smith | Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro[END_REF].

Human purified CAF-1 consists of three subunits: p150, p60 and p48 which copurify in a trimeric protein complex [START_REF] Stillman | Chromatin assembly during SV40 DNA replication in vitro[END_REF][START_REF] Smith | Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro[END_REF][START_REF] Kaufman | The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication[END_REF][START_REF] Verreault | Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4[END_REF][START_REF] Kaufman | Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I[END_REF]. Since 1989, functional homologues of CAF-1 have been identified in many other organisms such as mouse, Xenopus, Drosophila, Arabidopsis, chicken and yeasts. In fission yeast, CAF-1 complex is composed of three subunits, called Pcf1 (p150 homologue), Pcf2 (p60 homologue) and Pcf3 (p48 homologue). The function of CAF-1 in promoting DNA replication-dependent de novo nucleosome assembly is evolutionary conserved, thus enabling studies in model organisms to characterize CAF-1 features.

The largest subunit of CAF-1: p150

Human p150, Drospohila p180, budding yeast Cac1 and fission yeast Pcf1 share primary structure homology [START_REF] Kaufman | The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication[END_REF][START_REF] Enomoto | RLF2, a subunit of yeast chromatin assembly factor-I, is required for telomeric chromatin function in vivo[END_REF][START_REF] Kaufman | Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I[END_REF][START_REF] Tyler | Interaction between the Drosophila CAF-1 and ASF1 chromatin assembly factors[END_REF]. The largest subunit of human CAF-1, p150, interacts preferentially with newly synthesized histone through its KER and ED domains, also defined as "acidic domain" (Figure n.17A). These domains contain an internal cluster of negatively charged amino acids implicated in histone binding. Deletion of the acidic domain abrogates the binding of p150/p60 to H3 and H4, without affecting the interaction between p150 and p60 [START_REF] Kaufman | The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication[END_REF].

The C-terminal domain of p150 is involved in the dimerization of p150 and in the interaction with p60 [START_REF] Kaufman | The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication[END_REF][START_REF] Quivy | Dimerization of the largest subunit of chromatin assembly factor 1: importance in vitro and during Xenopus early development[END_REF]. The C-terminal domain contains a high affinity binding site for the p60 protein, which cannot be deleted without abolishing the chromatin assembly activity. The presence of an acidic cluster in this region could be involved in the interaction with other proteins or for the correct three- dimensional folding of the protein [START_REF] Kaufman | The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication[END_REF]. Human p150 oscillates between a dimeric and a monomeric state in vivo and its phosphorylation by the Cdc7-Dbf4 kinase (DDK) stabilizes p150 as a monomer. The phosphorylation of p150 favors its interaction with the homotrimeric sliding clamp PCNA (proliferating cell nuclear antigen), a component of the replication machinery [START_REF] Quivy | Dimerization of the largest subunit of chromatin assembly factor 1: importance in vitro and during Xenopus early development[END_REF][START_REF] Gerard | The replication kinase Cdc7-Dbf4 promotes the interaction of the p150 subunit of chromatin assembly factor 1 with proliferating cell nuclear antigen[END_REF]). Thus, the phosphorylation of p150 and its dimerization state is thought to be a control point regulating the chromatin assembly function of CAF-1.

The N-terminal of p150 is composed of the first 296 amino acids and is characterized by large clusters of acidic residues together with a region enriched in proline, glutamic acid, serine, threonine and aspartic acid residues (PEST region) [START_REF] Rogers | Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis[END_REF][START_REF] Kaufman | The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication[END_REF]. The PEST region can be a signal for the rapid degradation of the protein [START_REF] Rogers | Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis[END_REF]) but is dispensable for the chromatin assembly activity. The N-terminal of the p150 presents also a MIR domain containing three hydrophobic residues (consensus sequence: PxVxL) that are essential for its interaction with the Heterochromatin Protein 1 (HP1). The interaction between CAF-1 and HP1 is crucial for the maintenance of heterochromatin. However, in vitro, the MIR domain is dispensable for the replicationcoupled assembly activity of CAF-1 [START_REF] Murzina | Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins[END_REF].

The recruitment of CAF-1 at the replication fork involves a physical interaction between p150 subunit and PCNA [START_REF] Shibahara | Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin[END_REF][START_REF] Moggs | A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage[END_REF][START_REF] Krawitz | Chromatin assembly factor I mutants defective for PCNA binding require Asf1/Hir proteins for silencing[END_REF][START_REF] Ben-Shahar | Two fundamentally distinct PCNA interaction peptides contribute to chromatin assembly factor 1 function[END_REF]). The interaction between the largest subunit of CAF-1 and PCNA is crucial for the recruitment of CAF-1 at the replication fork and for its chromatin assembly function. PCNA is an essential processivity factor that encircles double-stranded DNA and acts as a sliding clamp to tether DNA polymerases to their substrate, during DNA synthesis. PCNA binds several replication enzymes, such as DNA ligase 1, RFC (replication factor C), DNA polymerase δ, FEN1 (Flap structure-specific endonuclease 1) [START_REF] Montecucco | DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories[END_REF][START_REF] Gomes | Two modes of FEN1 binding to PCNA regulated by DNA[END_REF][START_REF] Johansson | The Pol32 subunit of DNA polymerase delta contains separable domains for processive replication and proliferating cell nuclear antigen (PCNA) binding[END_REF]). Furthermore, PCNA also directly binds to enzymes involved in epigenetic inheritance, such as the DNA methyltransferase DNMT1 in human cells [START_REF] Chuang | Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1[END_REF][START_REF] Warbrick | PCNA binding through a conserved motif[END_REF]). As mentioned above, PCNA interacts also with CAF-1 to recruit this histone chaperone at DNA replication forks The first 31 residues of human p150 binds to PCNA in vitro and in vivo, through a particular motif termed PIP-box (for PCNA Interacting Protein) (Figure n.18) [START_REF] Shibahara | Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin[END_REF][START_REF] Moggs | A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage[END_REF][START_REF] Krawitz | Chromatin assembly factor I mutants defective for PCNA binding require Asf1/Hir proteins for silencing[END_REF]. Moreover, the mutation of the consensus sequence of the PIP-box has no impact on nucleosome assembly in vitro. A degenerated PCNA-interacting region within the KER domain of human p150 has been identified (PIP2) [START_REF] Moggs | A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage[END_REF][START_REF] Ben-Shahar | Two fundamentally distinct PCNA interaction peptides contribute to chromatin assembly factor 1 function[END_REF]). The PIP2 motif lacks a key conserved residue (Figure n.19).

Despite its weaker interaction with PCNA, the PIP2 motif is of crucial importance for replication-dependent nucleosome assembly in vitro and for targeting CAF-1 to DNA replication foci in vivo [START_REF] Ben-Shahar | Two fundamentally distinct PCNA interaction peptides contribute to chromatin assembly factor 1 function[END_REF].

The weak binding of p150 with PCNA is explained on the basis of the available crystal structures of canonical PIPs in complex with PCNA. In these structures, the carbonyl and the amino groups of the lateral chain of the highly conserved glutamine within the canonical PIP motif are involved in several interactions with PCNA. In addition, two positively charged residues of PCNA are in close proximity to the glutamine side chain. In the PIP2 motif of human p150, the conserved glutamine residue of canonical PIP boxes is replaced by a lysine.

Due to the loss of the multiple interactions mediated by the glutamine side chain and the proximity of positively charged PCNA residues, a lysine at that position would considerably weaken PCNA binding [START_REF] Gulbis | Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA[END_REF][START_REF] Bowman | Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex[END_REF]Bruning and Shamoo 2004;[START_REF] Chapados | Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair[END_REF]Hishiki, Hashimoto et al. 2009;[START_REF] Ben-Shahar | Two fundamentally distinct PCNA interaction peptides contribute to chromatin assembly factor 1 function[END_REF]). Molecular surface of the PCNA-PIP complex, with PCNA colored according to electrostatic potential and the PIP peptide showed in white. Regions of intense positive charge appear blue, and electronegative regions of the surface are red [START_REF] Gulbis | Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA[END_REF].

In budding yeast, the largest subunit of CAF-1, named Cac1, binds PCNA as well [START_REF] Zhang | PCNA connects DNA replication to epigenetic inheritance in yeast[END_REF]. This interaction occurs via a canonical PIP-box (PIP1) (Figure n.19). Indeed, yeast Cac1 exhibits only one canonical PIP-box motif, in contrast to human p150.

Mutations in the PIP-box of Cac1 abolish its interaction with PCNA, resulting in a severe impairment in nucleosome assembly in vitro and mild effects in gene silencing. Importantly, mutations of the PIP-box do not affect the assembly of CAF-1 complex, as Cac1 remains able to interact with Cac3, the third subunit (the budding yeast homologue of human p48) [START_REF] Krawitz | Chromatin assembly factor I mutants defective for PCNA binding require Asf1/Hir proteins for silencing[END_REF]. A second consensus PIP-box was identified in Cac2 subunit of CAF-1 (the budding yeast homologue of human p60). The function of this PIP-box remains unclear.

Nonetheless, a single mutation in the PIP-box of Cac1 is sufficient to abolish the interaction between CAF-1 and PCNA and to affect the chromatin assembly function in vitro. Thus, it appears that potential interactions between Cac2 and PCNA are not sufficient to promote a robust chromatin assembly activity [START_REF] Krawitz | Chromatin assembly factor I mutants defective for PCNA binding require Asf1/Hir proteins for silencing[END_REF].

The p60 subunit of CAF-1

The human p60, Drosophila p105, the budding yeast Cac2 and the fission yeast Pcf2 are members of protein family of tryptophan-aspartate (WD)-repeat [START_REF] Kaufman | The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication[END_REF][START_REF] Verreault | Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4[END_REF]. Such proteins contain four to eight WD repeated motifs; each one is approximately of 40 amino acids in length (Figure 17B). The human p60 has seven WD repeats, although the fifth and sixth repeats show significant divergences from canonical residues [START_REF] Verreault | Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4[END_REF]. WD repeats are proposed to mediate the interaction between p60 and p48 (the third subunit of CAF-1), which is also a member of (WD)-repeat protein family [START_REF] Komachi | The WD repeats of Tup1 interact with the homeo domain protein alpha 2[END_REF][START_REF] Kaufman | The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication[END_REF][START_REF] Kaufman | Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I[END_REF][START_REF] Tyler | Interaction between the Drosophila CAF-1 and ASF1 chromatin assembly factors[END_REF].

The p60 subunit physically interacts with the large subunit p150 and the deletion of p60 binding domain in the C-terminal part of p150 subunit impairs the nucleosome assembly activity [START_REF] Kaufman | The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication[END_REF]. The human p60 subunit also exhibits a C-terminal PEST domain and a B-domain-like motif that bind to Asf1 [START_REF] Malay | Crystal structures of fission yeast histone chaperone Asf1 complexed with the Hip1 B-domain or the Cac2 C terminus[END_REF]. The interaction between CAF-1 and Asf1 is crucial during replication-coupled nucleosome assembly to allow the transfer of histones from Asf1 to CAF-1 [START_REF] Mello | Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway[END_REF][START_REF] Tang | Structure of a human ASF1a-HIRA complex and insights into specificity of histone chaperone complex assembly[END_REF].

Another interesting aspect is that both p60 and p150 contains consensus target sites for various kinases [START_REF] Smith | Immunological characterization of chromatin assembly factor I, a human cell factor required for chromatin assembly during DNA replication in vitro[END_REF][START_REF] Kaufman | The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication[END_REF][START_REF] Martini | Recruitment of phosphorylated chromatin assembly factor 1 to chromatin after UV irradiation of human cells[END_REF]. The human p60 is phosphorylated by Cyclin/Cdk activities in vitro [START_REF] Keller | Requirement of Cyclin/Cdk2 and protein phosphatase 1 activity for chromatin assembly factor 1-dependent chromatin assembly during DNA synthesis[END_REF] and this could be a way to regulate CAF-1 activity. Indeed p60 phosphorylation correlates with the cell cycle dependent regulation of CAF-1 activity and localization [START_REF] Marheineke | Nucleosome assembly activity and intracellular localization of human CAF-1 changes during the cell division cycle[END_REF][START_REF] Martini | Recruitment of phosphorylated chromatin assembly factor 1 to chromatin after UV irradiation of human cells[END_REF].

The p48 subunit of CAF-1

Human p48, Drosophila p55, budding yeast Cac3 and fission yeast Pcf3 present a large domain containing WD repeats (Figure 17C) [START_REF] Tyler | The p55 subunit of Drosophila chromatin assembly factor 1 is homologous to a histone deacetylase-associated protein[END_REF][START_REF] Verreault | Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4[END_REF][START_REF] Kaufman | Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I[END_REF]. The human p48 subunit, in complex with p150 and p60, binds the H4 histone during DNA replication-coupled chromatin assembly [START_REF] Verreault | Nucleosomal DNA regulates the core-histonebinding subunit of the human Hat1 acetyltransferase[END_REF].

The p48 and the p46 proteins are homologue histone chaperones that play key roles in establishing and maintaining chromatin structure. Both p48 and p46 can interact with core histones H2A and H4 in vitro. The interacting domains between H4 and p48 and p46 were mapped in S. cerevisiae and Xenopus and data showed that p48 and p46 bind directly to helix1 of histone H4 [START_REF] Verreault | Nucleosomal DNA regulates the core-histonebinding subunit of the human Hat1 acetyltransferase[END_REF][START_REF] Vermaak | Functional analysis of the SIN3-histone deacetylase RPD3-RbAp48-histone H4 connection in the Xenopus oocyte[END_REF][START_REF] Murzina | Structural basis for the recognition of histone H4 by the histone-chaperone RbAp46[END_REF]). However, despite p46 and p48 present 90% identical at the amino acid sequence level, in both human and yeast cells p46 is found exclusively in complexes with histone acetyltransferase 1 (HAT1), whereas p48 is instead found in the CAF-1 complex [START_REF] Parthun | The major cytoplasmic histone acetyltransferase in yeast: links to chromatin replication and histone metabolism[END_REF][START_REF] Taunton | A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p[END_REF][START_REF] Martinez-Balbas | Drosophila NURF-55, a WD repeat protein involved in histone metabolism[END_REF][START_REF] Verreault | Nucleosomal DNA regulates the core-histonebinding subunit of the human Hat1 acetyltransferase[END_REF].

Although the interaction between p48 and the N-terminal of H4 occurs, it has been demonstrated that deletion of both H3 and H4 N-terminal domains does not affect CAF-1mediated nucleosome deposition onto newly synthesized DNA in vitro. Moreover, each of CAF-1 subunit binds the recombinant (H3-H4) 2 tetramers lacking the N-terminal domains of both H3 and H4 [START_REF] Shibahara | The N-terminal domains of histones H3 and H4 are not necessary for chromatin assembly factor-1-mediated nucleosome assembly onto replicated DNA in vitro[END_REF]. Thus, CAF-1 interacts with histone H3-H4 by multiple interactions. However, it is well known that in human cells and in budding yeast, that deletion of any one of CAF-1 subunit leads to a loss of function of the entire complex in vivo [START_REF] Kaufman | Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I[END_REF][START_REF] Linger | The yeast histone chaperone chromatin assembly factor 1 protects against double-strand DNA-damaging agents[END_REF].

The p48 protein was found in CAF-1 complex in stoichiometric amounts, but p48 was also found uncomplexed to p150 and p60 in cytosolic replication extracts [START_REF] Smith | Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro[END_REF][START_REF] Verreault | Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4[END_REF][START_REF] Marheineke | Nucleosome assembly activity and intracellular localization of human CAF-1 changes during the cell division cycle[END_REF]. Indeed, in a broad range of species, human p48 is present in several chromatin-related complexes involved in histone metabolism, including the histone deacetylase 1 complex (HDAC1), histone deacetylase 2 complex (HDAC2), the histone methyltransferase complex ESC-E(Z) and the ATP-dependent nucleosome remodeling complex NURF [START_REF] Parthun | The major cytoplasmic histone acetyltransferase in yeast: links to chromatin replication and histone metabolism[END_REF][START_REF] Taunton | A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p[END_REF][START_REF] Martinez-Balbas | Drosophila NURF-55, a WD repeat protein involved in histone metabolism[END_REF][START_REF] Verreault | Nucleosomal DNA regulates the core-histonebinding subunit of the human Hat1 acetyltransferase[END_REF][START_REF] Ahmad | WD repeats of the p48 subunit of chicken chromatin assembly factor-1 required for in vitro interaction with chicken histone deacetylase-2[END_REF].

Anti silencing function 1

The histone H3-H4 chaperone CIA/Asf1 (For CCG1-interacting factor A or Anti-silencing Factor 1) is the most conserved histone chaperone among eukaryotes. Asf1 was first identified from a genetic screening in budding yeast as a gene whose overexpression induces the expression of loci that are normally transcriptionally silent [START_REF] Le | Two new S-phase-specific genes from Saccharomyces cerevisiae[END_REF] Later on, the histone chaperone function of Asf1 was uncovered by Tyler and colleagues in Drosophila. The authors identified Asf1 in a complex with newly synthesized histone H3/H4 and showed that Asf1 acts as a factor with CAF-1 to synergize replication-coupled nucleosome assembly in vitro [START_REF] Tyler | The RCAF complex mediates chromatin assembly during DNA replication and repair[END_REF]. The most evolutionarily conserved region of Asf1 is the first 155 residues within the N-terminal [START_REF] Daganzo | Structure and function of the conserved core of histone deposition protein Asf1[END_REF][START_REF] Mousson | Structural basis for the interaction of Asf1 with histone H3 and its functional implications[END_REF]. This domain is characteristic of an immunoglobulin (Ig)-fold domain, in both yeast and human cells. The role of the Ig-fold domain is to bind histone H3/H4 and to stimulate CAF-1 dependent nucleosome assembly [START_REF] Umehara | Polyanionic stretch-deleted histone chaperone cia1/Asf1p is functional both in vivo and in vitro[END_REF][START_REF] Daganzo | Structure and function of the conserved core of histone deposition protein Asf1[END_REF]. The C-termini of Asf1 is rather divergent between species or human isoforms and could be a good candidate for the basis of distinct functions [START_REF] Umehara | Polyanionic stretch-deleted histone chaperone cia1/Asf1p is functional both in vivo and in vitro[END_REF][START_REF] Daganzo | Structure and function of the conserved core of histone deposition protein Asf1[END_REF].

During DNA replication, Asf1 regulates the histone turnover at the replication fork (see Introduction 3.2). Three-dimensional structure of Asf1 from S. cerevisiae, S. pombe and human has been resolved and these studies provide the molecular basis for the role of Asf1 in the assembly/disassembly of histones H3/H4 in vivo [START_REF] Umehara | Polyanionic stretch-deleted histone chaperone cia1/Asf1p is functional both in vivo and in vitro[END_REF][START_REF] Antczak | Structure of the yeast histone H3-ASF1 interaction: implications for chaperone mechanism, species-specific interactions, and epigenetics[END_REF][START_REF] English | Structural basis for the histone chaperone activity of Asf1[END_REF][START_REF] Agez | Structure of the histone chaperone ASF1 bound to the histone H3 C-terminal helix and functional insights[END_REF][START_REF] Natsume | Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4[END_REF][START_REF] Malay | Crystal structures of fission yeast histone chaperone Asf1 complexed with the Hip1 B-domain or the Cac2 C terminus[END_REF].

Newly synthesized H3/H4 histones are associated with Asf1 in the cytoplasm and are then imported into the nucleus [START_REF] English | Structural basis for the histone chaperone activity of Asf1[END_REF][START_REF] Natsume | Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4[END_REF]). Current models suggest that the N-terminal of Asf1 binds to the C-terminal of histone H3, thus physically blocking the formation of H3-H4 tetramer [START_REF] Agez | Structure of the histone chaperone ASF1 bound to the histone H3 C-terminal helix and functional insights[END_REF]). Asf1 also contacts and causes a conformational change of the C-terminus of histone H4, which otherwise interacts with H2A [START_REF] Natsume | Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4[END_REF]. Once in the nucleus, Asf1 binds other histone chaperones including CAF-1 and Rtt106 which mediate chromatin assembly following DNA synthesis [START_REF] Shibahara | Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin[END_REF][START_REF] Tyler | The RCAF complex mediates chromatin assembly during DNA replication and repair[END_REF][START_REF] Mello | Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway[END_REF]. Structural analysis demonstrated that Asf1 acts as a histone donor for CAF-1 with a molar ratio of approximately 1:1 between Asf1 and H3-H4 dimer [START_REF] Agez | Structure of the histone chaperone ASF1 bound to the histone H3 C-terminal helix and functional insights[END_REF][START_REF] Natsume | Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4[END_REF]).

In vertebrates, D. melanogaster, and S. pombe, Asf1 is essential for cell survival, with a critical role in maintaining genomic stability [START_REF] Groth | Human Asf1 regulates the flow of S phase histones during replicational stress[END_REF][START_REF] Sanematsu | Asf1 is required for viability and chromatin assembly during DNA replication in vertebrate cells[END_REF][START_REF] Schulz | The histone chaperone ASF1 localizes to active DNA replication forks to mediate efficient DNA replication[END_REF][START_REF] Tanae | Histone chaperone Asf1 plays an essential role in maintaining genomic stability in fission yeast[END_REF]. For example, the asf1-33 thermosensitive mutant, which lacks the N-terminal part of Asf1, is incapable of binding histone H3 and leads to a mislocalization of the Asf1-33 protein, in S. pombe. Cells harboring the asf1-33 mutation exhibit an alteration of overall chromatin structure, accompanied by degradation of chromosomal DNA, DNA-damage checkpoint activation, formation of recombination center (Rad22 foci) and impairment of heterochromatin structure [START_REF] Tanae | Histone chaperone Asf1 plays an essential role in maintaining genomic stability in fission yeast[END_REF].

Asf1 is also involved in gene silencing and heterochromatin maintenance [START_REF] Sanematsu | Asf1 is required for viability and chromatin assembly during DNA replication in vertebrate cells[END_REF][START_REF] Yamane | Asf1/HIRA facilitate global histone deacetylation and associate with HP1 to promote nucleosome occupancy at heterochromatic loci[END_REF][START_REF] Tanae | Histone chaperone Asf1 plays an essential role in maintaining genomic stability in fission yeast[END_REF]. In budding yeast, strains deleted for Asf1 display deficiencies in gene silencing at telomere and mating-type loci [START_REF] Le | Two new S-phase-specific genes from Saccharomyces cerevisiae[END_REF][START_REF] Tyler | The RCAF complex mediates chromatin assembly during DNA replication and repair[END_REF][START_REF] Meijsing | The silencing complex SAS-I links histone acetylation to the assembly of repressed chromatin by CAF-I and Asf1 in Saccharomyces cerevisiae[END_REF][START_REF] Sharp | Regulation of histone deposition proteins Asf1/Hir1 by multiple DNA damage checkpoint kinases in Saccharomyces cerevisiae[END_REF]. Asf1 function in gene silencing could be due to its interaction with HIRA (for HIstone cell cycle Regulation defective homolog A) another histone chaperone.

HIRA mediates chromatin assembly independently of DNA synthesis, prevalently during DNA repair, gene silencing and transcription [START_REF] Kaufman | Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I[END_REF][START_REF] Ray-Gallet | HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis[END_REF][START_REF] Blackwell | The Schizosaccharomyces pombe HIRA-like protein Hip1 is required for the periodic expression of histone genes and contributes to the function of complex centromeres[END_REF][START_REF] Tagami | Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis[END_REF][START_REF] Zhang | Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA[END_REF][START_REF] Fillingham | Chaperone control of the activity and specificity of the histone H3 acetyltransferase Rtt109[END_REF]. The interplay between Asf1 and HIRA was shown to be of crucial importance in fission yeast to maintain gene silencing, via the recruitment of the heterochromatin protein Swi6 at heterochromatic loci [START_REF] Yamane | Asf1/HIRA facilitate global histone deacetylation and associate with HP1 to promote nucleosome occupancy at heterochromatic loci[END_REF]). Moreover, it has been structurally demonstrated that the interaction of Asf1 to CAF-1 and HIRA involved the same interface, suggesting a mutually exclusive interaction [START_REF] Malay | Crystal structures of fission yeast histone chaperone Asf1 complexed with the Hip1 B-domain or the Cac2 C terminus[END_REF]. This exclusive interaction might represent a point of regulation for histone deposition associated or not with DNA synthesis. In addition, histone deposition appears also regulated by the selective binding of histone chaperone CAF-1 and HIRA to histone H3 variant present in the H3-H4 dimer [START_REF] Nakatani | Two distinct nucleosome assembly pathways: dependent or independent of DNA synthesis promoted by histone H3.1 and H3.3 complexes[END_REF][START_REF] Tagami | Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis[END_REF]).

The replication-coupled histone mark H3K56

Dynamic variations in the structure of chromatin influence DNA-related processes in eukaryotes, which are controlled in part by the post translational modifications of histones [START_REF] Krude | Chromatin assembly during DNA replication in somatic cells[END_REF]. Histones H4 deposited during S-phase are acetylated on lysines 5, 8 and 12 (H4K5Ac, H4K8Ac and H4K12Ac), a pattern highly conserved from yeast to humans.

Acetylation of newly synthetized histones is promoted by the acetyltransferase HAT1 [START_REF] Jackson | Modifications to histones immediately after synthesis[END_REF][START_REF] Sobel | Conservation of deposition-related acetylation sites in newly synthesized histones H3 and H4[END_REF][START_REF] Verreault | Nucleosomal DNA regulates the core-histonebinding subunit of the human Hat1 acetyltransferase[END_REF][START_REF] Imhof | Purification and properties of the Xenopus Hat1 acetyltransferase: association with the 14-3-3 proteins in the oocyte nucleus[END_REF]. The histone H3 is also acetylated within its amino terminal tail, but its acetylation pattern is more variable among organisms [START_REF] Sobel | Conservation of deposition-related acetylation sites in newly synthesized histones H3 and H4[END_REF][START_REF] Kuo | Transcription-linked acetylation by Gcn5p of histones H3 and H4 at specific lysines[END_REF]. The acetylation of H3 on lysine 56 (H3K56Ac) has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription [START_REF] Verreault | Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4[END_REF][START_REF] Chen | Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair[END_REF][START_REF] Li | Acetylation of histone H3 lysine 56 regulates replicationcoupled nucleosome assembly[END_REF][START_REF] Tjeertes | Screen for DNA-damage-responsive histone modifications identifies H3K9Ac and H3K56Ac in human cells[END_REF][START_REF] Yuan | Histone H3-K56 acetylation is important for genomic stability in mammals[END_REF][START_REF] Clemente-Ruiz | Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks[END_REF][START_REF] Su | Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106[END_REF]. All the studies performed to demonstrate the importance of the histone mark H3K56Ac in DNA metabolism were undertaken in yeast models because this mark is globally abundant in yeast and flies, but its presence has been uncertain in mammals. However, recent studies demonstrated the presence of this marker also in human cells [START_REF] Das | CBP/p300-mediated acetylation of histone H3 on lysine 56[END_REF][START_REF] Tjeertes | Screen for DNA-damage-responsive histone modifications identifies H3K9Ac and H3K56Ac in human cells[END_REF][START_REF] Xie | Histone h3 lysine 56 acetylation is linked to the core transcriptional network in human embryonic stem cells[END_REF].

In yeast, histone chaperones play a recurrent role in the biology of H3K56 acetylation: Asf1 binds and presents newly synthesized dimers H3-H4 to the histone acetyltransferase Rtt109, that in turns acetylates H3 on lysine 56 [START_REF] Recht | Histone chaperone Asf1 is required for histone H3 lysine 56 acetylation, a modification associated with S phase in mitosis and meiosis[END_REF][START_REF] Driscoll | Yeast Rtt109 promotes genome stability by acetylating histone H3 on lysine 56[END_REF][START_REF] Han | Acetylation of lysine 56 of histone H3 catalyzed by RTT109 and regulated by ASF1 is required for replisome integrity[END_REF][START_REF] Tsubota | Histone H3-K56 acetylation is catalyzed by histone chaperone-dependent complexes[END_REF][START_REF] Li | Acetylation of histone H3 lysine 56 regulates replicationcoupled nucleosome assembly[END_REF]. The histone mark H3K56Ac promotes S-phase specific chromatin assembly by enhancing the ability of histone chaperones CAF-1 and Rtt106 to bind histone H3 and assemble it with histone H4 into nucleosomes [START_REF] Li | Acetylation of histone H3 lysine 56 regulates replicationcoupled nucleosome assembly[END_REF][START_REF] Su | Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106[END_REF]). In S. cerevisiae, the absence of acetylation on lysine 56 of histone H3 (either by deleted asf1, rtt109 or expressing a nonacetylable form of H3, K56R) prevent co-purification of CAF-1 and Rtt106 with H3, indicating that H3 association with histone chaperones requires acetylation of H3K56 [START_REF] Huang | Rtt106p is a histone chaperone involved in heterochromatin-mediated silencing[END_REF][START_REF] Li | Acetylation of histone H3 lysine 56 regulates replicationcoupled nucleosome assembly[END_REF][START_REF] Su | Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106[END_REF]. The specific recognition of the acetylated form of H3 on K56 by CAF-1 and Rtt106 extends the "histone code" hypothesis [START_REF] Strahl | The language of covalent histone modifications[END_REF]. Indeed, as H3-H4 dimers harboring the H3K56Ac mark are likely to display concomitantly H4 marks (such as H4K5Ac, H4K12Ac), it is possible that multiple marks are necessary to recognize newly replicated DNA and to promote chromatin assembly [START_REF] Tyler | The RCAF complex mediates chromatin assembly during DNA replication and repair[END_REF][START_REF] Fillingham | A histone code for chromatin assembly[END_REF][START_REF] Li | Acetylation of histone H3 lysine 56 regulates replicationcoupled nucleosome assembly[END_REF]. Moreover, in S. cerevisiae, cells lacking Rtt109 or Asf1 present defects in MMS-induced HR while the HR induced by HO-induced DSBs does not require any of these genes. Indeed K56 acetylation may promotes sister-chromatid exchanges, considering that cells lacking Rtt109 or Asf1 present a dramatic decrease in SCE during S-phase [START_REF] Duro | Budding yeast Mms22 and Mms1 regulate homologous recombination induced by replisome blockage[END_REF].

During DNA replication, after nucleosome assembly in the rear of moving forks, histones have to be deacetylated by deacetylases as marks of "mature" chromatin. The interaction between Asf1 and the deacetylases Hst3/Hst4 in S. cerevisiae, promotes the deacetylation of H3K56 [START_REF] Maas | Cell cycle and checkpoint regulation of histone H3 K56 acetylation by Hst3 and Hst4[END_REF][START_REF] Yang | The Rtt109 histone acetyltransferase facilitates error-free replication to prevent CAG/CTG repeat contractions[END_REF][START_REF] Hachinohe | Hst3 and Hst4 histone deacetylases regulate replicative lifespan by preventing genome instability in Saccharomyces cerevisiae[END_REF]). An interesting theory is that the human p48 subunit of CAF-1 complex could play a role in histones deacetylation. There are many evidences suggesting that p48 is involved in alterations of the chromatin structure, based on deacetylation level of core histones. For example, human p48 associates with the catalytic subunit of the human deacetylase HD1 [START_REF] Taunton | A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p[END_REF]. Moreover, chicken p48 interacts with histone deacetylases chHDAC-1 and -2 in vivo and in vitro, probably through one or two N-terminal and Cterminal WD repeated motifs [START_REF] Ahmad | WD repeats of the p48 subunit of chicken chromatin assembly factor-1 required for in vitro interaction with chicken histone deacetylase-2[END_REF]). An intriguing hypothesis is that p48 subunit could have a role in chromatin maturation independently of the two other subunits of CAF-1. Indeed, p48 could switch from an interaction with CAF-1 to an interaction with enzymes involved in histone deacetylation and thus signaling the progression of the cell cycle.

Defects in the histone mark H3K56Ac during chromatin assembly lead to genetic instability which is linked to a higher susceptibility of fork stalling, damage or breakage [START_REF] Han | Acetylation of lysine 56 of histone H3 catalyzed by RTT109 and regulated by ASF1 is required for replisome integrity[END_REF][START_REF] Clemente-Ruiz | Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks[END_REF][START_REF] Su | Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106[END_REF]. The absence of H3K56Ac or the simultaneous knock-out of CAF-1 and Rtt106 affect the integrity of advancing replication forks, thus increasing homologous recombination (HR). An intriguing hypothesis is that H3K56Ac might recruit additional chromatin factors required for fork stabilization. Alternatively, fork instability may result from defective chromatin assembly and/or transfer of parental histones behind the fork (Clemente-Ruiz, Gonzalez-Prieto et al.

2011

). Defective chromatin assembly by partial depletion of H4 is rapidly followed by the collapse of replication forks, which are efficiently rescued by homologous recombination, suggesting that correct nucleosome deposition is required for replication fork stability [START_REF] Prado | Partial depletion of histone H4 increases homologous recombination-mediated genetic instability[END_REF]. The loss of integrity of advancing replication forks may end up in the collapse of some of them resulting in exposed ssDNA that is susceptible to be process by homologous recombination. HR participates in the rescue of these forks using the sister chromatid and, consistent with this, asf1-d strain shows an increase in sister chromatid exchange (SCE) [START_REF] Prado | The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange[END_REF][START_REF] Endo | Chromatin dynamics mediated by histone modifiers and histone chaperones in postreplicative recombination[END_REF][START_REF] Clemente-Ruiz | Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks[END_REF].

Beyond its role in marking newly replicated DNA, the histone mark H3K56Ac plays a crucial function in the DNA damage response, together with Asf1. In S. cerevisiae, the absence of Asf1 results in cell sensitivity to hydroxyurea (HU), camptothecin (CPT), cisplatin (CIS) and methyl methansulfonate (MMS). This sensitivity to DNA-damaging agents is shared by cells harboring the non acetylable form of histone H3 on K56 (mutation K56 to R56 leading to H3K56R), suggesting a common role for Asf1 and the histone mark H3K56Ac in genome stability [START_REF] Le | Two new S-phase-specific genes from Saccharomyces cerevisiae[END_REF][START_REF] Tyler | The RCAF complex mediates chromatin assembly during DNA replication and repair[END_REF][START_REF] Ramey | Activation of the DNA damage checkpoint in yeast lacking the histone chaperone anti-silencing function 1[END_REF][START_REF] Franco | Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C[END_REF][START_REF] Wurtele | Histone H3 lysine 56 acetylation and the response to DNA replication fork damage[END_REF]. The expression of H3 mutant (H3K56Q) that mimics the acetylation modification rescues the cell sensitivity to HU, CPT and MMS observed in the absence of Asf1, showing that the role of Asf1 in the DNA damage response is directly linked to its ability to promote the deposition of H3K56 onto DNA, either after DNA repair or during DNA replication [START_REF] Agez | Structure of the histone chaperone ASF1 bound to the histone H3 C-terminal helix and functional insights[END_REF]. Nonetheless, the links between H3K56Ac, the nascent chromatin structure and the DNA damage response remain unclear. In response to MMS, cells expressing H3K56R (the non-acetylable form) cannot complete DNA replication and accumulate recombination foci, suggesting that H3K56Ac helps to complete the repair of DNA lesions by homologous recombination [START_REF] Wurtele | Histone H3 lysine 56 acetylation and the response to DNA replication fork damage[END_REF]). One possibility is that both Asf1 and H3K56Ac are necessary to restore chromatin structure following DNA repair [START_REF] Chen | Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair[END_REF], an aspect developed in the next chapter.

Chromatin assembly at replication forks is essential to maintain genome stability

Several studies have pointed out an increased level of homologous recombination or general genome instability when replication-coupled chromatin assembly is impaired, as for example in the absence of CAF-1 or Asf1. While these data could be interpreted as a potential role for histones chaperones in repressing HR, alternative explanations such as impaired stability of replication forks and/or accumulation of DNA lesions are also possible.

As mentioned above, the absence of Asf1 results in an increased level of SCE. The hyperrecombinogenic effect of asf1-d is epistatic over cac1-d, suggesting that Asf1 and CAF-1 participate in the same pathway in the maintenance of genome integrity [START_REF] Prado | The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange[END_REF]). This is consistent with the fact that asf1 and cac1 mutants present an increased rate of gross chromosomal rearrangements (GCRs) [START_REF] Myung | Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability[END_REF]. Another evidence of increased level of spontaneous DNA lesions and genome instability in the absence of CAF-1 was provided by a study on post-replication repair by unequal sister chromatid recombination (uSCR), in budding yeast. Cells deficient for cac1 or cac2 display a higher level of HR during post-replication repair while the level of H3K56Ac remains unaffected. Others data suggest that the deposition of H3K56Ac onto newly synthesized DNA in CAF-1 deficient cells, even if delayed, is required for the induction of sister chromatin recombination (Tong, Evangelista et al. 2001;[START_REF] Pan | A DNA integrity network in the yeast Saccharomyces cerevisiae[END_REF][START_REF] Li | Acetylation of histone H3 lysine 56 regulates replicationcoupled nucleosome assembly[END_REF][START_REF] Endo | Chromatin dynamics mediated by histone modifiers and histone chaperones in postreplicative recombination[END_REF].

In plants, CAF-1 is also important for maintaining genome stability by suppressing illegitimate homologous recombination. In Arabidopsis thaliana, depletion of either subunit of CAF-1 complex increases the frequency of somatic homologous recombination of about 40

fold. This means that CAF-1 complex is responsible to maintain HR at low level in wild-type plants [START_REF] Endo | Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants[END_REF]. Furthermore, the frequency of transferred DNA (T-DNA)

integration was also elevated in CAF-1 mutants, and. T-DNA integration occurs mainly through non-HR mechanisms such as NHEJ. Increased level of illegitimate HR is probably due to an increased level of general genome instability, rather than enhanced expression of NHEJ pathway genes [START_REF] Endo | Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants[END_REF].

Several lines of evidences establish that chromatin assembly onto newly replicated DNA, especially the deposition of the histone mark H3K56Ac is crucial to maintain replication fork stability. In budding yeast, Asf1 interact with the replication factor RFC and defects in Asf1 results in loss of component of the replisome, including RFC, PCNA and DNA polymerase alpha [START_REF] Franco | Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C[END_REF]. Defects in histone deposition appear to destabilize the fork and thus trigger a need for repairing halted forks by homologous recombination. Replicative

Models for heterochromatin replication

During DNA replication, heterochromatin domains constitute a challenge for the replication machinery because of the high level of compaction. To allow the replisome to pass through these domains, there is the need for additional factors to confer flexibility to the heterochromatic DNA [START_REF] Taddei | Duplication and maintenance of heterochromatin domains[END_REF][START_REF] Loyola | Techniques used to study transcription on chromatin templates[END_REF][START_REF] Almouzni | HP1 and the dynamics of heterochromatin maintenance[END_REF]). In the meantime, a flow of epigenetic marks has to be established to guarantee the correct transfer of the epigenetic information behind the replication fork.

Heterochromatin domains such as those corresponding to the centromere are crucial for proper chromosome segregation, maintenance of epigenetic information and genome stability.

Hallmarks of heterochromatin domains in mammalian cells are the presence of epigenetic marks, such as DNA methylation, hypoacetylated histones, the trimethylation of histone H3 on lysine 9 (H3K9Me3), and the recruitment of the protein HP1 (Heterochromatin Protein 1), as well as the prevalence of the H3.1 histone variant in the core nucleosome [START_REF] James | Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene[END_REF][START_REF] Jeppesen | Antibodies to defined histone epitopes reveal variations in chromatin conformation and underacetylation of centric heterochromatin in human metaphase chromosomes[END_REF][START_REF] Tweedie | Methylation of genomes and genes at the invertebratevertebrate boundary[END_REF][START_REF] Peters | Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability[END_REF][START_REF] Bird | DNA methylation patterns and epigenetic memory[END_REF].

CAF-1, interacting with proteins involved in DNA replication and heterochromatin maintenance, plays a role in setting up the repressed state of chromatin, demonstrating that propagation of silenced chromatin is intimately linked to histone deposition process [START_REF] Taddei | Duplication and maintenance of heterochromatin domains[END_REF][START_REF] Quivy | A CAF-1 dependent pool of HP1 during heterochromatin duplication[END_REF][START_REF] Houlard | CAF-1 is essential for heterochromatin organization in pluripotent embryonic cells[END_REF]. This function seems to be conserved from yeast to multicellular organisms, indicating the importance of maintaining a correct epigenetic status, independently on the complexity of organism [START_REF] Eissenberg | The HP1 protein family: getting a grip on chromatin[END_REF][START_REF] Song | CAF-1 is essential for Drosophila development and involved in the maintenance of epigenetic memory[END_REF][START_REF] Dohke | Fission yeast chromatin assembly factor 1 assists in the replication-coupled maintenance of heterochromatin[END_REF][START_REF] Quivy | The HP1-p150/CAF-1 interaction is required for pericentric heterochromatin replication and S-phase progression in mouse cells[END_REF]Huang, Yu et al. 2010). In eukaryotes, the domains next to centromeres are rich in HP1, which would ensure the self-perpetuation of the epigenetic state of the domain [START_REF] James | Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene[END_REF]. Quivy and colleagues proposed a model in which human p150 could be used to facilitate the passage of the replication fork at heterochromatic regions, through its interaction with PCNA. This occurs by displacing chromatin bound HP1 ahead of the replication fork and transferring HP1 to the daughter strand, on H3K9Me3 [START_REF] Bannister | Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain[END_REF][START_REF] Lachner | Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins[END_REF][START_REF] Quivy | A CAF-1 dependent pool of HP1 during heterochromatin duplication[END_REF][START_REF] Quivy | The HP1-p150/CAF-1 interaction is required for pericentric heterochromatin replication and S-phase progression in mouse cells[END_REF][START_REF] Hayashi | The heterochromatin protein Swi6/HP1 activates replication origins at the pericentromeric region and silent mating-type locus[END_REF]). Such a mechanism could ensure the maintenance of the heterochromatin structure, and therefore the memory of the repressed state [START_REF] Quivy | A CAF-1 dependent pool of HP1 during heterochromatin duplication[END_REF][START_REF] Quivy | The HP1-p150/CAF-1 interaction is required for pericentric heterochromatin replication and S-phase progression in mouse cells[END_REF]. The interaction between CAF-1 and HP1 allows the replication machinery to progress efficiently through heterochromatin while the heterochromatic structure is destabilized. The disruption of p150-HP1 interaction prevents S-phase progression, without activation of the DNA damage checkpoint. This can be due to the presence of complexes formed by the interaction of HP1 with the DNA, which act as replication fork barriers [START_REF] Quivy | A CAF-1 dependent pool of HP1 during heterochromatin duplication[END_REF]).

An emerging theme for replication-coupled memory is the use of PCNA as a hub that couples chromatin reorganization to replication, establishing a connection between genetic and epigenetic inheritance [START_REF] Zhang | PCNA connects DNA replication to epigenetic inheritance in yeast[END_REF]. PCNA recruits a large number of chromatin-modulating enzymes to sites of DNA replication such as the methyltransferase DNMT1 [START_REF] Leonhardt | A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei[END_REF][START_REF] Chuang | Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1[END_REF], CAF-1 [START_REF] Shibahara | Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin[END_REF], the histone deacetylase 1 (HDAC) [START_REF] Milutinovic | Proliferating cell nuclear antigen associates with histone deacetylase activity, integrating DNA replication and chromatin modification[END_REF]) and the complex formed by the nucleosome-remodeling factor SNF2H and the Williams syndrome transcription factor WSTF (Poot, Bozhenok et al. 2004). Some of these enzymes can be considered as general chromatin-maturation factors but others operate in a domain specific manner such as DNMT1 [START_REF] Leonhardt | A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei[END_REF][START_REF] Chuang | Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1[END_REF].

It seems likely that the actions of DNMT1, WSTF-SNF2H and CAF-1 are coordinated to ensure efficient DNA methylation prior to chromatin maturation. DNMT1 and CAF-1 themselves have the ability to recruit a number of enzymes implicated in chromatin maturation. This interplay probably ensures that replication-coupled propagation of DNA methylation coincides with formation of a repressive chromatin state [START_REF] Leonhardt | A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei[END_REF][START_REF] Chuang | Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1[END_REF]. In this scenario, the role of CAF-1 will go beyond the mere HP1 mobilization. The interaction between CAF-1 with other chromatin maturation enzymes suggests that CAF-1 could be a direct player in epigenetic inheritance. Another intriguing possibility is that the transient presence of acetylated histones onto newly synthesized DNA could regulate the kinetics of nascent chromatin maturation. Histone-modifying enzymes interacting or not with CAF-1 could provide a window of opportunity to maintain or alter specific chromatin structures, before histone deacetylation [START_REF] Groth | Regulation of replication fork progression through histone supply and demand[END_REF]).

In the fission yeast S. pombe, spCAF-1 and Swi6 (the homologue of HP1) are involved in the maintenance of heterochromatin. The fission yeast centromeres are organized into distinct domains that consist of the central core (cnt) surrounding by large inverted-repeat structures (otr and imr). Therefore, S. pombe centromeres bear a close resemblance to higher eukaryotic centromeres. Moreover, centromeric heterochromatin is characterized by H3K9 methylation and by the recruitment of Swi6 heterochromatin binding protein [START_REF] Dohke | Fission yeast chromatin assembly factor 1 assists in the replication-coupled maintenance of heterochromatin[END_REF]. The depletion of spCAF-1 causes defects in silencing at centromeric and mating loci heterochromatin, accompanied with a decrease in Swi6 recruitment. These defects likely reflect the ability of CAF-1 to deliver HP1 to foci of heterochromatin replication in S-phase. Swi6 and when heterochromatin undergoes replication early in S-phase, the complex CAF-1-Swi6 is recruited to replication forks through an interaction with PCNA. At this moment, Swi6 is transferred onto daughter strands that retain K9 methylation. Chromatin bound Swi6 might recruit, directly or indirectly, H3K9 specific histone methyl transferase, Clr4 and HDACs to modify the newly assembled chromatin. The interaction between Swi6 and Clr4 has never been reported but, in mammalian cells, HP1 was shown to interact with the human homologue of Clr4, SUV39H [START_REF] Dohke | Fission yeast chromatin assembly factor 1 assists in the replication-coupled maintenance of heterochromatin[END_REF].

Epigenetic inheritance is necessary to ensure the proper genetic information and to maintain genome stability in daughter cells, after DNA replication. While the histone chaperone function of CAF-1 is conserved among all eukaryotes, CAF-1 appears to acquire novel important function in multicellular organisms. The three genes coding for the 3 subunits of CAF-1 are essential in multicellular eukaryotes including plants, mouse, chicken, Xenopus, Drosophila and human cells [START_REF] Hoek | Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo[END_REF][START_REF] Loyola | Histone chaperones, a supporting role in the limelight[END_REF][START_REF] Houlard | CAF-1 is essential for heterochromatin organization in pluripotent embryonic cells[END_REF]. In contrast, CAF-1 is not essential in unicellular eukaryotes (like yeasts).

It is possible that CAF-1 acquired novel functions for the necessity of specific developmental programs and for the determination of different tissues. Chromatin-based inheritance of the epigenetic information is instrumental in development, when the genome information must be used selectively to shape a highly complex organism. An evidence to corroborate this hypothesis is that the absence of CAF-1 in Arabidopsis leads to defects in plant structures organization, sustaining a critical role in proliferating tissues [START_REF] Endo | Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants[END_REF][START_REF] Ramirez-Parra | E2F regulates FASCIATA1, a chromatin assembly gene whose loss switches on the endocycle and activates gene expression by changing the epigenetic status[END_REF]. Moreover, the depletion of CAF-1 in mouse embryos leads to developmental arrest concomitant with severe alterations in heterochromatin organization [START_REF] Houlard | CAF-1 is essential for heterochromatin organization in pluripotent embryonic cells[END_REF]. In Drosophila, CAF-1 is an essential gene required to achieve a correct organism development, because homozygous mutants are unviable and heterozygous mutants show defects in repression of gene silencing at heterochromatic loci [START_REF] Song | CAF-1 is essential for Drosophila development and involved in the maintenance of epigenetic memory[END_REF]). In human cells, CAF-1 depletion results in a severe loss of viability, defects in S-phase progression, accompanied by spontaneous DNA damage and activation of the checkpoint kinase Chk1. Similar phenotypes were reproduced in human cells by expressing a dominant negative form of p150 [START_REF] Hoek | Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo[END_REF][START_REF] Loyola | Histone chaperones, a supporting role in the limelight[END_REF]. The accumulation of cells in early and mid S-phase could be caused by stalling of replication forks or by the activation of intra-S-phase checkpoint in response to chromatin defects, or because of specific defects in heterochromatin replication [START_REF] Houlard | CAF-1 is essential for heterochromatin organization in pluripotent embryonic cells[END_REF].

In mammalian cells, CAF-1 loads H3.1 onto replicated DNA, whereas H3.3 replaces H3.1 in particular regions of the genome, such as transcribed genes, telomeric and centromeric heterochromatin [START_REF] Mito | Genome-scale profiling of histone H3.3 replacement patterns[END_REF][START_REF] Jin | H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions[END_REF][START_REF] Wong | Histone H3.3 incorporation provides a unique and functionally essential telomeric chromatin in embryonic stem cells[END_REF][START_REF] Ray-Gallet | Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity[END_REF]. In yeast, the discrimination between the two histone variants does not exist because only the histone H3.3 is present. Thus, there is no need for histonesubstrate differentiation among the histone chaperones. The lack of histone variants in the yeast could be an evolutionary feature, due probably to the fact that most of the genome is transcribed [START_REF] Hoek | Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo[END_REF]. The absence of substrate differentiation among the histone chaperones suggests that, at least in yeast, the deficiency in CAF-1 could be overwhelmed by other histone chaperones, such as HIRA and/or Rtt106. Therefore, yeast cells do not present any proliferation issue in the absence of CAF-1.

Despite differences between multicellular and unicellular eukaryotes, the function of CAF-1 and Asf1 in the maintenance of genome stability in unicellular eukaryotes is undeniable. In yeast, disruption of CAF-1 results in gene silencing defects at telomeres and at the mating type loci and these phenotypes are enhanced when Asf1 is deleted [START_REF] Game | Role of Saccharomyces cerevisiae chromatin assembly factor-I in repair of ultraviolet radiation damage in vivo[END_REF]. Moreover, loss of CAF-1 and Asf1 genes, in conjunction with checkpoint genes such as tel1, chk1 and dpb11, leads to an increase in gross chromosomal rearrangements (GCRs) and in mutation rate. These GCRs includes translocations and deletion of chromosome arms with associated de novo telomere addition [START_REF] Myung | Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability[END_REF].

CHROMATIN RESTORATION DURING DNA REPAIR

The role of CAF-1 during DNA Repair

Beyond their role in replication-coupled nucleosome assembly during S-phase, histones chaperones such as Asf1 and CAF-1 are involved in the restoration of chromatin after DNA repair [START_REF] Gaillard | Chromatin assembly coupled to DNA repair: a new role for chromatin assembly factor I[END_REF][START_REF] Green | Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo[END_REF][START_REF] Lewis | Reduction of nucleosome assembly during new DNA synthesis impairs both major pathways of double-strand break repair[END_REF][START_REF] Schopf | Interplay between mismatch repair and chromatin assembly[END_REF]. During the process of chromatin assembly, CAF-1 deposits histones at sites of repair-associated DNA synthesis, therefore CAF-1 is a key protein for the maintenance of chromatin memory at sites of DNA repair [START_REF] Green | Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo[END_REF][START_REF] Ishii | Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1: a molecular link between recombination and chromatin assembly during meiosis[END_REF]. Moreover, recent evidences suggest that CAF-1 could have additional role during DNA repair and not be merely required to restore chromatin after the completion of DNA repair [START_REF] Baldeyron | HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair[END_REF]). An interesting hypothesis is that CAF-1 could act firstly in the recruitment or in the regulation of DNA repair enzymes at sites of DNA repair. Subsequently, CAF-1 function could switch toward a histone chaperone mode [START_REF] Ishii | Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1: a molecular link between recombination and chromatin assembly during meiosis[END_REF][START_REF] Baldeyron | HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair[END_REF][START_REF] Schopf | Interplay between mismatch repair and chromatin assembly[END_REF].

In this chapter, the role of histones chaperones in DNA repair and the DNA damage response are presented.

Role of CAF-1 during Nucleotide Excision Repair (NER)

Access to DNA lesions by the NER machinery required major remodeling of the chromatin structure [START_REF] Smerdon | Nucleosome rearrangement in human chromatin during UV-induced DNA-reapir synthesis[END_REF]. After completion of DNA repair, the chromatin has to be faithfully reassembled to restore the preexisting epigenetic information. Impairment in chromatin restoration might result in loss of epigenetic information and thus genetic instability [START_REF] Green | Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo[END_REF][START_REF] Polo | New histone incorporation marks sites of UV repair in human cells[END_REF].

Human CAF-1 promotes nucleosome assembly on damaged templates, both in vitro and in vivo during NER [START_REF] Gaillard | Chromatin assembly coupled to DNA repair: a new role for chromatin assembly factor I[END_REF][START_REF] Martini | Recruitment of phosphorylated chromatin assembly factor 1 to chromatin after UV irradiation of human cells[END_REF][START_REF] Mello | Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway[END_REF][START_REF] Green | Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo[END_REF]. In vivo, CAF-1 is locally recruited at sites of UV-induced DNA damages in a PCNA-dependent manner. Recruitment of both CAF-1 and PCNA occurs at damaged sites that have undergone the dual incision of the initial damaged lesion (Figure n.21) [START_REF] Moggs | A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage[END_REF][START_REF] Green | Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo[END_REF]. This data suggest that CAF-1 acts at late stages during NER, to restore locally chromatin structure at damaged-sites.

Chromatin restoration at repair sites might occur synergistically with Asf1, but Asf1 does not appear to be recruited at DNA lesion sites in vitro, neither it is able to promote nucleosome assembly on its own on a damaged template [START_REF] Mello | Human Asf1 and CAF-1 interact and synergize in a repair-coupled nucleosome assembly pathway[END_REF]. Moreover, CAF-1 could also act as a trap for histones evicted during DNA repair, therefore providing a hub to maintain epigenetic information [START_REF] Gaillard | Chromatin assembly coupled to DNA repair: a new role for chromatin assembly factor I[END_REF][START_REF] Green | Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo[END_REF][START_REF] Polo | Chromatin assembly: a basic recipe with various flavours[END_REF]. In support of the role of CAF-1 in chromatin restoration following NER, deletion of any of the three genes encoding CAF-1 subunits in S. cerevisiae and in Arabidopsis moderately increases sensitivity to UV irradiation [START_REF] Kaufman | Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I[END_REF][START_REF] Game | Role of Saccharomyces cerevisiae chromatin assembly factor-I in repair of ultraviolet radiation damage in vivo[END_REF][START_REF] Endo | Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants[END_REF][START_REF] Kirik | The chromatin assembly factor subunit FASCIATA1 is involved in homologous recombination in plants[END_REF][START_REF] Kim | Chromatin assembly factors Asf1 and CAF-1 have overlapping roles in deactivating the DNA damage checkpoint when DNA repair is complete[END_REF].

Role of CAF-1 during Mismatch Repair (MMR)

During DNA replication, DNA polymerases can mis-incorporate dNTP, thus creating a mismatch in the nascent strand. Histone depositions occurs around 250 bp behind the DNA synthesis point, in the rear of the moving fork. Therefore, the time window to correct potential mismatches is reduced and MMR pathway has to be tightly coordinated with chromatin assembly [START_REF] Sogo | Structure of replicating simian virus 40 minichromosomes. The replication fork, core histone segregation and terminal structures[END_REF]). After the DNA damage recognition, major remodeling of chromatin structure occurs, to allow the repair machinery to gain access to the DNA lesion. CAF-1 and PCNA are recruited at damaged sites that have undergone the dual incision stage of repair processing. After completion of DNA repair, CAF-1-dependent chromatin reassembly is essential to restore the preexisting epigenetic information (Adapted from [START_REF] Moggs | Chromatin rearrangements during nucleotide excision repair[END_REF].

The interplay between MMR and chromatin assembly seems to be regulated by the interaction between MutSα and p150 (Wilson, Carney et al. 1998;[START_REF] Schopf | Interplay between mismatch repair and chromatin assembly[END_REF].

MutSα is a heterodimer composed of MSH2 and MSH6 that act as a sliding clamp to initiate Exo1-dependent degradation of the error-containing DNA strand (Wilson, Carney et al. 1998;[START_REF] Genschel | Human exonuclease I is required for 5' and 3' mismatch repair[END_REF][START_REF] Wei | Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility[END_REF]. The crosstalk between MMR pathway and nucleosome assembly through CAF-1-MutSα interaction might imply a decreased affinity of CAF-1 to bind histones, or potential interference in the ability of CAF-1 to associate with the fork. Therefore, chromatin assembly would be delayed giving time to MMR to repair mismatches. Once the mismatch is repaired, MutSα no longer binds to DNA and CAF-1 can restore the chromatin organization [START_REF] Kadyrova | CAF-I-dependent control of degradation of the discontinuous strands during mismatch repair[END_REF][START_REF] Schopf | Interplay between mismatch repair and chromatin assembly[END_REF]. These data support a dual role for CAF-1 during MMR. Indeed, CAF-1 has an early role as MMR regulator, via its interaction with MutSα and a late role as histone chaperone for replication-coupled nucleosome deposition. In S. cerevisiae, inactivation of both CAF-1 and Asf1 reduces NHEJ efficiency and accuracy.

Role of CAF-1 during Double Strand Break (DSB) repair

However, Lewis and colleagues demonstrated that the effects on NHEJ, in CAF-1 depleted strains, are an indirect consequence of derepression of silencing at the mating locus [START_REF] Lewis | Reduction of nucleosome assembly during new DNA synthesis impairs both major pathways of double-strand break repair[END_REF]. Inactivation of both CAF-1 and Asf1 results in a 10-fold decrease in DSB-induced plasmid:chromosome recombination, and ionizing radiation-induced loss of heterozygosity was abolished in the mutants [START_REF] Lewis | Reduction of nucleosome assembly during new DNA synthesis impairs both major pathways of double-strand break repair[END_REF]. In Drosophila, the deletion of the largest subunit of CAF-1 (p180) affects the efficiency of gap repair by HR in vivo [START_REF] Song | CAF-1 is essential for Drosophila development and involved in the maintenance of epigenetic memory[END_REF]). Gap repair consists in the repair of a chromosomal DSB, using a non-chromosomal homologous template. Song and colleagues demonstrated that the efficiency of the gap repair is reduced by 2.5 fold in the absence of the largest subunit of CAF-1, though not completely abrogated. Altogether, these data suggest a role for CAF-1 in DNA repair by HR and that this function is conserved. Nonetheless, the exact function of CAF-1 during the different steps of HR remains unclear.

Further evidences involving a direct role of CAF-1 during DSB repair by HR were then highlighted. In human cells, a rapid and transient accumulation of all HP1 isoforms occurs at laser-induced DNA damage, both within euchromatin and heterochromatin, only 5 minutes after damage induction [START_REF] Ayoub | Mobilization and recruitment of HP1: a bimodal response to DNA breakage[END_REF][START_REF] Luijsterburg | Heterochromatin protein 1 is recruited to various types of DNA damage[END_REF][START_REF] Zarebski | Recruitment of heterochromatin protein 1 to DNA repair sites[END_REF][START_REF] Baldeyron | HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair[END_REF]. Baldeyron and colleagues focused on HP1α and they found that HP1α recruitment at DNA damage is rapid and transient. Importantly, HP1α recruitment depends on its ability to interact with p150 of CAF-1, but independently of heterochromatin marks. They also investigated KAP-1 dynamics at DNA lesions. KAP-1 is the transcriptional co-repressor KRAB-associated protein 1 and was found in complexes with HP1 [START_REF] Ryan | KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Kruppelassociated box-zinc finger proteins in heterochromatin-mediated gene silencing[END_REF]. Deletion of p150 impaired both HP1α and KAP-1 accumulation at damage sites while depletion of HP1α or KAP-1 reciprocally impaired their accumulation at laser-induced DNA lesions, without affecting p150 recruitment. Interestingly, HP1α and KAP-1 accumulation was not impaired by p60 deletion, showing that only p150, but not the function of CAF-1 in chromatin assembly, has a role in DNA repair [START_REF] Baldeyron | HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair[END_REF].

Depletion of either p150 or HP1α results in impaired recruitment of DSB repair proteins, including 53BP1, BRCA1 and Rad51 at laser-induced DNA lesions. While the depletion of HP1α leads to a clear defect in DSB repair by HR, depletion of p150 has only a modest effect on HR-mediated repair [START_REF] Baldeyron | HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair[END_REF]. The impact of p150 depletion on HR might be hidden by the fact that inactivation of p150 results in accumulation of cells in Sphase. Importantly, phosphorylation of RPA2, a single strand binding protein, was reduced when HP1α is depleted, as it is also observed in the absence of CtIP, a nuclease involved in end-resection. One possible explanation is that HP1α, together with p150, promote homologous recombination by stimulating the early step of end-resection necessary for Rad51 nucleation onto ssDNA. Considering that the recruitment of BRCA1, a CtIP partner, is affected in the absence of HP1α, the latter might have a role in promoting CtIP recruitment at damaged sites.

The p150 subunit of CAF-1 stays longer than HP1α and KAP-1 at damaged sites and a possible explanation is that the function of p150 switches toward an active histone chaperone mode, as part of the CAF-1 complex. By doing so, CAF-1 might ensure the restoration of chromatin organization after completion of DNA repair [START_REF] Green | Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo[END_REF][START_REF] Polo | Chromatin assembly: a basic recipe with various flavours[END_REF][START_REF] Polo | DNA damage leaves its mark on chromatin[END_REF][START_REF] Baldeyron | HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair[END_REF]. These results suggest a dual role for CAF-1 in HR: early function in the process of DNA repair, and a later function as histone chaperone.

Interestingly, direct interactions between CAF-1 and the meiotic recombinase Lim15/Dmc1 have been reported [START_REF] Ishii | Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1: a molecular link between recombination and chromatin assembly during meiosis[END_REF]. Programmed DSBs occur in meiosis to promote crossover events, leading to genetic variability among organisms. Chromatin organization following meiotic recombination is critical to ensure the correct segregation of homologous chromosomes into gametes and an involvement of histone chaperones in these processes is conceivable. Meiotic recombination between homologue chromosomes requires a meiosis-specific recombinase, Dmc1, in addition to Rad51. In the basidiomycete C. cinerea, after the formation of meiotic DSB, Lim15/Dmc1 coats ssDNA to perform strand invasion [START_REF] Namekawa | Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing[END_REF]Neale and Keeney 2006). After the strand invasion with the homologue chromosome, PCNA and DNA polymerases are recruited to ensure extension of the invading strand on the homologous template, allowing the pathway to progress toward chromatin assembly (Figure n.22) [START_REF] Sakaguchi | Meiosis in Coprinus: characterization and activities of two forms of DNA polymerase during meiotic stages[END_REF][START_REF] Sawado | A DNA polymerase alpha catalytic subunit is purified independently from the tissues at meiotic prometaphase I of a basidiomycete, Coprinus cinereus[END_REF]Namekawa, Hamada et al. 2003;Namekawa, Hamada et al. 2003). A physical and direct interaction between the largest subunit of CAF-1 and the meiosis-specific recombinase protein Lim15/Dmc1 has been reported [START_REF] Ishii | Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1: a molecular link between recombination and chromatin assembly during meiosis[END_REF]. Despite the lack of evidences for the function of this interaction, it was proposed that CAF-1 is recruited during extension of the D-loop to promote histones deposition (Figure n.22). This late role could allow chromatin assembly following meiotic recombination, contributing toward the establishment of the memory of recombination events [START_REF] Ishii | Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1: a molecular link between recombination and chromatin assembly during meiosis[END_REF]).

Figure n. 22 -Model of interaction between CAF-1 and the recombinase

Lim15/Dmc1. After DSB formation, Lim15/Dmc1 coats single strand end, during strand invasion. PCNA recruits the DNA polymerase to the site of Lim15/Dmc1. CAF-1 forms a complex with Lim15/Dmc1 and PCNA and this is necessary to deposits histones H3 and H4 or other factors, such as histone variants on the newly synthesized DNA [START_REF] Ishii | Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1: a molecular link between recombination and chromatin assembly during meiosis[END_REF].

The role of histone chaperones in the DNA damage response

Genome integrity is constantly threatened by exogenous and endogenous agents; therefore it is essential for cells to detect DNA lesions and to coordinate DNA damage response with cell cycle progression [START_REF] Foiani | S-phase DNA damage checkpoint in budding yeast[END_REF]Lambert and Carr 2005;[START_REF] Lambert | Arrested replication fork processing: interplay between checkpoints and recombination[END_REF][START_REF] Branzei | The checkpoint response to replication stress[END_REF][START_REF] Ciccia | The DNA damage response: making it safe to play with knives[END_REF]. The signalization cascade that leads to cell cycle arrest is called DNA Damage Response (DDR) and it consists of three main steps: a) sensing the presence of DNA damage by "sensor proteins" b) signaling to "transducer proteins" that amplify the signal c) activation of "effector checkpoint kinases", responsible for regulation of cell cycle progression, transcription, cell death, DNA repair.

Checkpoint pathways are essentials to arrest cell cycle progression, providing additional time for DNA repair and DNA replication completion, contributing to genome integrity (Qin and Li 2003;Lambert and Carr 2005;[START_REF] Lambert | Arrested replication fork processing: interplay between checkpoints and recombination[END_REF].

Once DNA damages are repaired, the checkpoint has to be turned OFF (checkpoint recovery) for cells resuming cell cycle progression. Completion of DNA repair implies not only the successful repair of initial DNA lesions, but also the restoration of the chromatin structure.

Importantly, chromatin restoration has been shown to be crucial to turn-off checkpoint signaling [START_REF] Tyler | The RCAF complex mediates chromatin assembly during DNA replication and repair[END_REF]. The restoration of chromatin implies: a) nucleosome reassembly involving either parental histones recycling or de novo histones deposition and b) restoration of locus-specific chromatin-structure, including epigenetic marks. Defects in epigenetic reinstatement at post-repair sites may contribute to epigenetic instability, leading to loss of epigenetic information and increased genetic instability [START_REF] Koundrioukoff | Interplay between chromatin and cell cycle checkpoints in the context of ATR/ATM-dependent checkpoints[END_REF]).

In S. cerevisiae, Asf1 might play a direct role in the inactivation of the DDR via its dynamic physical interaction with Rad53 [START_REF] Emili | Dynamic interaction of DNA damage checkpoint protein Rad53 with chromatin assembly factor Asf1[END_REF][START_REF] Hu | Asf1 links Rad53 to control of chromatin assembly[END_REF]). The checkpoint kinase Rad53 is part of the DDR but also prevents the accumulation of free histones in the cell, as a surveillance mechanism [START_REF] Gunjan | A Rad53 kinase-dependent surveillance mechanism that regulates histone protein levels in S. cerevisiae[END_REF]. The inactive form of Rad53 binds to Asf1 but upon phosphorylation of Rad53, Asf1 is released and cell cycle is arrested [START_REF] Emili | Dynamic interaction of DNA damage checkpoint protein Rad53 with chromatin assembly factor Asf1[END_REF][START_REF] Hu | Asf1 links Rad53 to control of chromatin assembly[END_REF][START_REF] Schwartz | FHA domain-mediated DNA checkpoint regulation of Rad53[END_REF]. Thus, the binding of Asf1 to Rad53 might be a signal for the checkpoint recovery (Figure n. 23).

Kim and colleagues demonstrated that CAF-1 and Asf1 cooperatively restore chromatin at DNA repair sites and mediate efficient deactivation of the checkpoint, especially upon robust checkpoint activation [START_REF] Kim | Chromatin assembly factors Asf1 and CAF-1 have overlapping roles in deactivating the DNA damage checkpoint when DNA repair is complete[END_REF]. The double mutant asf1-d cac1-d presents a defect in checkpoint recovery, while neither asf1-d nor cac1-d single mutant exhibits this phenotype. These data are not in agreement with those presented by Chen and colleagues, who found that the single deletion of asf1 was sufficient to impair checkpoint recovery [START_REF] Chen | Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair[END_REF].

Checkpoint deactivation following chromatin restoration requires the histone mark H3K56Ac. [START_REF] Masumoto | A role for cell-cycle-regulated histone H3 lysine 56 acetylation in the DNA damage response[END_REF]Collins, Miller et al. 2007;[START_REF] Driscoll | Yeast Rtt109 promotes genome stability by acetylating histone H3 on lysine 56[END_REF][START_REF] Han | Acetylation of lysine 56 of histone H3 catalyzed by RTT109 and regulated by ASF1 is required for replisome integrity[END_REF][START_REF] Tsubota | Histone H3-K56 acetylation is catalyzed by histone chaperone-dependent complexes[END_REF][START_REF] Wurtele | Histone H3 lysine 56 acetylation and the response to DNA replication fork damage[END_REF]). Asf1-dependent H3K56Ac occurs also outside of S-phase, suggesting that this histone mark may function in replication-independent chromatin assembly pathways, for example during DDR [START_REF] Rufiange | Genome-wide replication-independent histone H3 exchange occurs predominantly at promoters and implicates H3 K56 acetylation and Asf1[END_REF][START_REF] Li | Acetylation of histone H3 lysine 56 regulates replicationcoupled nucleosome assembly[END_REF]. Although cells deficient for Asf1 are able to efficiently repair DNA lesions caused by DNA damaging agents, they are highly sensitive to Inactive Rad53 is bound to Asf1 and upon checkpoint activation Rad53 becomes hyperphosphorylated, releasing Asf1. Asf1 promotes chromatin assembly indirectly, through the acetylation of H3 on K56, via Rtt109. K56 acetylation drives chromatin restoration after DSB and this is a signal for the completion of the repair. The mechanism is not understood, but it has been demonstrated that H3K56 is sufficient to turn off the DNA damage checkpoint, even in absence of Asf1 [START_REF] Chen | Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair[END_REF].

them. This defect in chromatin reassembly is correlated with a defect in the ability of cells to re-enter the cell cycle [START_REF] Tyler | The RCAF complex mediates chromatin assembly during DNA replication and repair[END_REF][START_REF] Wurtele | Histone H3 lysine 56 acetylation and the response to DNA replication fork damage[END_REF]. Cells lacking Asf1 perform normal resection and DNA repair, but their ability to reassemble chromatin at the site of DNA damage, after DNA repair is completed, is severely impaired [START_REF] Tyler | The RCAF complex mediates chromatin assembly during DNA replication and repair[END_REF][START_REF] Chen | Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair[END_REF][START_REF] Wurtele | Histone H3 lysine 56 acetylation and the response to DNA replication fork damage[END_REF]. Nevertheless, Asf1 is not the histone chaperone that directly deposits histones on repaired DNA but it promotes cell resistance to DNA damaging-agents by promoting H3K56Ac via Rtt109. Indeed, the presence of the histone mark H3K56Ac onto repaired chromatin is proposed to be the signal of repair completion and thus checkpoint recovery [START_REF] Chen | Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair[END_REF]. Importantly, in the absence of Asf1, no histones were detected at site of DNA repair after repair of the DSB.

Furthermore, the deletion of Rtt109 leads to similar phenotype that loss of Asf1, in term of checkpoint recovery. Mimicking the histone mark H3K56Ac (K56Q) bypasses the requirement for Asf1 in turning off the checkpoint, whereas mutations that prevent K56 acetylation (K56R) blocks chromatin reassembly after DSB-repair [START_REF] Recht | Histone chaperone Asf1 is required for histone H3 lysine 56 acetylation, a modification associated with S phase in mitosis and meiosis[END_REF][START_REF] Xhemalce | Regulation of histone H3 lysine 56 acetylation in Schizosaccharomyces pombe[END_REF][START_REF] Chen | Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair[END_REF]. In support for a role of H3K56Ac in promoting checkpoint recovery, the deacetylase Hst3 is transcriptionally repressed and degraded by the DNA damage checkpoint [START_REF] Maas | Cell cycle and checkpoint regulation of histone H3 K56 acetylation by Hst3 and Hst4[END_REF][START_REF] Thaminy | Hst3 is regulated by Mec1-dependent proteolysis and controls the S phase checkpoint and sister chromatid cohesion by deacetylating histone H3 at lysine 56[END_REF]). Consistent with this, H3K56Ac is massively present in the chromatin fractions undergoing DNA repair [START_REF] Masumoto | A role for cell-cycle-regulated histone H3 lysine 56 acetylation in the DNA damage response[END_REF].

The mechanism by which H3K56Ac-dependent chromatin assembly leads to checkpoint deactivation remains unclear. However, it seems likely that DNA integrity or cell cycle progression could be monitored by a mark at the chromatin level [START_REF] Chen | Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair[END_REF][START_REF] Fillingham | A histone code for chromatin assembly[END_REF].

OBJECTIVES: Investigating the role of CAF-1 in replication-induced recombination

Histones chaperones, such as CAF-1 and Asf1, appear to have a significant influence on homologous recombination pathways. In one hand, defects in replication-coupled chromatin assembly drives genome instability, especially at replication forks, thus creating a need for homologous recombination pathway to restart/repair stalled forks. This is likely to result in a hyper-rec phenotype. In contrast, analyzing DNA repair following induction of specific DNA lesions, such as DSBs, reveals a potential direct role, at least for CAF-1, in regulating HR.

The objectives of my PhD project were to investigate the role of CAF-1 in the regulation of replication-induced recombination in the fission yeast Schizosaccharomyces pombe. In addition to classical recombination assays available in fission yeast, I made use of conditional replication fork barriers. These genetic assays allow the arrest of a single replication fork at a specific locus to be induced. Fork arrest induces recombination events linked to the restart of the collapsed fork by homologous recombination. Importantly, these conditional fork arrest substrates allow the visualization of joints molecules (recombination intermediates) and the products of their resolution, thus permitting to investigate the role of various factors in the regulation of homologous recombination at a molecular level.

The fission yeast Schizosaccharomyces pombe as a model organism

The Importantly, the extremities of the chromosome III contain repeated transcription units of ribosomal RNA (rDNA) and are thus variable in size from one strain to another one. In addition to the three linear chromosomes, there is also a circular mitochondrial chromosome of 20 Kbp. About 60% of the fission yeast genome codes for proteins (4.970 protein-coding genes). The genome contains also around 450 non-coding RNAs. The gene density decrease in telomeric regions and genes are absent in centromeric regions.

The fission yeast CAF-1 complex is composed of three subunits encoded by the folloing genes:

 pcf1 (SPBC29A10.03c), on chromosome II, codes for the human homologue p150. It is an intron-free gene of 1.635 bp. The Pcf1 protein is composed of 544 amino acids with a mass of 62.2 kDa and an isoelectric point at pH 4.6.

 pcf2 (SPAC26H5.03), on chromosome I, codes for the human homologue p60. It is a gene containing 3 introns and the unspliced RNA length is of 1.740 bp, while the mRNA is of 1.539 bp. The Pcf2 protein is constituted of 512 amino acids with a mass of 57.4 kDa and an isoelectric point at pH 6.5.

 pcf3 (SPAC25H1.06), on chromosome I, codes for the human homologue p48 homologue.

It is a intron-free gene of 1.227 bp. The Pcf3 protein is composed of 408 amino acid, with a mass of 46.7 kDa and an isoelectric point at pH 6.5.

The RTS1-Replication Fork Barrier in S. pombe

The conditional replication fork barrier RTS1 is a polar RFB mediated by a DNA-bound protein. The binding of the Rtf1 protein to the RTS1 sequence causes the polar arrest of replication forks. The RTS1-RFB is naturally located closed to the mat locus on the chromosome II. Mating type switching requires the mat locus to be replicated in a unidirectional manner, by fork progressing from the telomere toward the centromere. By blocking replication forks progressing in the opposite direction (from telomere towards the centromere), the RTS1-RFB allows the mat locus to be replicated in a unidirectional manner.

Thus, the function of the RTS1-RFB is to coordinate replication dynamic at the mat locus, to ensure efficient mating type switching (Dalgaard and Klar 2001;[START_REF] Codlin | Complex mechanism of site-specific DNA replication termination in fission yeast[END_REF].

The RTS1 sequence if of 859 bp and is constituted of two regions: 25B). Indeed, the Rtf1 promoter was merged with the thiamine-repressible promoter nmt41.


Thereby, in the presence of thiamine, the nmt41 promoter is repressed, Rtf1 is not expressed and the RTS1 barrier is inactive (defined as "OFF" conditions). In contrast, in the absence of thiamine, the nmt41 promoter is induced, Rtf1 protein is expressed and the barrier is active (defined as "ON" conditions). One RTS1 barrier is bound by several Rtf1 proteins in association with protein-protein interactions. Efficient induction of Rtf1 expression requires

GENETIC AND CELL BIOLOGY TECHNIQUES

Growth media

Amino acids and bases: adenine hydrochloride (≥ 99 %, Sigma-Aldrich), uracil (≥ 99 %, Sigma-Aldrich), L-leucin (≥ 98 %, Sigma-Aldrich), L-arginin monohydrochloride (≥ 98 %, Sigma-Aldrich) and L-histidin monohydrochloride monohydrate (≥ 98 %, Sigma-Aldrich):

stock solution at 1.25 %, used at 0.0125% in media.

Strains and plasmids

Fission yeast strains were constructed using standard genetic techniques [START_REF] Moreno | Molecular genetic analysis of fission yeast Schizosaccharomyces pombe[END_REF][START_REF] Bahler | Heterologous modules for efficient and versatile PCRbased gene targeting in Schizosaccharomyces pombe[END_REF][START_REF] Hentges | Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe[END_REF] and are listed in Table n.4.

KF31 U-pcf1-check AGTCTATCAACATTCACCCAC KF32 L-pcf1-check GATATTCACTGTAACAAGAACCTCG KF42 F-pcf1 PIP* GGAAAGACAAGCATTGAAGGCAAACAACGCTGCCACCAAAGGAG KF43 R-pcf1 PIP* CTCCTTTGGTGGCAGCGTTGTTTGCCTTCAATGCTTGTCTTTCC
The final PCR product containing the entire pcf1 ORF with the desired mutations in the PIPbox, was used to transform a strain in which pcf1 gene was deleted by ura4 + . Clones resistant for 5-FOA (1.6 g/L) were selected (loss of ura4 + means integration of pcf1-PIP*)

S. pombe transformation

This technique consent the integration of a specific DNA fragment containing the marker cassette, into the fission yeast genome at a specific locus. The transformation consists in the permeabilization of the yeast cell wall, allowing the DNA molecule to enter into the cell.

Once in the nucleus, the bases complementarity between the DNA fragment and the flanking region of the target gene permits the integration of the exogenous sequence.

2.10 8 cells from an exponential culture of S. pombe were pelleted and washed with an equal volume of sterile water. After a second wash in 1 ml of sterile water, cells were resuspended in 1 ml LiAc-TE 1X (Sigma). Then, cells were spinned and resuspended in LiAc-TE 1X at a concentration of 2.10 9 cells/ml, mixed with 2 μl of herring sperm DNA (Invitrogen) at 10mg/ml and with 1 μg of the DNA fragment for the transformation. After 10 minutes of incubation at room temperature, 260μl of 40% PEG/LiAc-TE was added and then cells were incubated for 60 minutes at 30°C. 40μl of DMSO was added and heat shock was given at 90 42°C, during 5 minutes. Cells were pelleted and washed with 1 ml of sterile water. Then, cells were resuspended in 3 ml of recovery medium (EMM-Glu) for three hours, then spread on media containing antibiotics. Another option was that cells were not resuspendend in EMM-Glu, but in 0.5 ml of water and then plated on YE. The day after YE plates were replica-plated on selective plates containing Kan or Nat or Hyg antibiotics.

Serial dilution assay to study the response of fission yeast cells to particular growth conditions

The serial dilution assay was used to characterize the growth and the survival of cells experiencing different kinds of DNA replication challenges, such as the induction of the fork blockade or the exposure to a variety of genotoxic agents.

a) The genotoxic agents tested at different doses were hydroxyurea (Sigma), camptothecin (Sigma), mitomycin C (Sigma), cisplatin (Sigma), methyl methanesulfonate (Sigma) and tetrabenazine (Sigma). I also irradiated plates with UV-C Stratalinker (Stratagene).

To perform a serial dilution assay cells in the culture were counted and diluted in water, at different concentrations (1.10 7 to 1.10 b) The survival of different strains to the replication fork blockade was tested by a serial dilution assay. A single colony was inoculated in minimal media (EMM-Glu) with thiamine (pause OFF), to obtain a pre-culture. The pre-culture was washed twice in sterile water and an aliquot was inoculated in two liquid cultures, with or without thiamine (60 µM). The cells were grown for 24 hours (the fork arrest system is induced after 16-18 hours without thiamine). From these two cultures (with or without thiamine), 10 μl of 1/10th serial dilution (1.10 7 to 1.10 3 cells/ml) were dropped on EMM-Glu plates with (pause OFF) or without (pause ON).

Assay to score recombination events between dispersed repeated sequences

This assay allows the investigation of recombination events between the two RTS1 sequences integrated on both sides of ura4 locus and the RTS1 sequence naturally present on chromosome II. Recombination events lead to ura4 loss, including deletions or translocations.

Colonies growing on EMM-Glu plates without uracil were switched to EMM-Glu plates with uracil and with or without thiamine. Colonies growing on plates with and without thiamine were respectively inoculated in liquid EMM-Glu media containing or not thiamine, until saturation. From the saturated cultures, 1.

Assay to score recombination between sister chromatids and between homologous chromosomes.

To perform the investigation of allelic recombination in specific genetic backgrounds, we disposed of fission yeast haploid strains harbouring the construct to check spontaneous sister chromatid exchange [START_REF] Hartsuiker | Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair[END_REF]) and of fission yeast diploid strains harbouring the construct to score spontaneous homologous recombination and chromosome loss [START_REF] Hartsuiker | Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair[END_REF].

One colony, growing on complete media was disaggregated in 0.25 ml of ultra-pure water and dilutions were plated on EMM-Glu -ade, EMM-Glu -ade-ura and complete EMM-Glu for survival. After 5 days colonies were counted and the median calculated with the 95% confidence interval for deletions and gene conversion (sister chromatid exchange) and for homologous recombination and chromosome loss (diploid strains). The experiment to score sister chromatid exchanges was performed also in presence of CPT, to determine the rate of induced SCE. Therefore, colonies growing on complete media were switched in a liquid culture and the day after, treated with 20 µM of CPT for 4 hours or only with DMSO as control, before being plated on the selective plates, mentioned above.

Assay to score the repair of a site-specific polar DSB by recombination between sister-chromatids.

The strain h+ mat1-PΔ17 pcf1-d (-SSB + donors) was crossed with the h -mat1-M2,3∆

(+SSB, -donors) strain on a YE plate [START_REF] Roseaulin | Mus81 is essential for sister chromatid recombination at broken replication forks[END_REF]). After two days the patches were streaked onto G-plates (YE plates not implemented in amino acids) to select the white diploids. The selection of the diploid was possible because the two strains presented two ade6 alleles that can complement (ade6 M-210 and ade6 M-216). Such diploids were patched on SPA plates (specific plate for sporulation) and after two days, tetrads were dissected. Spores were analyzed by mating type colony PCR for the presence of the SSB (h -), the presence of the donors (h + ) and the deletion of pcf1.

Primers (Sigma -100 μM):

Fluorescence microscopy of living cells

A pre-culture of S. pombe cells was prepared in 10 ml of filtered EMM-Glu, implemented in amino acids. After 24 hours at 30°C, the cultures were diluted to obtain a maximum of cells in S-phase. The final cellular concentration should be between 5.10 6 and 1.10 7 cells/ml. If the recruitment of proteins in the presence of the fork blockade at RuraR has to be analyzed, the cells from the pre-culture have to be washed twice in sterile water and splitted into two cultures with or without thiamine, for 24 hours.

1 ml of the culture was centrifugated at low speed (1.500 rpm for 1 min) and the pellet was resuspendend in 1 ml of fresh filtered EMM-Glu. 1 µl of cells were dropped onto the microscopy agarose-slide, previously prepared. The slides present a layer of 1.4% agarose dissolved in filtered EMM-Glu. Cells were observed with a LEICA DMRXA microscopy equipped of an oil immersion 100X objective, with numerical aperture corresponding to 1.4

and coupled to a COOLSNAP HQ camera (Roper Scientific, USA). The filters we used on the microscope were FITC filter for GFP, CFP filter for CFP and the YFP filter for YFP. Photos were taken with the Z-stack (3D) parameterized at 15 slices and were analyzed using METAMORPH (Roper Scientific, USA) and Image J software.

MOLECULAR BIOLOGY TECHNIQUES

Colony PCR

Colony PCR was performed disaggregating a fission yeast colony in 30 μl of sterile water, in a PCR tube. Then the PCR tubes were heated in the microwave oven (900W) for 3 minutes.

For each tube 20 μl of PCR mix were added. The tubes were laid in the PCR machine and the appropriate PCR program was set.

The mix for one PCR tube was composed by: 1 μl of dNTPs (10 mM each), 0.5 μl Red Hot ®

Taq DNA Polymerase (ABgene), 0.25 μl for each primer (100μM), 5 μl of PCR buffer (10X), 9 μl of sterile water and 4 μl of MgCl 2 (25 mM).

Pulsed Field Gel Electrophoresis (PFGE)

Chromosomes can be separated depending on their size, by PFGE. This technic allows the detection of gross chromosomal rearrangements on the chromosome III, which are produced by the induction of the replication fork blockade at ura4 locus. The rearrangements were visualized via specific 32 P marked probes. Tris-HCl (pH 7.5), 1% SDS for 90 min at 55°C, following by twice 24 hours incubation in 1% lauryl sarcosine, 0.5 M EDTA (pH 9.5), 0.5 mg/ml proteinase K at 55°C (Euromedex).

Electrophoresis was performed using the Bio-Rad CHEF Mapper apparatus for 48 hr in TAE buffer 1X (Biorad) (0.8% agarose Biorad megabase, Pulse time: 1800 s, 2V/cm, angle: 100°, temperature: 14°C). Finally, agarose gel was stained in 0.5 μg/ml Ethidium bromide for 30 minutes and chromosomes were transferred on a nylon membrane. The chromosomes were probed with 32 P-rng3 probe (GE healthcare and Applied biosystem) and signals were screened with a Typhoon PhosphorImager. The intensity of the revealed bands was quantified by Imagequant software (Amersham Biosciences). The total chromosome III signal was calculated by summing up the 3.5Mb chromosome signal and the acentric signals (without the background signal of each one). The percentage of acentric chromosome was obtained doing the ratio between the acentric signal and the signal corresponding to the total chromosome III.

Restriction Fragment Length Analysis (RFLA)

Recombination outcomes due to template exchange reactions are visualized and quantified by RFLA.

Chromosomal DNA was extracted using standard procedures.

Replication intermediates (RIs) analysis: the 2D gel (2DGE)

2DGE is a powerful technique to visualize replication intermediates at the fork arrest site.

Chromosomal DNA of cells embedded in agarose plug was digested with 30 units of AseI ( 60units if DNA was previously cross-linked -See 2.5 for the DNA cross-link procedure), and RIs were enriched on BND cellulose columns using the following procedure: 2 g of BND (Sigma, B6385) was dissolved in 15 ml of 0.3 M NaCl, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA for at least 4 hr and then, equally splitting in Poly-Prep Chromatography Columns (Biorad), was washed three times with 1 ml of the same buffer and dried. DNA samples were equilibrated at 0.3 M NaCl and loaded on BND cellulose columns.

Most of the double-stranded DNA was eluted by addition of 3 ml of 0.8 M NaCl, Tris-HCl (pH 7.5), and 1 mM EDTA. DNA containing single-stranded regions was eluted by addition of 3 ml of 1 M NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 1.8% (w/v) caffeine.

Fractions containing RIs were precipitated and dissolved in 10 μl of 10 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 0.1 mM EDTA and were analyzed by 2D gels, run according to Brewer et al., (1992), using 0.35% and 0.9% agarose for the first and second dimensions, respectively. If DNA was cross-linked, before transfer onto a nitrocellulose membrane it was decross-linked 10 min at 280 nm.

Replication intermediates were visualized using 32 P-ura4 probe. Analysis and quantification of 32 P-probed blots (using positively charged membrane) were performed by phosphor-imager using Imagequant software (as for PFGE). Quantification of 2DGE: the volume signal (background subtracted) for each RI was collected. The relative level of joint molecules (JM) was calculated by comparing to stalled forks signal (% of JM/stalled forks = volume JM/(volume JM + volume stalled fork) × 100).

Crosslink with 365-nm UV lamp

The DNA of certain strains was cross-linked before the 2DGE analysis, to demonstrate that joint molecules instability occurs in vivo and they are not particularly liable to dissolution in vitro.

1 to 2.10 9 cells were washed twice in 20 ml ultra-pure ice-cold water and were resuspended in 20 ml ice-cold water. Then the cells were transferred into an 8.5 cm-diameter pre-cooled glass-petri dish and placed on ice. 1 ml of 200µg/ml trimethylpsoralen (TMP) was added (Sigma). The petri dishes were incubated for 5 min in the dark mixing every minute. The cross-link was made by using a "Sellamed system Dr. sellmeier 4000" platform, in which the flux was adjusted at 50 mW/cm 2 and the cells were irradiated for 90 seconds. In these conditions the cells were submitted to 45 KJ.

Chromatin immunoprecipitation (ChIP)

The ChIP allows the detection of the recruitment of a specific protein, in a defined region in the genome. ChIP was performed using a protocol modified from Strahl-Bolsinger and colleagues [START_REF] Strahl-Bolsinger | SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast[END_REF]. Anti-GFP antibody (Molecular Probe) was used 1:300 and precipitated with G protein Dynabeads. The relative amounts of DNA fragments were quantified by real-time PCR (qPCR) on 5 μl of each samples using QuantiTect SYBR Green PCR master mix (Qiagen).

Chromatin immunoprecipitation was performed as follow: 1.10 LiCl, 0,5% NP40, 0.5% NaDeoxycholate, 1 mM EDTA) for 5 min, and finally with 1 ml of TE for 1 min.

Immunoprecipitation was then eluted with 130 μl of elution buffer, following by 2 hr of incubation at 65°C. Input control was as well incubated in same condition and 20 μl of immunoprecipitation was kept for immunoblotting. Finally, DNA was recover using Qiagen PCR purification kit and eluted in 200 μl of water. The relative amounts of PCR products were quantified using real-time PCR (qPCR) performing using 5 μl of each samples (input control and immunoprecipitation) and QuantiTect SYBR Green PCR master mix (Qiagen).

Fold enrichments were calculated using previously described method [START_REF] Chakrabarti | Quantitative assessment of gene targeting in vitro and in vivo by the pancreatic transcription factor, Pdx1. Importance of chromatin structure in directing promoter binding[END_REF].

Total cellular extract (TCA) protocol

Add 1 ml of stop buffer (50 mM NaF, 10 mM NaN3 in PBS 1X) to 1.10 8 cells, mix and put on ice. Centrifuge 30 seconds 14.000 rpm, room temperature, and wash with 1 ml of stop buffer. Spin again and wash with 1 ml of TCA 20%. Spin, resuspend the pellet in 200 μl of TCA 20% and add glass beads to break the cell walls using a homogenizer machine (Precellys). The program: 6.000 rpm 3 times, 30 seconds with one minute of pause between the cycles. After the homogenization of cells, add 400 μl of TCA 5% and centrifuge 5 minutes 4.000 rpm at 4°C, to collect the surnatant in a new 1.5 ml tube. Centrifuge 5 minutes, 13.000 rpm at 4°C and, after the aspiration of the surnatant, dry well the pellet. Then, resuspend in 200 μl of loading blue (for 10 ml: 5 ml of 2X SDS loading buffer, 2 ml Tris-HCl 1M pH8 and 3 ml of sterile water) and heat 5 minutes at 100°C. Samples can be frozen at -20°C or loaded on acrylamide gel.

Protein complex immunoprecipitation (Co-IP)

Using the Co-IP technique we could investigate if the interaction of two proteins occurs in fission yeast cells and if this interaction is still present mutating particular amino acids residues of one of the two partners. Western Blot: Samples (Input, Unbound and Ip) were loaded and migrated using an acrylamide gradient gel 4-12% (NuPAGE® Gels). The proteins were transferred on nitrocellulose membrane (Amersham Biosciences) and stained with "Red Ponceau". Cut the membrane to incubate with anti-GFP/anti Myc antibody (Roche) and anti-PCNA antibody (Santa Cruz Biotech). The day after wash in TBSt 0.1% three times 15 min, incubate with appropriate secondary antibody (anti-rabbit for GFP/Myc tagged protein and anti-mouse for PCNA) for one hour and then wash again three times in TBSt 0.1%. The final step is the revelation with ECL-Plus kit (Amersham Biosciences).

CHARACTERIZATION OF STRAINS DEFICIENT FOR CAF-1

In an effort to investigate the role of CAF-1 in the DNA Damage Response (DDR) in fission yeast, Dr. Francesconi (UMR3348, Institut Curie) created three strains, each one deleted for one of CAF-1 subunits: pcf1-d, pcf2-d or pcf3-d. These three strains are viable, showing that CAF-1 is not an essential H3-H4 histone chaperone in fission yeast, in contrast to Asf1 [START_REF] Dohke | Fission yeast chromatin assembly factor 1 assists in the replication-coupled maintenance of heterochromatin[END_REF][START_REF] Tanae | Histone chaperone Asf1 plays an essential role in maintaining genomic stability in fission yeast[END_REF]).

Stability of CAF-1 subunits

In budding yeast, deletion of one of the three CAF-1 subunits results in a loss of function of CAF-1 and each deletion shares common phenotypes [START_REF] Kaufman | Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I[END_REF][START_REF] Linger | The yeast histone chaperone chromatin assembly factor 1 protects against double-strand DNA-damaging agents[END_REF]. These data suggest that, in budding yeast, the main function of each subunit is to assemble the active CAF-1 complex. This situation contrasts with the situation in mammals in which the largest subunit p150 has a function in the DDR, independently of the two other subunits. Moreover, the third subunit p48 interacts with chromatin modifier enzymes independently of CAF-1. Based on these data, I first analyzed the role of fission yeast CAF-1 subunits in the DDR. Moreover, as the absence of one subunit in the complex can destabilize the expression level or the stability of the others subunits [START_REF] Schnaitman | Regulation of outer membrane protein synthesis in Escherichia coli K-12: deletion of ompC affects expression of the OmpF protein[END_REF][START_REF] Rodgers | Deletion of the 6-kDa subunit affects the activity and yield of the bc1 complex from Rhodovulum sulfidophilum[END_REF], I investigated the stability of the largest subunit Pcf1 in the absence of the two other subunits.

The C-terminal domain of Pcf1 was fused to the yellow fluorescent protein tag (Pcf1-YFP).

The expression level of

Strains defective for CAF-1 are not sensitive to DNA-damaging and replication-blocking agents

Another key point was to investigate the implication of CAF-1 complex in the DDR. As previously mentioned (see Introduction, Chapter 4), CAF-1 is involved in DNA repair pathways, including DSBs repair, both by promoting chromatin restoration after completion of DNA repair and by acting at early steps to recruit repair enzymes. In support of this, lack of CAF-1 function leads to increase sensitivity to UV-C and DSB-inducing agents in budding yeast [START_REF] Kaufman | Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I[END_REF][START_REF] Linger | The yeast histone chaperone chromatin assembly factor 1 protects against double-strand DNA-damaging agents[END_REF]. Thus, I investigated the sensitivity of strains defective for CAF-1 (pcf1-d, pcf2-d and pcf3-d single mutant) to DNAdamaging and replication-blocking agents, in fission yeast. The following drugs were used:

 camptothecin (CPT), a replication-blocking agent that inhibits the topoisomerase I and results in torsional stress and replicative DSBs.

 hydroxyurea (HU), an inhibitor of the ribonucleotide reductase, which leads to an inhibition of bulk dNTP synthesis in S-phase and thus, to a strong slowing down of the replication forks progression.

 UV-C radiation, that produces two predominant types of DNA damage: cyclobutane pyrimidine dimers (CPD) and pyrimidine pyrimidone (6-4 photoproducts).

 methyl methanesulfonate (MMS), an alkylating agents leading to base alkylation and to a strong delay in S-phase progression.

 cisplatine (CIS) and mitomycin-C (MMC), two cross-linking agents. The main lethal lesions induced by cross-linking agents are inter-strands cross-links (ICLs) that compromise the unwinding of parental duplex DNA, ahead of advancing forks.

 tetrabenazine (TBZ) that inhibits microtubule polymerisation and compromise kinetochore attachment to the mitotic spindle.

Data showed that strains defective for CAF-1 are not more sensitive to these DNA-damaging and replication-blocking agents than the wt strain (Figures n. 30, 31B-C, 32). Importantly, sensitivities to these agents were analyzed both on rich (YE) and EMM-Glu media and similar data were obtained.

However, I found that strains deleted for each subunit of CAF-1 were highly resistant to MMS (Figure n. 31A). Indeed, the three strains pcf1-d, pcf2-d or pcf3-d exhibit at least a 10 fold greater resistance to MMS than the wt strain. These data suggest that CAF-1 defect might play a direct role in conferring MMS resistance. Another possible explanation is that chronic exposure to MMS (one week) results in a longer G1-phase in the absence of CAF-1, therefore increasing the resistance to MMS. These two hypotheses are not mutually exclusives.

In contrast to the data reported in S. cerevisiae [START_REF] Kaufman | Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I[END_REF][START_REF] Linger | The yeast histone chaperone chromatin assembly factor 1 protects against double-strand DNA-damaging agents[END_REF], my results suggest that none of CAF-1 subunits plays a major role in the DNA damage response. Probably, the differences observed between S. cerevisiae and S.

pombe are due to their cell cycle. Indeed, in fission yeast the longer phase of the cell cycle is the G2 phase (80% of the cell-cycle time), while it is not the case for the budding yeast. The higher sensitivity to DNA damaging agents occurs in S-phase, therefore in S. pombe the window of time in which cells can experience DNA damage is limited.

Genetic interactions with the histone chaperone HIRA

HIRA complex is a H3-H4 histone chaperone required for replication independent chromatin assembly, for example during gene transcription and gene silencing (See Introduction 3.2.3).

In S. pombe, the Hir complex (HIRA homologue) is composed of three subunits: Hip3 (gene: SPBC31F10.14c), Hip1 (gene: SPBC31F10.13c) and Slm9 (gene: SPBC15D4.03). Deletion of either hip1 or slm9 leads to cell cycle delay, poor recovery from nitrogen starvation, increased chromosome loss, sensitivity to DNA damaging agents, increased sensitivity to spindle damage and decreased transcriptional silencing in the outer centromere repeats. In addition, deletion of hip1 alone leads to derepression of core histone genes expression, outside S-phase. In S. cerevisiae, combining mutations in CAC genes (encoded the CAF-1 subunits)

and HIR genes results in increased sensitivity to DNA-damaging agents and in a synergistic decrease in gene silencing at both mating type and telomeric loci [START_REF] Kaufman | Hir proteins are required for position-dependent gene silencing in Saccharomyces cerevisiae in the absence of chromatin assembly factor I[END_REF][START_REF] Kanoh | Slm9, a novel nuclear protein involved in mitotic control in fission yeast[END_REF][START_REF] Sutton | Yeast ASF1 protein is required for cell cycle regulation of histone gene transcription[END_REF][START_REF] Blackwell | The Schizosaccharomyces pombe HIRA-like protein Hip1 is required for the periodic expression of histone genes and contributes to the function of complex centromeres[END_REF][START_REF] Greenall | Hip3 interacts with the HIRA proteins Hip1 and Slm9 and is required for transcriptional silencing and accurate chromosome segregation[END_REF][START_REF] Anderson | Silencing mediated by the Schizosaccharomyces pombe HIRA complex is dependent upon the Hpc2-like protein, Hip4[END_REF].

To test the genetic interactions between HIRA and CAF-1, I crossed a pcf1-d strain with two strains, respectively deleted for hip1 or slm9 genes ( Indeed, CAF-1 seems to play a role, directly or indirectly in transcription processes [START_REF] Kim | Potential role of the histone chaperone, CAF-1, in transcription[END_REF][START_REF] Heyd | The p150 subunit of the histone chaperone Caf-1 interacts with the transcriptional repressor Gfi1[END_REF]) and an excess of histones in the cell might have deleterious effects on cell survival.

CAF-1 IS DISPENSABLE FOR ALLELIC RECOMBINATION BUT NOT FOR ECTOPIC RECOMBINATION

In this chapter, I investigated the contribution of CAF-1 to mechanisms of homologous recombination. Therefore, I analyzed the impact of CAF-1 defects on both allelic recombination (recombination between sister-chromatids and homologous chromosomes) and on ectopic recombination (recombination between dispersed repeated sequences), both spontaneously or induced by replication-blocks.

Allelic recombination between homologous chromosomes

To achieve the understanding of the role of CAF-1 in homologous recombination, I used an assay to analyze allelic recombination between homologous chromosomes in a nonsporulating diploid. This assay allows the score of cross-over events between the two homologous chromosomes III. Moreover, this assay also permits to score the frequency of loss of chromosome III (Figure n. 34A) [START_REF] Hartsuiker | Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair[END_REF].

The non-sporulating diploid harbors two different alleles of ade6 on each chromosome III (ade6-M216 and ade6-M210) that allow an intra-allelic complementation. Thus, the diploid is prototroph for the adenine (Ade + ). On the opposite chromosome arm, one homologue harbors the wt ura4 + gene and the other one a deletion of ura4 + (ura4-D18). Thus, the diploid is prototroph for the uracil and sensitive to the 5-fluoroorotic acid drug (Ura + 5-FOA S ). A crossover event between the centromere and the ura4 locus with subsequent mitotic division, leads to a diploid harbouring ura4D-18 on the two chromosomes III, therefore leading to an Ade + 5-FOA R diploid. In contrast, loss of one homologue chromosome III leads to an aneuploid harbouring only one chromosome III and conferring an Ade -5-FOA R phenotype.

Both Ade + and Ade -cells can be selected on low-adenine media which confers white-color to Ade + diploid and red-color to Ade -diploid.

All diploids deleted for pcf1, pcf2 or pcf3 showed a rate of cross-overs between homologous chromosomes and a rate of chromosome loss similar to the ones of the wt strain (Figure n.

34B and C). Furthermore, I have found a small but not significant increase of chromosome loss in pcf1-d (1.7 fold over wt) but not in pcf2-d or pcf3-d strains. The values correspond to the mean of three independent rates, each one being median of 13 independent cultures. The confidence interval is calculated at 95% (enclosed in bracket) and the fold increase over wt is indicated for each strain.

Allelic recombination between sister-chromatids

To further investigate the role of CAF-1 in allelic recombination, I analyzed CAF-1 involvement in sister-chromatid exchange. For this purpose, I used a strain harboring two inactivated ade6 alleles orientated in direct repeats (ade6-L469 and ade6-M375 alleles), separated by the ura4 + marker [START_REF] Schuchert | The Ade6-M26 Mutation of Schizosaccharomyces Pombe Increases the Frequency of Crossing over[END_REF][START_REF] Hartsuiker | Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair[END_REF])

(Figure n. 35A).
A wild-type ade6 + gene can be restored by different intra or inter-chromatids recombination processes [START_REF] Schuchert | The Ade6-M26 Mutation of Schizosaccharomyces Pombe Increases the Frequency of Crossing over[END_REF]):

-Gene conversion that gives rise to ade6 + ura4 + cells. The gene serving as donor is either on the same chromatid (intra-sister recombination) or on the other sister chromatid (inter-sister recombination).

-Gene conversion associated to cross-over, that leads to ade6 + ura4 -cells.

-Intra-chromatid Single Strand Annealing (SSA), that gives rise to ade6 + ura4 -cells.

Strains deleted for pcf1, pcf2 or pcf3 did not present any significant differences in the rate of sister-chromatid exchange compared to wt strain, regardless the type of recombinant analyzed (gene conversion or deletion), (Figure n. 35B and C). Thus, I concluded that CAF-1 is dispensable for sister-chromatid recombination in S. pombe. These data are coherent with the fact that defects in CAF-1 do not lead to an increased sensitivity to DNA-damaging agents, suggesting that post-replication repair mediated by recombination between sister-chromatid remains functional in the absence of CAF-1.

To further reinforce this conclusion, I analyzed the frequency of sister-chromatid recombination induced by camptothecin (CPT). As previously reported by Ahn and colleagues, CPT-treatment induces a slight (1.5 fold over spontaneous events), but significant, increase in the frequency of gene conversion and a 3 fold increase in the frequency of deletions [START_REF] Ahn | Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast[END_REF] Therefore, I concluded that, in the absence of CAF-1, the potential reduction in nucleosome density behind the fork does not lead to an increased level of sister chromatid exchanges, even in response to replication stress.

Repair of a site-specific polar DSB by recombination between sisterchromatids

When the replication fork collides with an unrepaired single-strand break (SSB) or nick, a polar one-ended double-strand break (DSB) is formed. To repair such replicative DSB, homologous recombination uses the closer homologous sequence, the sister chromatid, thus restoring the fork structure and allowing replication to restart [START_REF] Arcangioli | Fate of mat1 DNA strands during mating-type switching in fission yeast[END_REF].

To confirm data obtained for the role of CAF-1 in the repair of replicative DSBs by homologous recombination, I made use of a genetic system that allows the repair of a unique broken replication fork at a specific locus to be analyzed [START_REF] Roseaulin | Mus81 is essential for sister chromatid recombination at broken replication forks[END_REF]). In S. pombe, a stable site-specific and strand-specific DNA lesion was identified at the mat1 locus [START_REF] Klar | Initiation of meiotic recombination by double-strand DNA breaks in S. pombe[END_REF][START_REF] Arcangioli | A site-and strand-specific DNA break confers asymmetric switching potential in fission yeast[END_REF]). The lesion was described as a Single Strand Break (SSB) with free 3′OH and 5′OH termini [START_REF] Kaykov | A programmed strand-specific and modified nick in S. pombe constitutes a novel type of chromosomal imprint[END_REF]. Arcangioli and colleagues showed that the SSB occurs on the neo-synthesized lagging strand during mat1

DNA replication. This SSB remains stable throughout the next cell cycle. During the following round of DNA replication, fork passage through the SSB converts the SSB into a polar blunt-ended DSB on the leading strand [START_REF] Kaykov | Formation, maintenance and consequences of the imprint at the mating-type locus in fission yeast[END_REF][START_REF] Holmes | Molecular and cellular dissection of mating-type switching steps in Schizosaccharomyces pombe[END_REF]. The mode of repair of the polar DSB depends on the presence or absence of homologous sequences for repair. In a wt strain harboring a switchable mat configuration, the two silent donor cassettes (mat2P and mat3M) are located in a heterocromatic region, and provides intact DNA templates for recombinational repair, thus allowing mating type switching [START_REF] Egel | Fission yeast mating-type switching: programmed damage and repair[END_REF]Roseaulin, Yamada et al. 2008) (Figure n. 36A). In the absence of silent cassettes (mat 2,3), the polar DSB is repaired by homologous recombination using the sister-chromatid. It was reported that inducing the strand-specific polar DSB at mat1 is sufficient to induce cell death in the absence of homologous recombination [START_REF] Roseaulin | Mus81 is essential for sister chromatid recombination at broken replication forks[END_REF]). Thus, this genetic system is a powerful tool to analyze genetic requirement to repair replicative DSBs, by sister-chromatid recombination.

To investigate the role of CAF-1 in the repair of a replicative DSB by sister-chromatid recombination, I created a strain mat1-PΔ17 (-SSB, + donors) deleted for pcf1. The PΔ17 corresponds to small deletion in mat1 that inhibits SSB formation, but leads to the expression of the P mating-type [START_REF] Arcangioli | A novel switch-activating site (SAS1) and its cognate binding factor (SAP1) required for efficient mat1 switching in Schizosaccharomyces pombe[END_REF][START_REF] Roseaulin | Mus81 is essential for sister chromatid recombination at broken replication forks[END_REF]. I crossed the h+ mat1-PΔ17 pcf1-d strain with the h -mat1-M2,3∆ (+SSB, -donors) strain. The resulted diploid was selected and spores were analyzed for the presence of the SSB (h -), the presence of the donors (h + ) and the deletion of pcf1.

The data showed that pcf1-d spores were viable, regardless of the presence or not of the SSB and the donors, indicating that CAF-1 is not essential to repair the polar DSB in the absence of donors (Figure n. 36B). Therefore, I concluded that CAF-1 is dispensable for the repair of replicative DSB by sister-chromatid recombination and these data are in agreement with the data presented above.

Altogether, the data obtained support that CAF-1 is dispensable for allelic recombination and for the repair of DSBs by sister-chromatid recombination.

Fork-arrest-induced ectopic recombination

Then, I investigated the role of CAF-1 in ectopic recombination. For this purpose, I made use of the RTS1-RFB genetic system that allows the site-specific block of a replication fork to be induced. The activity of the Replication Fork Barrier (RFB) RTS1 is mediated by the binding of the Rtf1 protein to the RTS1 sequence (see introduction, Replication forks arrested at the RTS1-RFB are restarted by homologous recombination (Lambert et al., 2010). Fork-restart by recombination occasionally results in ectopic recombination with the RTS1 sequence naturally located near the mat1 locus on chromosome II, thus leading to the loss of ura4 + marker ( 5 Therefore, these data establish that CAF-1 promotes ectopic recombination induced by the

RTS1-RFB.

Altogether, my investigations on the role of CAF-1 in homologous recombination lead to the conclusion that, while CAF-1 is dispensable for allelic recombination even in response to replication stress, it promotes ectopic recombination induced by fork-arrest. The role of CAF-1 during homologous recombination is of particular importance when the homology between the donor and the acceptor is limited. Importantly, this pro-recombinogenic function of CAF-

CAF-1 PROMOTES D-LOOP STABILIZATION

In this chapter, I investigated the impact of CAF-1 defects on homologous recombination at the molecular level. For this purpose, I used the RuraR system, based on the RTS1-RFB, which allows replication-induced recombination to be analyzed. Fork restart at the RTS1-RFB is proposed to be initiated by the unwinding of stalled nascent strands, on which Chromosomal rearrangements can be scored by restriction fragment length analysis (RFLA)

and pulse field gel electrophoresis (PFGE). Therefore, the RuraR system allows the different steps of replication-induced recombination to be analyzed: the stability of the arrested fork, the recruitment of recombination factors, the fork-recovery efficiency, the formation and stability of JMs, and the products of their resolution.

CAF-1 promotes replication-induced recombination by template exchange.

I

Are recombination factors properly recruited?

-Analysis of Rad22 protein recruitment at the RTS1-RFB by ChIP.

In which recombination pathways does CAF-1 act?

-Epistasis analysis with Rhp51, Rqh1 and Srs2

Are blocked forks stable and properly restarted by recombination?

-2DGE to investigate the direct fork restart either with EcoRV or AseI restriction enzymes. The digestion with EcoRV allows the analysis of acentric chromosome formation and the switch of ura4 orientation. The AseI digestion allows the investigation of the formation of acentric and dicentric chromosomes.

Chromosomal rearrangements were quantified as previously described (Lambert, Mizuno et al. 2010 and detailed in the material and methods) and presented as recombination events associated with one (acentric) or two cross-overs (switch of ura4 orientation). Chromosomal rearrangements accumulated in the wt RuraR strain, upon induction of the RTS1-RFB (Figures n. 42A and B), as previously reported [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF] To further confirm these results, I analyzed the formation of acentric iso-chromosome by PFGE in cells cultured with thiamine (OFF conditions) or at 24 h and 48 h after thiamine removal (ON conditions). As previously reported [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF] Collectively, these data establish that the products of resolution of JMs are decreased in the absence of CAF-1, whatever the type of events (associated with one or with two cross-overs).

Thus, I concluded that the decrease in JMs intensity in the absence of CAF-1 is not due to a faster resolution of JMs, but is rather due to defects in their formation or stability. Also, these data support the view that it is the histone chaperone function of CAF-1 that is essential to the formation or stability of JMs. However, further experiments are required to confirm this hypothesis.

Another important point is that defects in CAF-1 do not completely abolish replication-

Recruitment of Rad22 at arrested forks is not affected in the absence of CAF-1

To further understand the contribution of CAF-1 in replication-induced recombination, I studied the contribution of CAF-1 to early recombination steps, such as formation of recombination centers and Rad22 recruitment at arrested forks.

Firstly, I verified that the expression of Rad22 was not affected by defects in CAF-1. For this purpose, I used a strain in which the C-terminal of Rad22 was fused to the fluorescent tag Then, I investigated the formation of spontaneous recombination centers by analyzing Rad22 foci, using 3D-deconvolution fluorescence microscopy. In a wt asynchronous population (about 80% of G2 cells), around 10 % of cells show at least one Rad22 focus: 10 % of G2 cells and 10 % of S-phase cells (harbouring a septum as marker of S-phase cells). In the absence of each CAF-1 subunits, 20 to 25 % of cells showed at least one Rad22 focus, and this increased in recombination centers was particularly pronounced in S-phase cells, as 30 to 40 % of S-phase cells exhibited at least one Rad22 focus (Figures n. 44B an C). The accumulation of Rad22 foci, especially in S-phase, is coherent with data reported for other model organisms, in which defects in CAF-1 complex lead to impediments in S-phase progression [START_REF] Hoek | Chromatin assembly factor 1 is essential and couples chromatin assembly to DNA replication in vivo[END_REF][START_REF] Loyola | Histone chaperones, a supporting role in the limelight[END_REF]. Indeed, defective replication-coupled chromatin assembly was reported to cause accumulation of recombination centers in budding yeast, interpreted as accumulation of recombinogenic DNA lesions [START_REF] Clemente-Ruiz | Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks[END_REF]. Nonetheless, such accumulation of recombinogenic DNA structures does not cause cell cycle delay in S. pombe, as strains deleted for each CAF-1 subunit exhibit a generation time similar to that of a wt strain.

Moreover, these data indicate that defects in CAF-1 do not impede assembly of spontaneous recombination centers.

Afterwards, I concentrated on the recruitment of Rad22 at sites of replication forks arrested by the RTS1-RFB barrier, using the technique of chromatin immunoprecipitation (ChIP). As previously reported [START_REF] Lambert | Gross chromosomal rearrangements and elevated recombination at an inducible site-specific replication fork barrier[END_REF], Rad22-GFP was found enriched at the RuraR locus when the RTS1-RFB is activated, in a wt strain (Figure n. 44D). A similar level of Rad22-GFP recruitment was observed in pcf1-d or pcf2-d strains. Moreover, Rad22-GFP is significantly recruited up to 1.4 Kb behind arrested forks whatever the genetic background analyzed. Thus, Rad22 is recruited at arrested forks in the absence of CAF-1, to the same extend than the wild-type strain. Therefore, the decreased in replication-induced recombination efficiency cannot be explained by a defect in recruiting recombination factors at arrested forks, in the absence of CAF-1.

CAF-1 is dispensable for fork-stability and recovery by recombination

To further understand the role of CAF-1 in replication-induced recombination, I analyzed the stability of arrested forks and their ability to be recovered by recombination, in the absence of CAF-1. Indeed, an increased instability of arrested forks and/or defects in their recovery could also explain the observed decreased intensity of JMs in the absence of CAF-1.

For this purpose, I created strains deleted for each CAF-1 subunits and harbouring a single 45A-C). Therefore, I concluded that stabilization of arrested forks and their recovery by recombination is neither defective nor more efficient in the absence of CAF-1. Moreover, the data do not support the hypothesis that template exchange (in RuraR strain) is decreased in the absence of CAF-1 due to a more efficient engagement of fork restart on the initial template.

The aspect of stabilization of arrested forks in the absence of CAF-1 is a common theme in CAF-1 literature (Clemente- [START_REF] Clemente-Ruiz | Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks[END_REF][START_REF] Clemente-Ruiz | Chromatin assembly controls replication fork stability[END_REF]Introduction 3.2.4). A delay in histone deposition in the rear of advancing forks, due to CAF-1 defects, could result in a different chromatin context, favoring processes such as SCE and forks recovery by recombination. However, I demonstrated in S. pombe that CAF-1 defects do not affect in appreciable manner the stability of arrested forks. Moreover, I showed that replication-induced SCE are not significantly affected by the lack of CAF-1 function, confirming the marginal role of CAF-1 in maintaining integrity of impeded replication forks.

Altogether, these data suggest that CAF-1 is involved in the stability of JMs rather than in their formation. [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]). This effect results in an increased level of recombination products associated to two cross-overs (switch of ura4 orientation) at the expense of recombination products associated to one cross-over (acentric chromosome). Moreover, Rqh1 does not prevent chromosomal rearrangements by dissolution of JMs-B containing HJs, as these HJs were shown to be not branch-migrable in vitro. It was rather proposed that Rqh1 prevents chromosomal rearrangements by promoting D-loop dissociation [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]. As previously described, the level of acentric chromosome was reduced by 2 times in the rqh1-d RuraR strain, compared to wt level, upon induction of the RTS1-RFB. Interestingly, the level of acentric chromosome in pcf1-d rqh1-d and pcf2-d rqh1-d strains was similar to that of the wt strain (Figure n. 48B). Thus, the deletion of rqh1 rescues CAF-1 defects in promoting replication-induced recombination. Values correspond to the mean of three independent median rates (each one determined from 9 independent cultures using the median method). The error bars represent the standard deviation and the statistical significance was calculated using the non-parametric Mann Whitney test. Bottom panel: table representing the induction of ura4 loss by the RTS1-RFB in indicated strains (rate of ura4 loss in ON condition/ rate of ura4 loss in OFF condition), and the increase over wt. B) Examples of PCR analysis of 5-FOA R colonies obtained upon rtf1 induction (ON condition) in indicated strains. Primers used were: TLII and TLIII, Rng3 and Ura4 primers. C) Top: The % of deletion events, as determined by the PCR assay, was used to balance the rate of ura4 loss. The values reported are means of 3 independent median rates. Error bars correspond to the standard deviation. Bottom: The % of translocation events, as determined by the PCR assay, was used to balance the rate of ura4 loss. The values reported are means of 3 independent median rates. Error bars correspond to the standard deviation. Right tables: induction of genomic deletions or translocations by the RTS1-RFB (rate of genomic deletions or translocations in ON conditions/rate of genomic deletions or translocations in OFF conditions) and the relative increase over wild-type.

THE CHROMATIN ASSEMBLY FUNCTION OF CAF-1 IS REQUIRED TO STABILIZE D-LOOP INTERMEDIATES

In this chapter, I investigated by which function CAF-1 promotes D-loop stabilization. One hypothesis was that CAF-1 promotes histone deposition during the elongation of D-loop intermediates, thus counteracting their dissolution by Rqh1.

The silencing / heterochromatin function of CAF-1 is dispensable for D-loop stabilization

The RTS1-RFB is based on a direct interaction between the Rtf1 protein and the RTS1 sequence, to block the progression of replication forks. It has been recently reported that replication stress arising from tight protein-DNA complexes favours heterochromatin formation. This occurs through the ectopic recruitment of the SIR complex which promotes gene silencing and possible changes in subnuclear localization [START_REF] Dubarry | Tight protein-DNA interactions favor gene silencing[END_REF].

To investigate if gene silencing occurs at the RTS1-RFB, I took advantage of the counterselectable marker ura4 to select for ura4 loss (resistance to 5-FOA) after short induction of fork-arrest (in contrast to the ectopic recombination assay that requires prolonged induction of fork-arrest). The strains used were a wt and a pcf1-d harbouring the RuraR construct. As positive control I used a strain deleted for ura4 (ura4-D18), which can grow on 5-FOA. I tested also a swi6-d strain harbouring the construct RuraR. In S. pombe, Swi6 (SPAC664.01c) is involved in heterochromatin maintenance. It directly interacts with Pcf1, to be displaced from the heterochromatin ahead the replication fork and delivered to methylated histones, behind the replication fork, at the centromeric region. This transfer is necessary to facilitate the progress of the replication fork through heterochromatin and to ensure the selfperpetuation of the epigenetic state of the domain [START_REF] Dohke | Fission yeast chromatin assembly factor 1 assists in the replication-coupled maintenance of heterochromatin[END_REF]Introduction 3.3).

After 72 hours of induction of the RTS1-RFB, there was no significant increase in the amount of cells resistant to 5-FOA, whatever the genetic background analyzed (wt, pcf1-d or swi6-d) andB). Thus, these data do not support a potential gene silencing effect upon fork-arrest, in the RTS1-RFB system. In mammals, HP1 is the homologue of Swi6 and it has similar functions, interacting with the largest subunit of CAF-1 (p150). Depletion of HP1 leads to defects in HR-mediated repair and it also compromises the recruitment of factors involved in the DNA damage response [START_REF] Baldeyron | HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair[END_REF]). Thus, it seems likely that HP1 is involved in recombination processes and I decided to test if the role of CAF-1 in promoting replication-induced recombination could be linked to its Swi6-dependent function in maintaining heterochromatin.

(Figure n. 51A
To achieve my purpose, I studied if a swi6-d strain could phenocopy strains defective for CAF-1, using previously described recombination assays. The strain deleted for swi6 presented a slow growth phenotype compared to the wt strain and a slight increase in UV-C sensitivity, as previously reported (Figure n. 52A) [START_REF] Ekwall | Fission yeast mutants that alleviate transcriptional silencing in centromeric flanking repeats and disrupt chromosome segregation[END_REF]. The values represent the mean of three independent rates, each one being median of 11 independent cultures. The confidence interval is calculated at 95% (enclosed in bracket). Fold increase over wt is indicated for each strain. C) Analysis of recombination between homologous chromosomes: the rate of recombination events and rate of chromosome loss is reported for indicated strains. The values correspond to the mean of three independent rates, each one being median of 13 independent cultures. The confidence interval is calculated at 95% (enclosed in bracket) and the fold increase over wt is indicated for each strain.

I also analyzed the contribution of Swi6 to sister-chromatid exchange and found that the strain swi6-d exhibited no particular defect or increase in SCE, whatever the type of recombinant scored (gene conversion or deletion) (Figure n. 52B). In contrast, the rate of cross-overs between homologue chromosomes was decreased by 3 times in swi6-d, compared to wt strain.

This decrease in allelic recombination between homologue chromosomes was accompanied by a 20 fold increase in chromosome loss (Figure n. 52C). The significant increase in chromosome loss could be due to a defect in maintaining centromeric heterochromatin, which could explain also the increased sensitivity to TBZ reported in the literature [START_REF] Ekwall | Fission yeast mutants that alleviate transcriptional silencing in centromeric flanking repeats and disrupt chromosome segregation[END_REF]). As mentioned above, Swi6 has a critical role in supporting accurate chromosome segregation, through heterochromatin maintenance at the centromere. Indeed, it has been demonstrated that localization of cohesins at the centromeric repeats is Swi6-dependent [START_REF] Nonaka | Recruitment of cohesin to heterochromatic regions by Swi6/HP1 in fission yeast[END_REF]. 151 C) Top: The % of deletion events, as determined by the PCR assay, was used to balance the rate of ura4 loss. The values reported are means of 3 independent median rates. Error bars correspond to the standard deviation. Bottom: The % of translocation events, as determined by the PCR assay, was used to balance the rate of ura4 loss. The values reported are means of 3 independent median rates. Error bars correspond to the standard deviation. On the right, tables reporting the induction of genomic deletion or translocation by the RTS1-RFB (rate of genomic deletion or translocation in ON conditions/ rate of genomic deletion or translocation in OFF conditions) in indicated strains. Decrease over the wild-type is reported.

Thus, these data establish that the interaction between Pcf1 and PCNA is crucial to promote replication-induced recombination and D-loop stabilization. This supports the hypothesis that CAF-1 promotes D-loop stabilization via its replication-coupled chromatin assembly function.

CAF-1 PROMOTES THE STABILIZATION OF TOXIC RECOMBINATION INTERMEDIATES

Exploiting the RTS1-RFB-based system, data obtained support the view that CAF-1 stabilizes D-loop intermediates by counteracting the anti-recombinase activity of Rqh1 helicase. CAF-1 function is achieved by promoting chromatin assembly during DNA synthesis, associated to homologous recombination.

Defects in processing JMs and/or in the disassembly of Rad51-filaments result in accumulation of toxic recombination intermediates, explaining the severe slow growth phenotype of the double mutant srs2-d rqh1-d. This negative genetic interaction is suppressed by the deletion of rhp51, as Rhp51 is responsible for JMs formation which are normally resolved by either Srs2 or Rqh1 [START_REF] Chanet | Semidominant mutations in the yeast Rad51 protein and their relationships with the Srs2 helicase[END_REF][START_REF] Gangloff | Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases[END_REF][START_REF] Doe | The involvement of Srs2 in post-replication repair and homologous recombination in fission yeast[END_REF][START_REF] Burgess | Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo[END_REF]).

To confirm the conclusion that CAF-1 regulates homologous recombination by acting in the As reported in the literature, defects in replication-coupled chromatin assembly lead to the instability of advancing forks and a need for homologous recombination to process them. This results in a general increase in genome instability (increased level of recombination and

A B

genome rearrangements) caused by the deficiency of two main histones chaperones involved in DNA metabolism: CAF-1 and Asf1 [START_REF] Myung | Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability[END_REF][START_REF] Prado | The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange[END_REF][START_REF] Endo | Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants[END_REF][START_REF] Clemente-Ruiz | Chromatin assembly controls replication fork stability[END_REF][START_REF] Clemente-Ruiz | Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks[END_REF]. Moreover, defects in the deposition of the histone mark H3K56Ac during chromatin assembly lead to genetic instability due to a higher susceptibility of fork stalling, damage or breakage. The absence of H3K56Ac or the simultaneous knock-out of CAF-1 and Rtt106 affect the integrity of advancing replication forks, increasing the level of homologous recombination [START_REF] Han | Acetylation of lysine 56 of histone H3 catalyzed by RTT109 and regulated by ASF1 is required for replisome integrity[END_REF][START_REF] Clemente-Ruiz | Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks[END_REF][START_REF] Su | Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106[END_REF]. Thus, to investigate the truthfulness of the supposition that forks arrested at the RTS1-barrier are unstable and more liable to restart on the initial template, I analyzed the level of recombination between sister chromatids and the stability of halted replication forks, in the absence of CAF-1. Defects in CAF-1 histone chaperone activity do not lead to a general genomic instability, as it was reported for S. cerevisiae, Arabidopsis and human cells. [START_REF] Myung | Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability[END_REF][START_REF] Prado | The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange[END_REF][START_REF] Endo | Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants[END_REF][START_REF] Clemente-Ruiz | Chromatin assembly controls replication fork stability[END_REF][START_REF] Clemente-Ruiz | Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks[END_REF]. Strains deleted for pcf1, pcf2 or pcf3 accumulate spontaneously Rad22 foci in S-phase, suggesting that recombinogenic DNA structures arise in the absence of CAF-1. Nonetheless, the level of spontaneous or replication-stress-induced SCE is not affected by the absence of CAF-1. Moreover, strains defective for CAF-1, do not present an increased sensitivity to replication blocking agents. I also found that arrested forks are stable and prone to recombination-dependent restart: restart of arrested forks on the initial template is neither defective nor more efficient, in the absence of CAF-1. Thus my data exclude that the deficiency of CAF-1 could threaten the stability of the arrested fork and channel a preferential recovery of the fork on the same template to the detriment of template exchange.

The second hypothesis was that CAF-1 could be involved directly in promoting template exchange reactions (Figure n. 59B). Indeed, I found that, in the absence of CAF-1, both JMs and the product of their resolution (chromosomal rearrangements) were reduced. I supposed that CAF-1 complex could be involved at different steps in template exchange pathway, therefore I investigated:

1. an eventual role of CAF-1 in the recruitment of Rad22, during early steps of homologous recombination.

2. the possibility that JMs could be solved faster in the absence of CAF-1.

CAF-1 stabilizes D-loop intermediates by counteracting their dissolution by Rqh1 activity.

CAF-1 appears to be part of an equilibrium that regulates the stability/dissociation of early steps of recombination events, acting as an antagonist of the DNA helicase Rqh1 (Figure n. 60). My data evidenced that, in the absence of both CAF-1 and Rqh1, JMs and the product of their resolution are properly formed. Moreover, the deletion of rqh1 gene rescues the decreased level of fork-arrest induced ectopic recombination due to CAF-1 defects. Rqh1 but not Srs2 helicase, was shown to antagonize CAF-1 function during homologous recombination mechanism.

Both Srs2 and Rqh1 are DNA helicases proposed to act during early steps of homologous recombination, by either dismantling the Rad51 filament or by dissolving D-loop intermediates, thus promoting the SDSA pathway [START_REF] Fabre | Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication[END_REF][START_REF] Aylon | Molecular dissection of mitotic recombination in the yeast Saccharomyces cerevisiae[END_REF][START_REF] Ira | Srs2 and Sgs1-Top3 suppress crossovers during doublestrand break repair in yeast[END_REF][START_REF] Robert | Mrc1 and Srs2 are major actors in the regulation of spontaneous crossover[END_REF][START_REF] Oh | BLM ortholog, Sgs1, prevents aberrant crossing-over by suppressing formation of multichromatid joint molecules[END_REF][START_REF] Oh | RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, resolve aberrant joint molecules during meiotic recombination[END_REF][START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]see Introduction 2.3.2). In addition, Rqh1 plays a crucial role in preventing mitotic cross-overs by promoting the dissolution of double Holliday-junctions [START_REF] Doe | Partial suppression of the fission yeast rqh1(-) phenotype by expression of a bacterial Holliday junction resolvase[END_REF].

Considering the results obtained, it will be of interest to test the DNA helicase Fml1 (SPAC9.05), which deletion phenotype is viable. Fml1 is an orthologoue of human FANCM, a family of DNA helicases/translocases playing key roles in homologous recombination (HR)

and DNA repair in archaea and eukaryotes [START_REF] Whitby | The FANCM family of DNA helicases/translocases[END_REF]. FANCM family members have a general ability to unwind and/or branch migrate DNA junctions that resemble intermediates of HR, in particular to branch migrate Holliday junctions (HJs) and unwinding of D-loops structures that are formed by Rad51-mediated strand invasion [START_REF] Prakash | Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination[END_REF][START_REF] Sun | The FANCM ortholog Fml1 promotes recombination at stalled replication forks and limits crossing over during DNA double-strand break repair[END_REF][START_REF] Zheng | Processing of DNA structures via DNA unwinding and branch migration by the S. cerevisiae Mph1 protein[END_REF]. It is proposed that Fml1 prevents crossing-over events during DSB repair by its ability to dissociate D-loop intermediates [START_REF] Lorenz | The fission yeast FANCM ortholog directs noncrossover recombination during meiosis[END_REF][START_REF] Sun | The FANCM ortholog Fml1 promotes recombination at stalled replication forks and limits crossing over during DNA double-strand break repair[END_REF], thus it could be a good candidate to counteract the stabilization function of CAF-1. Similarly, the orthologoue of Fml1 in S.

cerevisiae, Mph1, is thought to channel recombination intermediates into a non-crossover pathway by dissociating D-loop structures [START_REF] Prakash | Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination[END_REF].

Fission yeast rqh1 mutants are hypersensitive to agents such as UV light, CPT and MMS, that cause replication forks to stall [START_REF] Boddy | Damage tolerance protein Mus81 associates with the FHA1 domain of checkpoint kinase Cds1[END_REF][START_REF] Stewart | rqh1+, a fission yeast gene related to the Bloom's and Werner's syndrome genes, is required for reversible S phase arrest[END_REF]. It has been proposed by Lambert and colleagues that Rqh1 helicase regulates Rhp51dependent recombination at the RTS1 barrier, and this limits the likelihood of rearrangements without affecting the efficiency of fork restart [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]). This might be achieved by Rqh1 function in processing the D-loop [START_REF] Stewart | rqh1+, a fission yeast gene related to the Bloom's and Werner's syndrome genes, is required for reversible S phase arrest[END_REF][START_REF] Doe | Mus81-Eme1 and Rqh1 involvement in processing stalled and collapsed replication forks[END_REF] The fact that CAF-1 stabilizes Rhp51-dependent JMs, could also provide an explanation for MMS resistance of strains deleted for pcf1 or pcf2 or pcf3. MMS methylates DNA on N 7deoxyguanine and N 3 -deoxyadenine and causes a variety of DNA damages, including stalling of replication forks, where HR is essential for fork restart [START_REF] Lundin | Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks[END_REF].

Therefore, a Another interesting point to explore is whether other histone chaperones are also involved in D-loop stabilization. Other H3-H4 and H2A-H2B histone chaperones could be involved in Dloop stabilization. In S. pombe, other histone chaperones could be tested:

-Histone chaperone Rtt106-like (H3-H4) is encoded by mug183 gene (SPAC6G9.03c) and the strain deleted for this gene is viable. Rtt106 acts in replication-coupled chromatin assembly, therefore it could be a good candidate.

-Histone chaperone Asf1 (H3-H4) is encoded by cia1 gene (SPCC663.05c), but the strain deleted for this gene is inviable. Termosensitive mutants were created in S.

pombe [START_REF] Tanae | Histone chaperone Asf1 plays an essential role in maintaining genomic stability in fission yeast[END_REF]) and also a mutant that cannot interact with HIRA and histone H3 and H4 [START_REF] Yamane | Asf1/HIRA facilitate global histone deacetylation and associate with HP1 to promote nucleosome occupancy at heterochromatic loci[END_REF]. However, Asf1 is known to donate H3-H4 tetramers to Rtt106, CAF-1 and HIRA and not to load them directly on the DNA.

-HIRA histone chaperone complex (H3-H4), which is composed of three subunits encoded by: hip1 (SPBC31F10.13c) which deletion phenotype is viable; slm9 (SPBC15D4.03) which deletion phenotype is viable and hip3 (SPBC31F10.14c) which deletion phenotype is viable. However, HIRA is known to act during DNA replication-independent chromatin assembly pathways, such as during transcription and gene silencing.

160

-CENP-A chaperone Scm3 encoded by scm3 gene (SPAPB1A10.02), but the deletion phenotype is inviable. Scm3 is involved in replication-coupled chromatin assembly at centromeric regions.

-Nap1 (SPCC364.06) is a H2A-H2B histone chaperone, which deletion phenotype is viable. However this histone chaperone is involved in replication-independent chromatin assembly (transcription-coupled nucleosome assembly).

-Nap2 (SPBC2D10.11C) is a H2A-H2B histone chaperone, which deletion does not affect the viability of the cells. This histone chaperone acts in replication-coupled chromatin assembly.

-Histone chaperone Chz1 (H2A-H2B) is encoded by chz1 gene (SPAC4G9.06c) and the strain deleted for this gene is viable. Chz1 histone chaperone has overlapping functions with Nap1.

CAF-1 promotes D-loop stabilization via its replication-coupled chromatin assembly function.

My data also established that a tight interaction between Pcf1 and PCNA is crucial to promote replication-induced recombination and D-loop stabilization. This supports the hypothesis that CAF-1 promotes D-loop stabilization via its replication-coupled chromatin assembly function (Figure n. 60). Indeed, it has been shown that the interaction between PCNA and the largest subunit of CAF-1 is necessary to promote histone deposition during DNA replication [START_REF] Krawitz | Chromatin assembly factor I mutants defective for PCNA binding require Asf1/Hir proteins for silencing[END_REF].

After Rad51-filament invasion, a delay of 40 minutes is observed before extension of the Dloop, in budding yeast [START_REF] Hicks | Real-time analysis of double-strand DNA break repair by homologous recombination[END_REF]Introduction 2.3.2). During this timeframe, PCNA is recruited together with the DNA polymerases delta and eta to promote initiation of DNA synthesis from D-loop intermediates. At this step, PCNA could be also responsible for CAF-1 recruitment, necessary for the chromatinization of the extended Dloop. However evidences reported in the literature suggest that, PCNA might have an early role in homologous recombination during the formation of the D-loop [START_REF] Hashimoto | RAD51-and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks[END_REF]. Therefore the recruitment of CAF-1 could be achieved in two steps, as suggested from Baldeyron and colleagues, in human cells (2011). Indeed, they propose that the larger subunit of CAF-1 could have a role independent on its function of histone chaperone during early steps of homologous recombination and, after the DNA damage repair is completed, 1::his3 + H3.3/H4.3::arg3, H3.2K56R -Xemalce, Miller et al. 2007). The strain will be crossed with a second one, harbouring the RuraR construct at ura4 locus to investigate if the absence of acetylation on K56 could mimics defects in D-loop stabilization, such as those due to the loss of CAF-1 complex. My hypothesis is that, D-loop dissociation by Rqh1 might be sensitive to histone acetylated on H3K56Ac and not to deposition of histones per se.

This model implies that CAF-1, as reported in the literature, could be a hub to couple genetic and epigenetic inheritance during DNA repair, as it does during DNA replication [START_REF] Polo | Chromatin assembly: a basic recipe with various flavours[END_REF]Groth, Rocha et al. 2007). Indeed, the histone deposition onto D-loop intermediates undergoing DNA synthesis could facilitate the maintenance of the memory of the epigenetic state. Parental histones harbouring specific marks could be evicted ahead of the replication fork and, together with newly synthesized histones, might be deposited onto the Dloop. Therefore, CAF-1 could be responsible for the maintenance of specific chromatin states during DNA repair and, especially, homologous recombination. During DNA replication, PCNA interacts with several enzymes involved in epigenetic inheritance, such as the DNA methyltransferase DNMT1 in human cells [START_REF] Chuang | Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1[END_REF][START_REF] Warbrick | PCNA binding through a conserved motif[END_REF]. A further speculation is that the interaction between PCNA and such enzymes could facilitate the role of CAF-1 in maintaining the epigenetic memory, in mammals. Indeed, parental histones might be used as fingerprint, to promote specific modifications on newly synthesized histones before being taken in charge by CAF-1.

The role of CAF-1 in channelling the recombination pathway choice

As detailed in the introduction, homologous recombination is a very dynamic process, in Paradoxically, even if CAF-1 could be considered as a sort of genome-keeper due to its function in DNA metabolic processes, it promotes recombination pathways associated with genome rearrangements. Thus, my data reveal a novel role for CAF-1, which could be considered as the first component of a regulator factor class, orchestrating ectopic but not allelic recombination.

Introduction

Maintenance of genome stability requires the faithful and accurate replication of the genetic material. Genome instability is a hallmark for most types of cancer and it is strongly associated with predisposition to cancer in many human syndromes (for a review, see [START_REF] Aguilera | Genome instability: a mechanistic view of its causes and consequences[END_REF][START_REF] Branzei | Maintaining genome stability at the replication fork[END_REF]). Genome instability is manifest at two levels: at the nucleotide level, resulting in base-substitutions, frame-shifts or in micro-insertions/deletions (referred to herein as mutations); and at the chromosomal level, resulting in duplications, deletions, inversions and translocations (referred to herein as gross chromosomal rearrangements or GCRs).

Genome instability during cancer development and in other human genomic disorders may be consequences of failures in chromosome replication (for a review, see [START_REF] Halazonetis | An oncogene-induced DNA damage model for cancer development[END_REF][START_REF] Zhang | Complex human chromosomal and genomic rearrangements[END_REF]). Origin spacing has recently been shown to cause chromosomal fragility at some human fragile sites [START_REF] Letessier | Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site[END_REF][START_REF] Tallec | Molecular profiling of common fragile sites in human fibroblasts[END_REF]. Impediments to replication fork movements per se may also cause genome instability [START_REF] Petermann | Pathways of mammalian replication fork restart[END_REF][START_REF] Hastings | A microhomology-mediated break-induced replication model for the origin of human copy number variation[END_REF][START_REF] Weinert | The replication fork's five degrees of freedom, their failure and genome rearrangements[END_REF]. Indeed, both slowing down and blockages to fork progression can lead to chromosomal fragilities or GCRs in human cells and yeast models [START_REF] Lemoine | Chromosomal translocations in yeast induced by low levels of DNA polymerase a model for chromosome fragile sites[END_REF][START_REF] Mizuno | Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism[END_REF][START_REF] Bester | Nucleotide deficiency promotes genomic instability in early stages of cancer development[END_REF][START_REF] Ozeri-Galai | Failure of origin activation in response to fork stalling leads to chromosomal instability at fragile sites[END_REF][START_REF] Paek | Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast[END_REF]. However, how a blocked replication fork leads to genetic instability remains poorly understood.

In eukaryotes, DNA replication is initiated at numerous origins along linear chromosomes, and impediments to fork progression appear unavoidable during each S-phase (for a review, see [START_REF] Weinert | The replication fork's five degrees of freedom, their failure and genome rearrangements[END_REF][START_REF] Blow | How dormant origins promote complete genome replication[END_REF]). Impediments to fork progression can be caused by DNA lesions, by non-histone proteins tightly bound to DNA, by sequencecaused secondary structures such as cruciform structures and possibly G-quadruplexes, by nucleotide pool imbalance and by conflicts with transcription machinery (for a review, see [START_REF] Mirkin | Replication fork stalling at natural impediments[END_REF][START_REF] Lambert | Arrested replication fork processing: interplay between checkpoints and recombination[END_REF]). In case of failures in fork progression, DNA replication can be completed either by the recovery of the arrested fork by forkrestart mechanisms, or as a result of the progression of a converging fork which can be ensured by activation of dormant origins [START_REF] Petermann | Pathways of mammalian replication fork restart[END_REF][START_REF] Blow | How dormant origins promote complete genome replication[END_REF][START_REF] Kawabata | Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression[END_REF]. Fork restart is presumably essential in unidirectional replication regions, such as the rDNA locus, in regions of low densities of origins, such as some human fragile sites, and when two converging forks are both impeded [START_REF] Letessier | Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site[END_REF][START_REF] Murray | Smc5/6: a link between DNA repair and unidirectional replication?[END_REF][START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF].

To ensure faithful and complete DNA replication, cells coordinate DNA synthesis restart with specific pathways, including DNA replication checkpoint and homologous recombination mechanisms [START_REF] Lambert | Arrested replication fork processing: interplay between checkpoints and recombination[END_REF]. The integrity of replication forks is guaranteed by the DNA replication checkpoint that maintains the replisome in a replication-competent state to keep DNA polymerases at the site of nucleotide incorporation [START_REF] Katou | S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex[END_REF]. It remains unclear how the DNA replication checkpoint modulates replisome activities to maintain its function [START_REF] Katou | S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex[END_REF][START_REF] De Piccoli | Replisome stability at defective DNA replication forks is independent of s phase checkpoint kinases[END_REF]. The DNA replication checkpoint also regulates nuclease activities (e.g. Exo1 or Mus81) which contribute to preserving the integrity of stalled forks [START_REF] Froget | Cleavage of stalled forks by fission yeast Mus81/Eme1 in absence of DNA replication checkpoint[END_REF][START_REF] Cotta-Ramusino | Exo1 processes stalled replication forks and counteracts fork reversal in checkpoint-defective cells[END_REF]. If replisome function is lost or the replisome dissociates at broken replication forks, the resumption of DNA synthesis appears to require the replisome to be rebuilt. In E.coli, restart of a collapsed fork involves homologous recombination and the PriA helicase that allows replisome components to be loaded de novo on joint-molecule structures [START_REF] Heller | Replisome assembly and the direct restart of stalled replication forks[END_REF][START_REF] Michel | Recombination proteins and rescue of arrested replication forks[END_REF]. In eukaryotes, the restart of collapsed or broken replication forks is dependent upon homologous recombination, but the mechanism of origin-independent loading of the replisome remains to be described [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF][START_REF] Hashimoto | RAD51-and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks[END_REF][START_REF] Roseaulin | Mus81 is essential for sister chromatid recombination at broken replication forks[END_REF][START_REF] Moriel-Carretero | A postincision-deficient TFIIH causes replication fork breakage and uncovers alternative Rad51-or Pol32-mediated restart mechanisms[END_REF][START_REF] Lydeard | Break-induced replication requires all essential DNA replication factors except those specific for pre-RC assembly[END_REF]. It has been proposed that the repair of a double-strand break (DSB) by recombination (breakinduced replication, BIR) in budding yeast similarly involves the assembly of a replication fork (for a review, see [START_REF] Lydeard | Break-induced replication requires all essential DNA replication factors except those specific for pre-RC assembly[END_REF][START_REF] Llorente | Break-induced replication: what is it and what is it for?[END_REF][START_REF] Mceachern | Break-induced replication and recombinational telomere elongation in yeast[END_REF]). When BIR occurs outside S-phase, recombination-dependent replication fork assembly can synthesise hundreds of kilobases (Kb). However, this DNA synthesis is highly inaccurate due to frequent template switching of nascent-strands and frame-shift mutations [START_REF] Smith | Template switching during breakinduced replication[END_REF][START_REF] Deem | Breakinduced replication is highly inaccurate[END_REF].

We previously reported a system that displays replication fork arrest at a specific locus in the fission yeast S. pombe. The system is a polar replication fork barrier (RFB) regulated by the Rtf1 protein binding to its RTS1 binding site [START_REF] Lambert | Gross chromosomal rearrangements and elevated recombination at an inducible sitespecific replication fork barrier[END_REF]. The RTS1-RFB causes fork arrest because of a non-histone protein complex binding to the DNA. As proposed for other polar RFBs, the RTS1-RFB is thought to block fork progression by directly (contact between proteins and the replisome) or indirectly (topological constraint) affecting the replicative helicase activity and thereby preventing DNA unwinding [START_REF] Eydmann | Rtf1mediated eukaryotic site-specific replication termination[END_REF][START_REF] Kaplan | Mechanisms of polar arrest of a replication fork[END_REF]. Recovery of the arrested fork occurs by a DSB-independent mechanism and involves the recruitment of recombination proteins at the RTS1-RFB site. We proposed that recombination proteins associate with unwound nascent strands that then anneal with the initial template to allow DNA synthesis to restart [START_REF] Mizuno | Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism[END_REF][START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]. The causative protein barrier then has to be removed either by DNA helicase or by the recombination machinery itself to allow fork-progression to resume [START_REF] Mcinerney | Replisome fate upon encountering a leading strand block and clearance from DNA by recombination proteins[END_REF][START_REF] Sabouri | DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase[END_REF][START_REF] Steinacher | The DNA helicase Pfh1 promotes fork merging at replication termination sites to ensure genome stability[END_REF]. Occasionally, the unwound nascent strand can mistakenly anneal with a homologous template in the vicinity of the collapsed fork, resulting in the restart of DNA synthesis on non-contiguous template. This incorrect template switch of nascent strands results in inversions and iso-acentric and dicentric chromosomes in ,2-3% of cells/generation [START_REF] Mizuno | Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism[END_REF][START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]. Error-free template switching between sister-chromatids provides an efficient mechanism for filling-in single-stranded gaps left behind damage-induced stalled forks [START_REF] Branzei | SUMOylation regulates Rad18-mediated template switch[END_REF]. Inverted chromosome fusions in yeast and rare-genome rearrangements in human genomic disorders, may both be consequences of template switching between ectopic repeats associated with impeded replication forks [START_REF] Hastings | A microhomology-mediated break-induced replication model for the origin of human copy number variation[END_REF][START_REF] Paek | Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast[END_REF].

Here, we used the RTS1-RFB to investigate the consequences of fork collapse on genome instability. We report that recovery from a collapsed fork is associated with a high frequency of instability, with a single fork arrest increasing the rates of mutation, deletion and translocation by 10, 40 and 5 fold, respectively. We show that genetic instability associated with fork arrest is dependent on homologous recombination. Fork-arrest-induced GCRs (deletion and translocation) result from inappropriate ectopic recombination at the site of the collapsed fork. We also demonstrate that restoration of fork progression by homologous recombination results in error-prone DNA synthesis due to frequent replication slippage between short tandem repeats. We investigated the molecular mechanisms of this replication slippage and found that post-replication repair, including ubiquitination of PCNA or translesions-synthesis (TLS) DNA polymerases, is not involved in forkarrest-induced replication slippage. Micro-deletions/insertions flanked by micro-homology associated with copy number variations (CNVs) in cancer cells or in response to replication stress may therefore be scars left following the restoration of forks progression by homologous recombination.

Results

The conditional replication fork barrier RTS1

We generated fork arrest constructs by manipulating the polar RTS1-RFB (Figure 1A). We introduced the RTS1 sequence on the centromere-proximal (cen-proximal) side of the ura4 locus, 5 kb away from the strong replication origin (ori) 3006/7 on chromosome III. This created the t-ura4,ori locus, in which ''t'' and ''ori'' refer to the telomere and the origin 3006/7, respectively; and '','' and'' .''refer to the RTS1-barrier and its polarity that is whether it blocks replication forks travelling from the ori 3006/7 towards the telomere or forks travelling from the telomere towards the ori 3006/7, respectively. We previously confirmed that forks moving from ori 3006/7 towards the telomere (tel) are efficiently blocked by the RTS1-RFB at the t-ura4,ori locus [START_REF] Lambert | Gross chromosomal rearrangements and elevated recombination at an inducible sitespecific replication fork barrier[END_REF]. In this model system, fork arrest is activated by inducing the expression of rtf1 + gene that is under control of the thiamine repressible promoter nmt41. Thus, the RTS1-RFB is inactivated by adding thiamine to the media and it is activated in thiamine-free media. Efficient induction of Rtf1 expression requires incubation for 12-16 hours in thiamine-free media. Replication intermediates were analysed by native 2-dimensional gel electrophoresis (2DGE). In conditions of Rtf1 expression, more than 95% of replication forks were blocked by the RTS1-RFB at the t-ura4,ori locus (see black arrow on Figure 1B, t-ura4,ori ON). Arrested forks were not detected without Rtf1 induction (Figure 1B, t-ura4,ori OFF) [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF].

Conditional fork-arrest constructs to investigate forkarrest-induced genome instability

The RTS1 sequence was inserted on the tel-proximal side of ura4 creating the t,ura4-ori locus. 2DGE analysis of this construct revealed a strong fork arrest signal on the descending large Y arc (Figure 1A and 1B, t,ura4-ori ON). The ura4 + gene, used in this system as a reporter to score genetic instability, is located behind the arrested fork when the RTS1-RFB is active at the t,ura4-ori locus and ahead of the arrested fork at the t-ura4,ori locus. This explains the distinct position of the arrested fork signal on the Y arc. Inversion of the RTS1 sequence at the tel-proximal side of ura4 created the t.ura4-ori locus and no fork arrest signal was detected for this construct by 2DGE when Rtf1 was expressed (Figure 1A and 1B, t.ura4-ori ON). Thus, RTS1 behaves as a polar RFB at the ura4 locus, and replication across this locus is strongly unidirectional due to the relative positions of the origins [START_REF] Miyabe | The major roles of DNA polymerases epsilon and delta at the eukaryotic replication fork are evolutionarily conserved[END_REF].

Introducing a second RTS1 sequence, such that the two RTS1 sequences are inverted repeats (IRs), created t.ura4,ori and t,ura4.ori loci (Figure 1A and 1B, t.ura4,ori and t,ura4.ori ON). Given the orientation of the polar RTS1-RFB in the t,ura4.ori strain, converging forks cannot be blocked. Whereas block of converging forks can virtually occur in the t.ura4,ori strain, 2DGE in this construct revealed that forks arrested on the cen-proximal side of ura4 were efficiently recovered by recombination before forks are arrested on the tel-proximal side. Indeed, joint-molecules (JMs) resulting from recombination between RTS1 repeats were detected by 2DGE (see red arrows on Figure 1B, t.ura4,ori and t,ura4.ori ON). Resolution of these JMs gives rise to chromosomal rearrangements [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]. In the absence of homologous recombination (i.e. in a rad22-d mutant), JMs were not detected and termination signals accumulated (see green arrow on Figure 1B, t.ura4,ori rad22-d strain). Similarly, termination signals accumulated in the rad22-d t-ura4,ori strain (see green arrow on Figure 1B, t-ura4,ori rad22-d), showing that, when arrested forks are not restarted by homologous recombination, the RTS1-RFB behaves as a hot spot for replication termination [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF].

A single fork arrest induces genomic deletions

We investigated fork-arrest-induced genome instability by selecting for cell resistance to 5-FOA R , the result of loss of ura4 + function. Inducing fork-arrest at t-ura4,ori increased ura4 loss 3 fold (Table 1). Rtf1 expression in the t-ura4-ori and t.ura4-ori strains did not cause site-specific fork-arrest at ura4 as assessed by 2DGE and did not increase the rate of ura4 loss. Thus, ura4 loss results from the RTS1-RFB activity and not simply from the presence of RTS1 and/or Rtf1 expression (Table 1). To investigate the nature of this genetic instability, primers were designed to amplify the ura4 coding sequence and, as a control, the essential rng3 gene, mapping 30 kb tel-proximal to ura4, that should not be rearranged (Figure 2A and2B) [START_REF] Lambert | Gross chromosomal rearrangements and elevated recombination at an inducible sitespecific replication fork barrier[END_REF]. The absence of ura4 amplification was classified as a deletion event; sequencing of amplified ura4 sequence was used to identify point mutation events (Figure 2B).

A single arrested fork at the t-ura4,ori locus was sufficient to increase the rate of genomic deletion up to 40 times over spontaneous events (i.e. in the t-ura4-ori strain, p = 0.006) (Figure 2C and Figure S1A). Fork-arrest-induced deletion was recombinationdependent. Spontaneously (i.e. when the RTS1-barrier was inactivated), the rate of genomic deletion in rad22-d or rhp51-d strains was higher than that in the wild-type strain (Figure S1B). Nonetheless, no further increase in the rate of genomic deletion was observed in the surviving rad22-d or rhp51-d cells upon activation of the RTS1-barrier (Figure S1B, t-ura4,ori). Frequent spontaneous genomic deletion in the rad22-d or rhp51-d strains is consistent with previous reports showing that mutations in recombination genes are associated with an increase level of GCRs [START_REF] Paek | Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast[END_REF][START_REF] Myung | Multiple pathways cooperate in the suppression of genome instability in Saccharomyces cerevisiae[END_REF][START_REF] Chen | Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants[END_REF]. Deleting the natural RTS1 sequence from chromosome II abolished deletion events at collapsed forks, indicating that fork-arrest-induced deletion was also mediated by inter-chromosomal recombination (Figure 2C and t-ura4,ori RTS1-d on Figure S1A). Thus, these data are consistent with the view that homologous recombination makes a major contribution to suppressing genome instability, but can occasionally drive non allelic recombination events leading to GCRs [START_REF] Lambert | Gross chromosomal rearrangements and elevated recombination at an inducible sitespecific replication fork barrier[END_REF][START_REF] Putnam | Specific pathways prevent duplication-mediated genome rearrangements[END_REF].

We detected no fork-arrest-induced deletion in the t,ura4-ori strain, in contrast to the t-ura4,ori strain (Figure S1A and Figure 2C). The ura4 marker is located behind and ahead of collapsed forks in the t,ura4-ori and t-ura4,ori strains, respectively (Figure 1A). Therefore, replicated regions, located behind collapsed forks, do not display instability, and fork-arrest-induced deletion occurs within unreplicated regions immediately in front of arrested forks. Overall, our data establish that genomic deletion at collapsed forks results from inappropriate recombination between ectopic sequences during the process of fork recovery by recombination proteins.

Inverted repeats stimulate fork-arrest-induced deletion by promoting inter-and intra-chromosomal recombination

Inverted repeats (IRs) are structural elements often associated with genome rearrangements [START_REF] Mizuno | Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism[END_REF][START_REF] Paek | Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast[END_REF][START_REF] Voineagu | Replication stalling at unstable inverted repeats: interplay between DNA hairpins and fork stabilizing proteins[END_REF][START_REF] Carvalho | Inverted genomic segments and complex triplication rearrangements are mediated by inverted repeats in the human genome[END_REF]. We investigated the effects of IRs in the vicinity of the RTS1-RFB on fork-arrestinduced genomic deletion. We first compared the t.ura4,ori strain (IRs flanking ura4) to the t-ura4,ori strain (no IRs near the RTS1-RFB). The rate of fork-arrest-induced genomic deletion was 200 times higher in the t.ura4,ori than that in the t-ura4,ori strain (p = 0.009, Figure 2C and Figure S1A). Thus, intrachromosomal ectopic recombination permitted by the RTS1 sequence on the tel-proximal side of ura4 accounted for 99.5% of the genomic deletions observed in the t.ura4,ori strain (Figure 2C, compare with t-ura4.ori). Preventing inter-chromosomal recombination by deleting RTS1 from the chromosome II (t.ura4,ori RTS1-d) abolished 90% of deletion events (Figure 2C and Figure S1A). Thus, genomic deletions induced by fork-arrest near IRs are due to inter-and intra-chromosomal recombination events. In support of this, stimulation of fork-arrest-induced deletion by IRs is mediated by homologous recombination. Indeed, the rate of genomic deletion was not increased upon induction of the RTS1-RFB in the surviving population of t.ura4,ori rad22-d and rhp51-d strains (Figure S1B). These data indicate that IRs favour genomic deletion at collapsed forks by promoting inappropriate inter-and intra-chromosomal recombination during fork recovery by recombination proteins.

We verified that our data were not influenced by the orientation of IRs or by rare blocking of converging forks in the t.ura4,ori strain. We analysed the t,ura4.ori construct in which RTS1 repeats are in the opposite orientations relative to the t.ura4,ori construct, such that forks converging towards ura4 cannot be blocked (Figure 1). The rate of fork-arrest-induced genomic deletion was 1,000 times higher in the t,ura4.ori than that in the t,ura4-ori strain, that does not contain IRs near the RTS1-RFB (p = 0.008, Figure 2C and Figure S1A). Thus, intra-chromosomal recombination, permitted by the RTS1-RFB sequence on the cenproximal side of ura4, accounted for nearly 100% of the genomic deletions observed in the t,ura4.ori strain (Figure 2C, compare with t,ura4-ori). Preventing inter-chromosomal recombination by deleting RTS1 from the chromosome II (t,ura4.ori RTS1-d) abolished 90% of deletion events (Figure 2C and Figure S1A).

Importantly, the deletion rates for the t,ura4.ori and t.ura4,ori strains were not significantly different (Figure 2C), showing that IRs cause genomic deletion at collapsed forks irrespective of their orientations and independently of blockage of converging forks.

A single collapsed fork induces translocations that are stimulated by IRs

Fork-arrest at t.ura4,ori results in translocations between ectopic RTS1 repeats on chromosomes II and III [START_REF] Lambert | Gross chromosomal rearrangements and elevated recombination at an inducible sitespecific replication fork barrier[END_REF]. We investigated the influence of IRs on fork-arrest-induced translocation. We designed primers to amplify the predicted translocation junction between chromosomes II and III (TLII and TLIII on Figure 2A and2B). A single arrested fork at the t-ura4,ori locus was sufficient to increase the translocation rate to 5 times higher than the spontaneous rate (p = 0.002, Figure 2D and Figure S1C). The translocation rate for the t.ura4,ori construct (containing IRs) was 1,500 fold higher than that for the t-ura4,ori strain that does not contain IRs near the RTS1-RFB (p = 0.009, Figure 2D and Figure S1C). Thus, intra-chromosomal recombination accounted for nearly 99% of translocation events observed in the t.ura4,ori construct (Figure 2D and Figure S1C, compare with t-ura4,ori). No translocation events were detected when interchromosomal recombination was prevented by deleting RTS1 from the chromosome II (t.ura4,ori RTS1-d on Figure 2B). Therefore, as reported for genomic deletions, fork-arrest-induced translocation associated with IRs is due to inter-and intrachromosomal ectopic recombination. No translocations were detected in the t,ura4.ori strain (data not shown), so we cannot formally exclude the possibility that fork-arrest-induced translocations in the t.ura4,ori strain was caused by blocking of converging forks. However, as no translocation event occurred in the absence of Rad22 Rad52 or Rhp51 Rad51 , it is most likely that translocations occur during fork recovery by recombination (Figure 2B and [35]). Overall, our data indicate that recovery of a single collapsed fork causes translocations and IRs near the site of fork-arrest stimulate translocations by promoting inappropriate inter-and intra-chromosomal recombination. Fork-arrest-induced GCRs are caused by inter-and intrachromosomal recombination. We noticed a slightly greater contribution of intra-than inter-chromosomal recombination (Figure 2C). This is consistent with ectopic recombination preferentially occurring at the most proximal homologous sequence, as previously reported [START_REF] Lichten | Position effects in ectopic and allelic mitotic recombination in Saccharomyces cerevisiae[END_REF]. Nonetheless, the rate of fork-arrest-induced deletion in the t.ura4,ori strain (8.4 10 27 ) was not simply the sum of the rates of intra-chromosomal recombination events (9.9 10 28 in the t.ura4,ori RTS1-d strain) and inter-chromosomal recombination events (4 10 29 in the t-ura4.ori strain). Similar reasoning can be applied for the t,ura4.ori strain. Thus, independent intra-and inter-recombination events cannot themselves explain high rate of GCRs induced by fork arrest near IRs. Therefore, we infer that there is interplay between inter-and intra-chromosomal recombination such that fork-arrest-induced GCRs may involve recombination between three homologous sequences (tri-parental recombination).

The RecQ helicase Rqh1 prevents GCRs at collapsed forks

To confirm that fork-arrest-induced GCRs are the result of inappropriate ectopic recombination during fork recovery, we analysed the involvement of the RecQ helicase Rqh1. We previously reported that Rqh1 limits inappropriate template switching of stalled nascent strands without affecting the efficiency of fork restart [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]. In the t-ura4,ori construct (in which only interchromosomal recombination is possible), fork-arrest-induced deletion and translocation rates were 31 and 109 times higher in the rqh1-d strain than that in the wild-type control, respectively (p = 0.0003, Figure 2D-2E and Figure S1C). For the t.ura4,ori construct (containing IRs near fork-arrest), fork-arrest-induced deletion and translocation rates were 5 times higher in the rqh1-d than that in the wild-type control (p = 0.0007, Figure 2D-2E and Figure S1C). Thus, Rqh1 limits GCRs at collapsed forks by preventing inappropriate ectopic recombination during the process of fork recovery by recombination proteins.

A single fork arrest induces mutations

We analysed the effects of collapsed forks on the mutation rate. We sequenced the ura4 coding sequence from 5-FOA R isolated cells and identified base-substitutions, frame-shifts and small insertions and duplications between short tandem repeats (Table 2). A single collapsed fork in the t-ura4,ori strain increased the overall mutation rate up to 10 times over spontaneous events (Figure 3A, p = 0.003). Similar increases in the overall mutation rate were found for the strains with IRs near the arrested fork and those with RTS1 deleted from chromosome II (Figure 3A and Figure S2A). Thus, fork-arrest-induced mutation is not mediated by inappropriate ectopic recombination. Induction of the RTS1-RFB in the t,ura4-ori strain did not increase the mutation rate of the ura4 gene. Thus, as for GCRs, replicated regions behind arrested forks are not prone to mutation. This observation rules out the hypothesis that fork-arrest-induced mutation is a consequence of the accumulation of damaged single-stranded DNA behind collapsed forks (see discussion). Our data suggest that recovery from collapsed forks results in error-prone DNAsynthesis.

Collapsed forks result specifically in replication slippage

We then analysed the spectra of mutations found in the ura4 ORF by sequencing the PCR products. The rates of basesubstitutions and frame-shifts were not significantly increased by the RTS1-RFB activity over spontaneous events (i.e. compare to t-ura4-ori strain, Figure 3C and Table 2). In contrast, the rate of deletions and duplications (Del/Dup) flanked by short homology was increased by 7 times over spontaneous events in the t-ura4,ori strain, but not in the t,ura4-ori strain (Figure 3C and Table 1). These data further confirm that fork-arrest does not promote mutation events behind collapsed forks.

We used reversed mutation assays to test if fork-arrest at the RTS1-RFB specifically induced Del/Dup mutations. We made use of strains harbouring a single mutation within the ura4 ORF: either a single base-substitution or a 21 frame-shift in homonucleotide (Figure S2B). We also studied strains harbouring either a duplication of 20 or 22 nt flanked by 5 or 4 bp of microhomology, respectively (defined as ura4-dup20 and ura4-dup22, Figure S2B). These non-functional ura4 2 alleles were inserted in front of the RTS1-RFB in the t-ura4,ori configuration and we then tested whether fork arrest could restore a functional ura4 + gene. Activation of the RTS1-RFB at ura4 increased the frequency of Ura + revertants up to 15 and 7 times in strains harbouring ura4-dup22 and ura4-dup20, respectively (Figure 3D and Figure S2B). Thirty Ura + colonies were studied by PCR and all gave a product of the same size as the wild-type ura4 + gene: they had therefore lost the duplication (Figure 3E and data not shown). Sequencing the full ura4 ORF confirmed that Ura + revertants contained an intact ura4 + sequence, showing that the reversion of these alleles was due solely to the precise deletion of 20 or 22 nt (Figure 3F and data not shown). In contrast, activation of the RTS1-RFB did not increase the frequency of Ura + revertants of strains harbouring ura4 alleles with a single base-substitution or a 21 frame-shift (Figure 3D and Figure S2B). Thus, collapsed forks tend to induce deletion events between short tandem repeats rather than base-substitution or frame-shift mutations.

Among Del/Dup events, deletions represented the two-third of events in the t-ura4,ori strain (Table 2). The median size of Del/ Dup events was 24 and 22 nt respectively, and Del/Dup occurred between short direct repeats 1 to 10 nt long (Figure S3). Thus, the ura4-dup20 and ura4-dup22 alleles used in the reverse mutation assay were representative of the Del/Dup events observed. Del/ Dup flanked by micro-homology result from intra-molecular template switching mechanisms in which nascent strands dissociate from the template and misalign with the template when restarting the elongation step. This leads to loop formation, either in the nascent strand or in the template, resulting in duplication or deletion events, respectively [START_REF] Tran | Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes[END_REF]. Consequently, we will hereafter refer to Del/Dup as replication slippage. Replication slippage was observed all along the ura4 ORF and up to 1.2 kb ahead of the arrested fork, even if a hot spot of deletion was present 500 bp away from the RTS1-RFB (Figure 3G and Figure S3B). Thus, our data suggest that the DNA synthesis is prone to replication slippage at least for the first 1,200 nt synthetized during the recovery of collapsed forks. Inaccuracy of DNA synthesis on further distances was not directly addressed.

Replication slippage results from error-prone DNA synthesis during fork recovery

To confirm that replication slippage occurs as forks recover, and not behind the fork in the DNA already replicated, we inserted the ura4-dup20 or the ura4-dup22 allele either behind (in the t,ura4-ori configuration) or in front of the RTS1-RFB (in the t-ura4,ori configuration) (Figure 4). This allows the analysis of the same event of replication slippage behind and ahead of collapsed forks. In the t-ura4,ori configuration, induction of the RTS1-RFB resulted in a 8 and 16 fold increases in the replication slippage frequency for the ura4-dup20 and ura4-dup22 alleles, respectively (Figure 4A and4B). Similar increases in the rate of replication slippages were observed (Figure 4C). In contrast, in the t,ura4-ori background, the frequency of replication slippage was induced by only 2-3 fold A. Effect of intra-and inter-chromosomal recombination between RTS1 repeats on fork-arrest-induced mutation rates (base-substitutions, frame-shifts and small insertions or deletions between short tandem repeats). RTS1-RFB activity and ura4 location with respect to the RFB are given for each construct. The % of mutation events, as determined by the PCR assay and sequencing, was used to balance the rate of ura4 loss. Then, the RFB-induced mutation rate was calculated by subtracting the rate obtained in the presence of thiamine (Rtf1 being repressed) from the rate obtained in the absence of thiamine (Rtf1 being expressed). The values reported are means of at least 3 independent median rates. Error bars correspond to SE. (Refer to Figure S2 for corresponding rates of mutation when Rtf1 is expressed or not). B. Rate of mutation for indicated strains; ON and OFF refers to the RTS1-RFB being active or not, respectively. The % of mutation events, as determined by the PCR assay and sequencing, was used to balance the rate of ura4 loss. The values reported are means of at least 3 independent median rates. Error bars correspond to SE. Statistically significant fold differences in mutation rates between the ''OFF'' and ''ON'' conditions are indicated with an *. C. Spectra of mutation events in indicated strains upon RFB induction (refer to Table 2 for exact numbers and to Figure S3 for mapping of deletions/duplications and their features). D. Strains harbouring the ura4 alleles with a single base-substitution or frame-shift or duplication of 20 or 22 nt, together with the RTS1-RFB in the t-ura4,ori configuration were streaked onto the indicated media after cell growth with (RFB ''OFF'') or without (RFB ''ON'') thiamine. The bottom diagram indicates strain positions and the mutation events required to obtain Ura + revertants. E. PCR analysis of Ura + revertants isolated from the t-ura4-dup20,ori strain (duplication of 20 nt in ura4) after RFB induction. With the primers used, a 106 bp fragment is amplified from the ura4 + strain and a 126 bp fragment is amplified from the t-ura4-dup20,ori strain. F. Sequence alignments of ura4-dup22, ura4-dup20, ura4 + alleles and corresponding Ura + revertants. Micro-homologies are indicated in grey and duplicated sequences are underlined in black. The phenotype of each allele is indicated on the figure. G. Map of deletion and duplication events within the ura4 ORF. doi :10.1371/journal.pgen.1002976.g003 by the RTS1-RFB (Figure 4B-4C). These data confirm that DNA located ahead of collapsed forks is more prone to replication slippage than replicated DNA adjacent to arrested forks, further evidence that replication slippage arises during fork recovery.

Replication slippage results from forks restarted by recombination

Replication slippage occurs in DNA in front of (and not behind) the arrested fork, this DNA being replicated only after restart of the fork. Thus, a defect preventing fork recovery would be expected to abolish the error-prone DNA synthesis during restart. We analyzed fork-arrest-induced mutation in recombination mutants in which collapsed forks at the RTS1-RFB cannot recover, resulting in cell death. Induction of the RTS1-RFB did not increase the overall mutation rate in the surviving populations of t.ura4,ori or t-ura4,ori rad22-d and rhp51-d strains (Figure 3B). In addition, only 7% of mutation events in the survivors of the rad22-d t-ura4,ori strain were Del/Dup mutations, compared to 40% in the wild-type strain (Figure 3C and Table 1). We currently cannot assess mutation events associated with defects in fork recovery because this appears to be lethal in the absence of recombination. Nevertheless, our data are consistent with forkarrest-induced replication slippage being dependent on homologous recombination. The rad22-d and rhp51-d strains are themselves spontaneously mutagenic. Consequently, any small increase in the fork-arrest-induced mutation rate might be masked by the high frequency of spontaneous 5-FOA R cells in rad22-d and rhp51d strains. We therefore used a more specific mutation assay, based on the ura4-dup20 allele, to determine the rate of replication slippage induced by the RTS1-RFB over spontaneous events.

Strains carrying mutations in recombination genes grow slowly, so replication slippage was scored as a function of the number of generations following thiamine removal (i.e. generations subject to fork arrest at ura4) (Figure 4D and4E). In the wild-type strain, fork arrest at the RTS1-RFB resulted in a 10 fold-increase in the frequency of replication slippage, as expected. In recombination mutants (rad50-d, rhp51-d and rad22-d), fork-arrest at the RTS1-RFB increased the frequency of replication slippage by only 2 times over spontaneous events: therefore, replication slippage occurs less frequently in survivors from recombination mutants than those from the wild-type strain (Figure 4D-4F). Based on 2DGE analysis, fork-restart is severely impaired in the absence of a the following nomenclature is used: ''t'' and ''ori'' refer to the telomere and the replication origin 3006/7, respectively; '','' and ''.'' indicate the RTS1-RFB and its polarity (, blocks replication forks moving from the ori3006/7 towards the telomere, and . blocks replication forks moving from the telomere towards the origin 3006/ 7). b number of events. c event/cell/division 610 28 6 standard error. The % of each mutation events was used to balance the rate of mutation. d value not significantly different from those for the t-ura4-ori strain (spontaneous events). doi:10.1371/journal.pgen.1002976.t002

Rad22 Rad52 (Figure 1B and [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]), such that even the two-fold induction in replication slippage by fork arrest in the rad22-d strain was surprising. The rad22-d strain accumulates suppressors involving the Fbh1 helicase that limits Rhp51 Rad51 -dependent recombination at blocked replication forks [START_REF] Lorenz | Fbh1 limits Rad51-dependent recombination at blocked replication forks[END_REF][START_REF] Osman | The F-Box DNA helicase Fbh1 prevents Rhp51-dependent recombination without mediator proteins[END_REF]. Therefore, we analyzed replication slippage in the rad22-d rhp51-d double mutant in which no homologous recombination event occurs. In this background, there was no detectable fork-arrest-induced replication slippage (Figure 4D-4F). Thus, complete defect in fork restart results in a complete abolition of fork-arrest-induced replication slippage in the surviving population. Overall, our data establish that replication slippage results from inaccurate DNA synthesis during the restart of collapsed forks by recombination.

Replication stress leading to fork collapse induces replication slippage

We investigated the effects of replication stress, other than the replication block imposed by the RTS1-RFB, on replication slippage. Strains harbouring ura4 2 alleles (base-substitutions, 21 frame-shift, and ura4-dup20) were exposed to replication-blocking A. Serial tenfold-dilutions from indicated strains (t-ura4-dup20-ori associated or not with the RTS1-RFB) were spotted onto the indicated media after cell growth with (Rtf1 -, repressed) or without (Rtf1 +, expressed) thiamine. RTS1-RFB activity is given for each construct and condition. B. Frequency of Ura + revertants in indicated strains after cell growth with (Rtf1 repressed) or without (Rtf1 expressed) thiamine. The RTS1-RFB activity is given for each construct and condition. Values correspond to the mean of at least three independent experiments and error bars correspond to the standard error of the mean (SEM). C. Rate of replication slippage in the indicated strains and conditions. The rate of Ura + revertants was calculated using the method of the median from at least 11 independent cultures. Values in brackets indicate the 95% confidence interval. Statistical significance was detected using the nonparametric Mann-Whitney U test. D. Serial tenfold-dilutions from the strains indicated spotted onto the media indicated after cell growth without thiamine. RTS1-RFB activity ''-'' refers to the strain t-ura4-dup20-ori and ''+'' refers to the strain t-ura4-dup20,ori. E. Kinetics of Ura + revertants frequency for the strains indicated as a function of the number of generations after thiamine removal. RTS1-RFB activity ''-'' refers to the strain t-ura4-dup20-ori and ''+'' refers to the strain t-ura4-dup20,ori. The values reported are the means of two experiments. F. The rate of replication slippage/generation for the strains indicated with (t-ura4-dup20,ori) or without (t-ura4-dup20-ori) the active RTS1-RFB. The rate was calculated from the slope of the curves presented in panel F. The values reported are means of three independent experiments and error bars correspond to SE. doi :10.1371/journal.pgen.1002976.g004 agents or UV-C-induced DNA damages and the frequency of Ura + revertants was scored. Three hours of treatment with either the topoisomerase I inhibitor camptothecin (CPT) or mitomycin C (MMC), an inter-strand cross-linking agent (ICls), increased the frequency of Ura + revertants by 3 to 4 fold in the ura4-dup20 strain (Figure 5A and 5B). At equivalent survival (70-90%), DNAdamages induced by a dose of 100 J/m 2 of UV-C did not increase the frequency of Ura + revertants in the ura4-dup20 strain. Increasing the UV-C dose (150 J/m 2 ) resulted in an increased reversion effect. The other ura4 alleles exhibited an opposite behaviour pattern. As expected, UV-C-induced DNA damages, but not CPT or MMC treatment, increased the frequency of Ura + revertants of the basesubstitution and the 21 frame-shift mutants (Figure 5A). Thus, replication slippage, unlike other point mutations, appears to be a mutation event specifically induced by replication stress.

Hydroxyurea (HU) that prevents the bulk of dNTP synthesis during S-phase by inhibiting the ribonucleotide reductase, results in a slow-down of fork progression which did not induce replication slippage (Figure 5A). In contrast, CPT and MMC treatments that lead to replication stress by causing fork collapse induced replication slippage. Homologous recombination is repressed during HU treatment and recombination proteins are recruited to collapsed but not stalled forks [START_REF] Alabert | Differential regulation of homologous recombination at DNA breaks and replication forks by the Mrc1 branch of the S-phase checkpoint[END_REF][START_REF] Lisby | Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins[END_REF][START_REF] Meister | Temporal separation of replication and recombination requires the intra-S checkpoint[END_REF]. Consistent with this, we found that the rad22-d mutant is highly sensitive to acute exposure to CPT, but not to HU (Figure S4). Thus, acute exposure to HU results in stalled forks that recover without recombination, whereas recombination may be required for restarting forks that have collapsed due to CPT or MMC treatment. We confirmed that CPT-induced replication slippage results from collapsed forks and was thus S-phase specific: the ura4-dup20 strain was synchronized in early S-phase by HU treatment and released into S-phase with or without CPT. HU-synchronization and release into DMSO (used as vehicle for CPT) did not induce replication slippage. In contrast, the release of cells into S-phase in the presence of CPT stimulated replication slippage up to 12 fold (Figure 5C). These data indicate that CPT-induced fork collapse results in error-prone DNA synthesis characterized by replication slippage. These experiments further support the view that replication slippage results from recovery of collapsed forks by recombination and point out that the RTS1-barrier is representative of collapsed forks restarted by homologous recombination.

TLS-DNA polymerases are not involved in fork-arrestinduced replication slippage

To investigate the inaccuracy of the DNA synthesis occurring immediately following the restart of collapsed forks, we analysed the involvement of TLS-DNA polymerases. In fission yeast, TLS pathways require either mono-or poly-ubiquitination of the clamp loader PCNA on lysine 164 [START_REF] Coulon | Rad8Rad5/Mms2-Ubc13 ubiquitin ligase complex controls translesion synthesis in fission yeast[END_REF]. We found that mutating this lysine to arginine residue did not affect replication slippage induced by the RTS1-RFB. None of Rev1, Rev3 or DinB DNA polymerases were required for fork-arrest-induced replication slippage (Figure S5). Therefore, the error-prone DNA synthesis associated with fork recovery by recombination does not rely on TLS DNA polymerases activity.

The mismatch repair pathway does not counteract forkarrest-induced replication slippage

The mismatch repair (MMR) pathway is temporally coupled to DNA replication, and MMR components are associated with replication centres [START_REF] Hombauer | Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates[END_REF]. The heterodimer Msh2/Msh6 recognises mispaired DNA and Msh2/Msh3 recognises small DNA loops up to 31 bases long, arising from replication slippage [START_REF] Tran | The prevention of repeatassociated deletions in Saccharomyces cerevisiae by mismatch repair depends on size and origin of deletions[END_REF]. The failure to repair small DNA loops results in more frequent insertions and deletions [START_REF] Sia | Genetic control of microsatellite stability[END_REF]. Therefore, MMR activity could potentially lead to an underestimation of the extent of fork-arrest-induced replication slippage. However, replication slippage induced by the RTS1-RFB activity was as frequent in msh2-d, msh6-d and msh3-d strains as in wild-type control. Also, spontaneous replication slippage at ura4 (without RTS1-RFB) was unaffected by the absence of MMR proteins (Figure S5). Therefore, there is no evidence that MMR repairs small DNA loops (20 nt) in fission yeast and fork-arrestinduced replication slippage is not counteracted by MMR in our model system.

Discussion

Using conditional fork arrest constructs, we studied the consequences for genome instability of impediments to replication forks progression. A single fork arrest results in large-scale genomic changes and mutations that occur during recombination-mediated fork recovery (Figure 6). Inappropriate ectopic recombination at arrested forks results in GCRs, whereas appropriate restarting of the fork on the initial template results in error-prone DNA synthesis. GCRs and mutations at collapsed forks are genetically separable: Rqh1 limits fork-arrest-induced GCRs but not mutations (Figure 2D and Figure 3B). We demonstrate here that collapsed forks whose progression resumes by recombination lose accuracy during DNA synthesis, resulting in frequent intratemplate switches. Thus, homologous recombination contributes to completion of DNA replication when forks progression is impeded but also fuels genome modifications both at the chromosomal and nucleotide level. Non allelic homologous recombination (NAHR) between low copy number repeats (LCR) contributes to recombinationmediated GCRs in mitosis and meiosis. NAHR is responsible for translocations, deletions, inversions and loss of heterozygosity [START_REF] Liu | Mechanisms for recurrent and complex human genomic rearrangements[END_REF]. Ou et al. predicted 1143 LCR pairs in the human genome liable to mediate recurrent translocations [START_REF] Ou | Observation and prediction of recurrent human translocations mediated by NAHR between nonhomologous chromosomes[END_REF]. In budding yeast, a single DSB is sufficient to mediate recombination-dependent translocation [START_REF] Bosco | Chromosome break-induced DNA replication leads to nonreciprocal translocations and telomere capture[END_REF]. Here, we report that a single collapsed fork increases the rate of genomic deletion 40 fold, and that of translocation 5 fold. Fork-arrest-induced GCRs are mediated by NAHR between heterologous chromosomes. It is not clear whether fork arrest on both homologous repeats contributes to fork-arrest-induced GCRs. We could not address this question in our model system, because the RTS1 sequence close to the mat1 locus on chromosome II has a low RFB activity [START_REF] Dalgaard | A DNA replication-arrest site RTS1 regulates imprinting by determining the direction of replication at mat1 in S. pombe[END_REF]. Also, the recruitment of recombination proteins at the RTS1 sequence near the mat1 locus is not regulated by the level of Rtf1 expression, showing that regulating Rtf1 expression was in itself insufficient to regulate the RTS1-RFB activity at the mat1 locus [START_REF] Lambert | Gross chromosomal rearrangements and elevated recombination at an inducible sitespecific replication fork barrier[END_REF].

Inverted repeats (IRs) are structural elements that contribute to genome instability. Impediments to replication forks progressing through IRs favour their fusion to generate acentric and dicentric inverted chromosomes [START_REF] Mizuno | Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism[END_REF][START_REF] Paek | Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast[END_REF]. IRs in humans can also trigger the formation of inverted genomic segments and complex triplication rearrangements by a replication-based mechanism [START_REF] Carvalho | Inverted genomic segments and complex triplication rearrangements are mediated by inverted repeats in the human genome[END_REF]. Here, we report that IRs near collapsed forks can increase the rate of GCRs by up to 1,500 fold. This high level of GCRs cannot be explained merely by the addition of independent inter-and intra-chromosomal recombination events. Rather, our analyses suggest that IRs may stimulate tri-parental recombination events induced by template switching of nascent strands at collapsed forks, such that three homologous sequences are involved. Similarly, recombination-dependent translocations induced by a single DSB in budding yeast is proposed to be the consequence of tri-parental recombination [START_REF] Ruiz | Chromosomal translocations caused by either pol32-dependent or pol32-independent triparental breakinduced replication[END_REF][START_REF] Schmidt | Control of translocations between highly diverged genes by Sgs1, the Saccharomyces cerevisiae homolog of the Bloom's syndrome protein[END_REF]. One possible mechanism is that IRs-induced GCRs result from successive template switches initiated by nascent strands at collapsed forks, reminiscent of the multiple template switches during breakinduced-replication (BIR) in budding yeast [START_REF] Smith | Template switching during breakinduced replication[END_REF]. Interestingly, Rqh1 prevents fork-arrest-induced GCRs by limiting both inter-and intrachromosomal recombination without affecting fork restart efficiency. Thus, tri-parental recombination might correspond to multiple and successive template switches between homologous repeats.

The high accuracy of DNA replication is compromised by impediments to fork progression, and recombination-mediated fork recovery results in decreased processivity of DNA synthesis (Figure 6). Recombination-induced mutations associated with DSBs or impeding DNA replication have been described previously. The formation of damaged single-stranded DNA during the resection of DSBs favours base-substitutions [START_REF] Yang | Hypermutability of damaged single-strand DNA formed at double-strand breaks and uncapped telomeres in yeast Saccharomyces cerevisiae[END_REF]. We detected the recruitment of the single-strand binding protein RPA up to 1.4 kb behind, but not ahead of the RTS1-arrested fork (data to be published), showing that fork-arrest-induced mutation is not correlated with damaged single-stranded DNA exposed behind collapsed forks. Nevertheless, there were rare replication slippage occurred behind RTS1-arrested forks (in the t,ura4-ori construct), suggesting that resumption of DNA synthesis can in some cases occur at a position behind the site of the collapsed fork. Recombination-dependent DNA synthesis occurring outside S-phase is also highly inaccurate during gene conversion and BIR, resulting in either template switches, basesubstitutions or frame-shifts [START_REF] Smith | Template switching during breakinduced replication[END_REF][START_REF] Deem | Breakinduced replication is highly inaccurate[END_REF][START_REF] Hicks | Increased mutagenesis and unique mutation signature associated with mitotic gene conversion[END_REF]. Elevated dNTP pools, due to activation of the DNA-damage checkpoint in G2 cells, contributes to the generation of mutations when hundreds of kbs are synthesised during BIR [START_REF] Deem | Breakinduced replication is highly inaccurate[END_REF]. Here, we demonstrate that during normal S-phase progression, a single collapsed fork, restored by recombination, results in replication slippage up to 1.2 Kb away from the initial restarting point.

Recombination-induced replication slippage has been reported previously.

In fission yeast, a defect in pol alpha (swi7-H4 mutant) is associated with a recombination-mediated mutator phenotype characterized by an increased frequency of base-substitutions and Del/Dup between short direct repeats [START_REF] Kai | Replication checkpoint kinase Cds1 regulates Mus81 to preserve genome integrity during replication stress[END_REF]. DNA-polymerase kappa (DinB) and zeta (Rev3) are responsible for the increased base-substitution rate, but the DNA-polymerases involved in Del/ Dup mutations were not identified [START_REF] Kai | Checkpoint activation regulates mutagenic translesion synthesis[END_REF]. In budding yeast, a defect in polymerase delta (pol3-t mutant) results in an increased level of replication slippage, mediated by homologous recombination [START_REF] Tran | Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes[END_REF]. In contrast, the increased rate of replication slippage in the absence of the accessory subunit of polymerase delta, Pol32, does not depend on a functional recombination pathway [START_REF] Huang | POL32, a subunit of the Saccharomyces cerevisiae DNA polymerase delta, defines a link between DNA replication and the mutagenic bypass repair pathway[END_REF]. Here, we report that recovery of collapsed forks by recombination is specifically associated with replication slippage. Nonetheless, spontaneous replication slippage events are also increased in strains mutated for recombination genes (Figure 4D-4F). Pol32 is required for BIR and replication-induced template switches leading to segmental duplication [START_REF] Payen | Segmental duplications arise from Pol32-dependent repair of broken forks through two alternative replication-based mechanisms[END_REF][START_REF] Lydeard | Break-induced replication and telomerase-independent telomere maintenance require Pol32[END_REF]. Recombination is responsible for only half of these segmental duplications. Thus, it is possible that fork-restart mechanisms dependent on Pol32 and homologous recombination are prone to replication slippage and that in the absence of these pathways alternative micro-homology mediated mechanisms are revealed.

We suggest that at least two steps of the recombination-based fork recovery mechanism can compromise genome stability (Figure 6). At an initial stage, recruitment of recombination proteins on stalled nascent strands favours both fork recovery and ectopic template switches leading to GCRs. At a later stage, once the replisome has been reconstituted and the fork has resumed its progression, the nascent strands are prone to intra-template switching leading to replication slippage. The reasons for the inaccuracy of DNA synthesis associated with restarted forks during scheduled DNA replication (i.e. in S-phase) remain to be determined. One possibility is that one or more factors are missing in the rebuilt replisome during recovery by recombination. Oncogene-induced replication stress results from unbalanced DNA replication that contributes to genome instability in precancerous cells [START_REF] Bester | Nucleotide deficiency promotes genomic instability in early stages of cancer development[END_REF]. Completion of DNA replication in such stress conditions is likely to rely on recombination-mediated fork recovery that in turn generates genome instability. Insertions/deletions flanked by micro-homology, responsible for copy number variations (CNVs), have been identified both in cancer cells and also in response to replication inhibition [START_REF] Colnaghi | The consequences of structural genomic alterations in humans: Genomic Disorders, genomic instability and cancer[END_REF][START_REF] Arlt | Replication stress and mechanisms of CNV formation[END_REF]; their reported sizes are between 1 Kb and several tens of mega bases, but the analysis of these features has been limited by the resolution of array-based genomics approaches. Sub-microscopic insertions/deletions flanked by micro-homology have been also described at loci in which replication origins are scarce, including the human fragile site FRA3B, the instability of which is a consequence of replication stress [START_REF] Durkin | Replication stress induces tumor-like microdeletions in FHIT/FRA3B[END_REF]. Interestingly, homologous recombination contributes to the stability of fragile sites by facilitating complete replication or by repairing gaps and breaks at these sites. Thus, we propose that micro-homology-mediated CNVs could be viewed as scars left by error-prone replication forks restarted by recombination.

Materials and Methods

Standard genetic and molecular biology

Strains used were constructed by standard genetic techniques and are listed in Table S1. 2DGE was performed as previously described [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]. To create ura4-dup20 and ura4-dup22 alleles associated or not with the RTS1-RFB, genomic DNA was isolated from selected 5-FOA R cells containing duplications identified by sequencing. A PCR fragment containing duplications within the ura4 ORF was amplified using the following primers: TTC-TGTTCCAACA-CCAATGTTT and TCACGTTTATTTTCA-AACATCCA. The PCR products were purified and used to transform strains SL206 (ura4 + ), SL350 (t-ura4,ori) and SL504 (t,ura4-ori). Transformants were selected on 5-FOA-containing plates. Appropriate replacement of ura4+ by ura4-dup20 or ura4-dup22 was verified by PCR and sequencing.

ura4 loss assay

A minimum of 11 independent single colonies from appropriate strains growing with or without thiamine were inoculated in 10 ml of non-selective media (with or without thiamine) and grown to stationary phase. Appropriate dilutions were plated on supplemented YEA to determine plating efficiency and on 5-FOAcontaining plates. Colonies were counted after 5-7 days of incubation at 30uC. The rate of ura4 loss was determined with the method of the median and data are presented on Table 1. Statistical significance was detected using the nonparametric Mann-Whitney U test.

PCR assays and sequencing to determine the rates of genomic deletion, translocation, and mutation At least 200 5-FOA R colonies per strain and condition were subjected to PCR analysis with the following primers: AAAA-CAAACGCAAACAAGGC and GTTTAACTATGCTTCGT-CGG to amplify ura4 ORF, TGAATCCTCCGTTCAGTAGG and AAGGACTGCGTTCTTCTAGC to amplify rng3 and TTTCCTTTCACGGCTAACCC (TLII) and TGTACCCAT-GAGCAAACTGC (TLIII) to amplify the translocation junction. The amplified ura4 fragments were then sequenced on both strands, with the primers used to amplify the ura4 ORF. Only mutations present on both strands were considered to determine mutation spectra. Deletions, mutations and translocations were scored as percentages of all events and these values were used to balance the rates of ura4 loss to determine the respective rates of deletion, mutation and translocation (see Figure S1 for deletion and translocation rates and Figure S2 for mutation rates).

The fork-arrest-induced deletion, translocation and mutation rates (Figure 2C-2E and Figure 3A) were calculated by subtracting the rate obtained in presence of thiamine (Rtf1 being repressed, OFF) from the rate obtained in the absence of thiamine (Rtf1 being expressed, ON). This method allows the spontaneous instability of IRs and the leakiness of the RTS1-RFB activity to be disregarded to determine strictly the rate of events induced by fork-arrest. The nonparametric Mann-Whitney U test was used to test for statistically significant differences.

Reverse mutation assay

Exponentially growing cells were treated with 20 mM of HU, 40 mM of CPT or 1 mM of MMC. At indicated times, samples were taken and appropriate dilutions were plated on supplemented minimal media to determine plating efficiency and on uracil-free plates. Colonies were counted after incubation at 30uC for 5-7 days and the frequency of Ura + colonies was determined.

Replication slippage assay using ura4-dup20 and ura4-dup22 strains

For strains showing a slow growth phenotype (recombination mutants), the frequency of Ura + revertants was determined as a function of the number of generations experiencing fork arrest at ura4. Exponentially growing 5-FOA R cells were washed twice in water and used to inoculate uracil-containing media without thiamine. Every 24 hours, cells were counted to determine the number of generations, and appropriate dilutions were plated on supplemented minimal media and on uracil-free plates. Colonies were counted after incubation at 30uC for 5-7 days and the frequency of Ura + colonies was determined. The slope of the curves presented on Figure 4F corresponds to the rate of replication slippage/generation. For strains showing similar growth to wild-type cells, a single 5-FOA R colony was grown on uracil-containing plates with or without thiamine for 2-3 days, and then grown in uracil-containing media with or without thiamine for 2 days at 30uC. Appropriate dilutions were plated on supplemented minimal media and on uracil-free plates. Colonies were counted after incubation at 30uC for 5-7 days and the frequency of Ura + colonies was determined.

  ABBREVIATIONS

Figure

  Figure n.1: DNA repair pathways according to the nature of DNA lesions (Hoeijmakers 2001).

Figure

  Figure n.3: Mechanism of BIR. A) Extension of the invading strand results in the synthesis of one strand that is displaced from the D-loop, thus converted into a DSB. The newly synthetized strand is then used as a template for the synthesis of the second strand. B) Extension of the invading strand results a unidirectional replication fork that progresses to the end of the donor chromosome. Resolution of the HJ results in two semiconservative replicated products. C) Branch migration of the HJ displaces the two newly synthetized strand. (Mc Eachern and Haber 2006).

  (Figure n.4B). SSA does not require a strand invasion step, therefore SSA is genetically independent of the recombinase Rad51, and RMPs that promote D-loop formation such as Rad54 and Rad55/Rad57 complex (McDonald and Rothstein 1994). After the annealing is completed, non-homologous 3' flap are cleaved by the Rad1/Rad10 nuclease complex (Figure n.4C)

Figure

  Figure n.4: Model of Single Strand Annealing. A) End-resection in the 5'-3' direction until the homology is revealed as ssDNA. B) The long 3' ssDNA tail is bound by the RMPs Rad52 and Rad59 that promote the annealing of complementary strands. C) Non-homologous 3'flap are cleaved by the Rad1/Rad10 nuclease complex.

Figure n. 6 :

 6 Figure n.6: Four strand exchange reaction by Rad51. After strand invasion, the ability of Rad51 to promote branch migration of the HJ results in the extension of the heteroduplex and complete exchange between duplex-duplex DNA (Murayama, Kurokawa et al. 2008).

Figure n. 7 :

 7 Figure n.7: Overview of obstacles causing RFB. Typical obstacles causing RFBs (DNA-bound proteins, chromatin organization, at risk sequences motif, DNA-damage) and interference between replication and transcription all have the potential to interfere with the progression of replication forks and its associated DNA synthesis. Global replication-stress and impediments to fork progression result in the activation of dormant origins that helps to complete DNA replication. In region of paucity in replication origins, completion of replication relies on long-traveling forks which are thus particularly sensitive to fork arrest and replication inhibition.

Figure n. 8 :

 8 Figure n.8: Three main mechanisms by which HR acts as a fork-escort pathway to ensure faithful genome duplication: repair of broken forks (left panel), repair of ssDNA gaps at damagedforks (middle panel) and fork protection when fork progression is slowing down (right panel).

Figure n. 9 :

 9 Figure n.9: Recombination outcomes associated to crossing-over. Arrows indicate repeated sequences and their orientations. 1) Copy number variation after inequal CO between sister chromatids (allelic recombination). 2) Segmental deletion after CO between flanking repeated sequences (NAHR).3) Sequence inversion after CO between inversed repeated sequences (NAHR). 4) Interchromosomal CO between repeated sequences leads to reciprocal translocation (NHAR). 5) Acentric and dicentric chromosomes formation after CO between interchromosomal inverted repeats[START_REF] Grabarz | Initiation of DNA double strand break repair: signaling and single-stranded resection dictate the choice between homologous recombination, non-homologous end-joining and alternative end-joining[END_REF].

Figure n. 10 :

 10 Figure n.10: Loss of Heterozygosity (LOH) by gene conversion. 1) Gene conversion between two alleles can lead to LOH (allelic recombination).2) A gene conversion event between a pseudo-gene and a functional gene can result in LOH and the subsequent inactivation of the functional gene[START_REF] Grabarz | Initiation of DNA double strand break repair: signaling and single-stranded resection dictate the choice between homologous recombination, non-homologous end-joining and alternative end-joining[END_REF]).

Figure n. 11 :

 11 Figure n.11:Model of FoSTes (Fork Stall and Template Switch. 1) after replication fork stalling, the lagging strand (red lines) disengages from the replicon and anneals to a second fork (green lines) on the basis of micro-homology.2) DNA synthesis occurs on a non-contiguous template. 3) Several template exchange reactions can occur between multiple stalled forks. 4) then the resumption of replication occurs on the initial template[START_REF] Gu | Mechanisms for human genomic rearrangements[END_REF]

Figure n. 12 :

 12 Figure n.12: Model of replication-induced genome instability by homologous recombination, either homology or micro-homology-mediated.

Figure n. 13 :

 13 Figure n. 13: Different levels of chromatin organization: from the DNA to the metaphase chromosome (Freeman et al., 2005).

Figure

  Figure n. 16: Scheme of a replication fork and histones turnover. Nucleosomes are evicted ahead of the fork by Asf1 and FACT, both interacting with the MCM complex. Asf1 transfer H3-H4 dimers to CAF-1.CAF-1 deposits H3-H4 dimers on leading and lagging strand, via its interaction with PCNA. Histone chaperone NAP1 loads H2A-H2B dimers on the (H3-H4) 2 tetramer, to form a complete nucleosome[START_REF] Groth | Replicating chromatin: a tale of histones[END_REF].

Figure n. 17 :

 17 Figure n.17: Features of p150, p60 and p48 proteins, the three subunits of human CAF-1. Amino acids are numbered in bold. A) Structure of human p150 protein. The colored lines represent the different domain of the protein. The p150 presents a MIR domain that interacts with heterochromatin proteins, two PCNA binding motifs (PIP1 and PIP2), an acidic domain that interacts with histones and a p60 binding domain. The pink line represents the fragment of p150 necessary to ensure chromatin assembly coupled to DNA synthesis. B) Structure of human p60 protein with 7WD repeats (white squares) and a PEST domain. C) Structure of human p48 protein with 7WD repeats and the H4 interacting domains (Ridgway and Almouzni 2000; Dohke, Miyazaki et al. 2008).

  Mammalian p150 subunit presents two PIP-box motifs: PIP1 exhibits a canonical sequence (Qxx(I/L/V)xx(F/Y)(F/Y); while PIP2 exhibits a degenerated sequence. In human cells, mutations in the canonical PIP-box (PIP1) decrease the ability of p150 to interact with PCNA but have a modest contribution on the targeting of p150 to DNA replication foci in vivo.

Figure n. 18 :

 18 Figure n.18: View of a PCNA bound by three p21 PIPs.Molecular surface of the PCNA-PIP complex, with PCNA colored according to electrostatic potential and the PIP peptide showed in white. Regions of intense positive charge appear blue, and electronegative regions of the surface are red[START_REF] Gulbis | Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA[END_REF].

Figure

  Figure n. 19: the PIP-box motifs of CAF-1: PIP1 and PIP2. A) Sequence alignment for PIP1 and PIP2 in different model organisms. Hs is Homo sapiens, Mm is Mus musculus, Xl is Xenopus laevis, Sc is S. cerevisiae. Red residues are conserved, while blue residues are not. B) Scheme of human p150, Cac1 and Pcf1 and their relative PIP boxes. PIP1 is a canonical PIP box (red circles) and PIP2 is a degenerated PIP box (blue circle). (Adapted from (Rolef Ben-Shahar, Castillo et al. 2009).

  stress induces a complex DNA damage response in which S-phase checkpoints aim to maintain the stability of stalled replication forks and provide time to repair or tolerate the damage[START_REF] Lopes | The DNA replication checkpoint response stabilizes stalled replication forks[END_REF]. In cells defective for replication-coupled chromatin assembly pathway, the integrity of advancing but not stalled replication forks is compromised, which leads to checkpoint activation and accumulation of recombinogenic DNA damage[START_REF] Clemente-Ruiz | Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks[END_REF]. Thus H3K56Ac/CAF1/Rtt106dependent chromatin assembly provides a mechanism for the stabilization of replication forks, whose deficiency causes a "hyper-rec" phenotype probably associated with the generation of DSBs (Clemente-[START_REF] Clemente-Ruiz | Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks[END_REF]. Furthermore, the partial depletion of histone H4 due to defects in histone chaperone activity, leads to recombinogenic DNA damage, as well. Indeed, the depletion of H4 is followed by the collapse of unperturbed and stalled replication forks, even when the S-phase checkpoint remains functional (Clemente-[START_REF] Clemente-Ruiz | Chromatin assembly controls replication fork stability[END_REF]. This has been demonstrated in budding yeast by a reduction in the amount of replication intermediates and by an increase of recombination intermediates such as HJ-like structures, likely reflecting fork restart by HR (Clemente-Ruiz and Prado 2009).To summarize data described above, defects in chromatin assembly at replication forks due to deficiencies in histone chaperones activities lead to abnormal recombination phenotypes.Several explanations can be proposed:a) The general decrease in nucleosome density could lead to a novel chromatin context, being more liable to DNA lesions or facilitating DNA access to DNA repair enzymes, including HR. In support of this, recent data showed that a reduction in histone H3 and H4 gene dosage results in cell resistance to all common DNA damaging agents in S. cerevisiae.Cell resistance results from an increased efficiency in DNA repair, especially by HR, suggesting that histones might indeed act as a barrier to DNA repair enzymes from accessing DNA lesions[START_REF] Liang | Histone dosage regulates DNA damage sensitivity in a checkpoint-independent manner by the homologous recombination pathway[END_REF].Liang and colleagues data might support the initial hypothesis that reducing nucleosome density in the absence of chromatin assembly facilitates recombination efficiency. b) The collapse of replication forks occurs more frequently when H3K56Ac deposition is impaired, while a general decrease in nucleosome deposition per se might not increase the instability of advancing replication forks. Restart or repair of collapsed forks requires HR, resulting in a hyper-rec phenotype (Clemente-Ruiz and Prado 2009; Clemente-Ruiz, Gonzalez-Prieto et al. 2011). c) Histone chaperones could have a direct role in repressing HR. However, this explanation is unlikely because recent data that have highlighted the role of CAF-1 during DNA repair by HR rather support a role for CAF-1 in promoting HR.

  Dohke and colleagues (2008) proposed a model for the implication of CAF-1 in heterochromatin maintenance, similar to Quivy's one, suggesting an interaction among yeast HP1 (Swi6), p150 (Pcf1) and p60 (Pcf2), with consequent Swi6 delivery to H3K9 histones, in the wave of the moving fork(Figure n. 20).

Figure n. 20 :

 20 Figure n.20: Swi6 dynamics during heterochromatin replication, in fission yeast.The dissociation of Swi6 from heterochromatin during mitosis is induced by the phosphorylation of the serine 10 on histone H3. Swi6 does not dissociate from subtelomeric heterochromatin in G1/S, suggesting that the regulation of these regions is different from the centromeric heterochromatin. At the G1/S transition, CAF-1 interacts with released Swi6 and when heterochromatin undergoes replication early in S-phase, the complex CAF-1-Swi6 is recruited to replication forks through an interaction with PCNA. At this moment, Swi6 is transferred onto daughter strands that retain K9 methylation. Chromatin bound Swi6 might recruit, directly or indirectly, H3K9 specific histone methyl transferase, Clr4 and HDACs to modify the newly assembled chromatin. The interaction between Swi6 and Clr4 has never been reported but, in mammalian cells, HP1 was shown to interact with the human homologue of Clr4, SUV39H[START_REF] Dohke | Fission yeast chromatin assembly factor 1 assists in the replication-coupled maintenance of heterochromatin[END_REF]).

Figure n. 21 :

 21 Figure n. 21: Chromatin remodeling during NER.After the DNA damage recognition, major remodeling of chromatin structure occurs, to allow the repair machinery to gain access to the DNA lesion. CAF-1 and PCNA are recruited at damaged sites that have undergone the dual incision stage of repair processing. After completion of DNA repair, CAF-1-dependent chromatin reassembly is essential to restore the preexisting epigenetic information (Adapted from[START_REF] Moggs | Chromatin rearrangements during nucleotide excision repair[END_REF].

CAF- 1

 1 appears to have a role in DSB repair, both in non-homologous end-joining (NHEJ) and homologous recombination. While the impacts of CAF-1 defects on NEHJ are rather indirect, some data point out a direct role for CAF-1 during DSB repair by HR[START_REF] Lewis | Reduction of nucleosome assembly during new DNA synthesis impairs both major pathways of double-strand break repair[END_REF][START_REF] Nabatiyan | Induction of CAF-1 expression in response to DNA strand breaks in quiescent human cells[END_REF][START_REF] Ramirez-Parra | E2F regulates FASCIATA1, a chromatin assembly gene whose loss switches on the endocycle and activates gene expression by changing the epigenetic status[END_REF].

Figure n. 23 :

 23 Figure n. 23: Model for the role of chromatin restoration in deactivation of the DNA-damage checkpoint.During DSB response, the DNA damage checkpoint is activated and the chromatin is disassembled. Although chromatin remodeling is required for recruitment of the resection machinery, DNA resection itself is sufficient to disassemble the chromatin in the vicinity of a DSB. Inactive Rad53 is bound to Asf1 and upon checkpoint activation Rad53 becomes hyperphosphorylated, releasing Asf1. Asf1 promotes chromatin assembly indirectly, through the acetylation of H3 on K56, via Rtt109. K56 acetylation drives chromatin restoration after DSB and this is a signal for the completion of the repair. The mechanism is not understood, but it has been demonstrated that H3K56 is sufficient to turn off the DNA damage checkpoint, even in absence of Asf1[START_REF] Chen | Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair[END_REF]).

  model organism Schizosaccharomyces pombe (fission yeast), commonly used in biology, derives from a strain isolated in 1921 in Switzerland, by Osterwalder. The same strain was isolated in 1893 by Lindner from East African millet beer. The fission yeast name "Schizosaccharomyces" is derived from the latin "saccharum" which means sugar and from the greek "mukes" which means mushroom. Lindner specified the name of the species which is the Swahili word for beer (pombe) (Figure n. 24A).The S. pombe strain isolated by Osterwalder was firstly used as model organism in the 1950s, by Leupold for genetic studies and by Mitchison for cell cycle studies. Fission yeast cells present features that are common to multi-cellular organisms included conserved cellular pathways. Moreover, the rapidity of the cell cycle and the lack of pathogenicity mark the fission yeast as an ideal model organism[START_REF] Yanagida | The model unicellular eukaryote, Schizosaccharomyces pombe[END_REF].S. pombe is a unicellular eukaryote with rod-shaped cells of 3.5 micrometers in diameter and 7-15 micrometers in length (Figure n. 24B). The name fission yeast indicates that cells divide by medial fission producing a septum. The cells maintain their shape during cell cycle and they grow only to the tips, dividing by medial fission and producing daughter cells of the same size.The cell cycle of the fission yeast is prevalently haploid. Cells of opposite mating type (h+ or h-) undergo conjugation, induced by nitrogen starvation. The conjugation of the two cells leads to unstable diploid zygote that rapidly undergoes into meiosis to generate four haploid spores (tetrad). These four spores, in favorable conditions, can proliferate to form haploid colonies. The mating type of S. pombe can be divided in two categories: homothallic strains (h 90 ) that switch mating type and thus undergo conjugation among them, leading to tetrads formation; heterothallic strains (either h+ or h-) that are stab le in their mating type and thus undergo conjugation when h+ cells mate h-cells.The cell cycle of S. pombe is rapid, around 3 hours, and varies according to culture media and temperature. The mitotic phase (M-phase) is constituted of chromosome segregation (nuclear division) and cytokinesis (cell division). A characteristic of yeast models is that nuclear envelope does not break down during mitosis, and proteins responsible for microtubule organization remain associated with the nuclear membrane. The G1 phase follows M-phase and is extremely fast in S. pombe, thus mitosis is rapidly followed by the entry into S-phase, while cytokinesis is not yet completed. Because of this, cells in S-phase are easily recognized in an asynchronous population, as cells having a septum (septated cells). Subsequent to Sphase, the G2 phase is very long and it occupies 80% of the cell cycle timing (Figure n. 24C).

Figure

  Figure n. 24: Features of the model organism S. pombe. A) Biologic classification of the model organism S. pombe B) Rod-shaped cell. In the picture, it is possible to notice cells in different phases of the cell cycle: 1.S-phase cell 2.late Sphase cell 3.G2-phase. C) Cell cycle of S. pombe: the 80% of the time yeasts cell are in G2 phase while M, G1 and Sphase are rapid.

  The region B of 450 bp is essential for the activity of the RFB. It contains four repetitions of a 55 bp motif binds by the Rtf1 protein. The current view is that Rtf1 binds to RTS1 and by interacting with additional proteins, as Rtf2, mediates polar fork arrest  The region A of about 60 bp, rich in purine, which is thought to reinforce the RFB activity of the region B. The proteins Swi1, Swi3 and Rtf2 can bind the region A[START_REF] Dalgaard | swi1 and swi3 perform imprinting, pausing, and termination of DNA replication in S. pombe[END_REF][START_REF] Codlin | Complex mechanism of site-specific DNA replication termination in fission yeast[END_REF][START_REF] Lee | Biochemical interactions between proteins and mat1 cis-acting sequences required for imprinting in fission yeast[END_REF]).The RTS1-RFB causes fork arrest because of a non-histone protein complex binding to the DNA. As proposed for other polar RFBs, the RTS1-RFB is thought to block fork progression by directly (contact between proteins and the replisome) or indirectly (topological constraint) affecting the replicative helicase activity and thereby preventing DNA unwinding[START_REF] Eydmann | Rtf1-mediated eukaryotic site-specific replication termination[END_REF] Kaplan and Bastia 2009).Lambert and colleagues exploited the RTS1-RFB to design conditional fork arrest substrates in order to decipher the impact of impediments to fork progression on genome stability and molecular mechanisms of fork-restart by homologous recombination (Figure n. 25A). It was initially established that fork arrested by the RTS1-barrier are hot spots of recombination, and of chromosomal rearrangements and that the HR pathway, but not the checkpoint pathway, is required to ensure full cell viability upon induction of the RTS1-RFB[START_REF] Lambert | Gross chromosomal rearrangements and elevated recombination at an inducible site-specific replication fork barrier[END_REF]). Importantly, it was then established that HR promotes efficient recovery of fork arrested at the RTS1-barrier by a DSB-independent mechanism[START_REF] Mizuno | Nearby inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism[END_REF][START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]. The polar RTS1 barrier was introduced on the centromereproximal (cen-proximal) side of the ura4 locus, 5 kb away from the strong replication origin (ori) 3006/7 on chromosome III. This created the uraR locus (Figure n. 25A). The activity of the polar RTS1-barrier is dependent on its interaction with the protein Rtf1 which the expression is controlled by the presence or the absence of thiamine in the media (Figure n.

Figure n. 25 :

 25 Figure n.25: An inducible site-specific fork arrest assay in S. pombe. A) RTS1-RFB sequences are integrated at one side or both sides of ura4 gene, blocking the incoming replication fork. Centromereproximal and telomere-proximal regions are represented in black and grey, respectively. Strong or putative replication origins (ori) and the centromere are indicated by red, green and black circles, respectively. Blue arrows indicate the polarity of the RTS1-RFB. The transcription direction of ura4 + gene is indicated by a red arrow. On the bottom, representations of the primary arrested fork structure are given for each construct (Lambert, Watson et al., 2005). B) Scheme of fork arrest substrate. C) Condition of Rtf1 expression. D) left panels: analysis of replication intermediates by 2DGE in wt strain in OFF (Rtf1 being repressed) and ON (Rtf1 being expressed) conditions, and in rad22-d strain (ON condition). The red arrow indicates accumulation of termination signal, a sign of defective fork-restart in the absence of homologous recombination. Right panels: scheme of replication intermediates observed in uraR strains within the AseI restriction fragment (Lambert, Mizuno et al. 2010).

Figure n. 26 -

 26 Figure n.26 -Model of fork restart by recombination. A) Blue arrows indicate RTS1-RFB and its polarity. The ura4 gene is indicated in red and black arrow indicates its transcription orientation.Centromere-proximal and telomere-proximal regions are represented in black and grey, respectively. Centromere is represented by a black circle. The model propose that unwinding of stalled nascent strands allows the recruitment of Rhp51 (yellow circle) and Rad22 (green circle). In the RuraR system, two mechanisms have been reported: the restart occurs either on the initial template (A, direct restart, left panel) or by template exchange of stalled nascent strands thus allowing resumption of DNA synthesis on a non-contiguous template (B, right panel). Template exchange between the two RTS1 repeats result in formation of joint-molecules visualized by 2DGE and whose resolution leads to chromosomal rearrangements (switch of ura4 orientation, acentric and dicentric iso-chromosome). B) Scheme of replication intermediates and joint-molecules observed by 2DGE in the RuraR strain within the AseI restriction fragment. C) 2DGE in indicated strains and conditions. Red circle indicates jointmolecules that are proposed to be extended D-loop and green circle indicates joint-molecules that are proposed to be Holliday-junctions-like structures (HJs-like)[START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]).

Figure n. 27 :

 27 Figure n.27: Chromosomal rearrangements accumulate upon fork arrest within inverted repeats. A) Scheme of the RuraR parental construct. Blue squares with black lines inside represent RTS1 sequences and their polarity.Centromere-proximal and telomere-proximal regions are represented in black and grey, respectively. The centromere is indicated by a black circle. The transcription direction of ura4 + gene is indicated by a black arrow. Fork arrest at RTS1-RFB within inverted repeats results in chromosomal rearrangements including the switch of ura4 orientation (resolution of HJ-like structure in one crossing over direction), and acentric and dicentric iso-chromosomes (resolution of HJ-like structure in two crossing over direction)[START_REF] Lambert | Gross chromosomal rearrangements and elevated recombination at an inducible site-specific replication fork barrier[END_REF][START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]). B) Restriction Fragment Length Analysis (RFLA) of the RuraR locus in indicated strain and conditions. wt and rad22-d strains. Note that no chromosomal rearrangements are observed in the rad22-d strain(Lambert, Mizuno et al. 2010). C) Pulse Field Gel Analysis (PFGE) followed by Southern-blot of chromosome III in indicated strains harboring the RuraR construct and in indicated conditions.

  glutamate (EMM-Glu): Edinburgh minimal medium (MP Biomedicals), Glutamate 27.14 g/L. b) Yeast Extract (YE): yeast extract (DIFCO) Solid media contain bacto agar (BD) 20 g/L.Culture media, growth conditions and basis protocols of fission yeast manipulation //biosci.osu.edu/~nile/nurse_lab_manual.pdf). Fission yeast cultures realized in liquid media were performed in Yeast Extract (YE) (BD Biosciences) a rich medium containing 2% final concentration of glucose or Edinburgh Minimum Media L-Glutamate (EMM-Glu) (MP Biomedicals), a minimal medium in which the nitrogen source is glutamate and not ammonium chloride (as for others common minimal media). Liquid culture media were supplemented in amino acids, necessary for strains growth depending on their genotype (adenine, leucine, histidine, arginine and uracil at 12.5 mg/mL -Sigma).For fission yeast growth on solid media, 2% of agar (BD Biosciences) was added. Antibiotic selection was realized on solid media by adding: a) 200mg/L of geneticine (G418) (Invitrogen) for Kan (kanamycin) resistant strains, b) 200mg/L of nourseothricin (Werner Bioagents) for Nat resistant strains c) 200mg/L of hygromycin (Invitrogen) for Hyg resistant strains.Regarding strains harbouring the fork arrest system at ura4 locus, the activity of the replication fork barrier is regulated by the conditional addition of thiamine. The expression of Rtf1 protein is under the control of the inducible nmt41 promoter, which was repressed by 60µM of thiamine. Therefore, depending on the necessity of repression (OFF condition) or induction (ON condition) of the replication fork barrier, media were respectively supplemented or not in thiamine at a final concentration of 60 μM (Calbiochem).

  Gene deletion was carried out by gene targeting, using PCR products containing homology with flanking region of the gene of interest. To achieve this purpose, primers were designed with two segments: the first one was 100 bp complementary to flanking region of the gene and the second one is 20 bp complementary to flanking region of antibiotic marker on pFA6a-derived plasmid (Bahle,[START_REF] Bahler | Heterologous modules for efficient and versatile PCRbased gene targeting in Schizosaccharomyces pombe[END_REF][START_REF] Hentges | Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe[END_REF]. By PCR, deletion cassettes were obtained, containing the antibiotic marker and 100 bp corresponding to the complementary flanking region of the target gene. For gene replacement by a kanamycin, nourseothrycin or hygromycin marker, PCR were respectively performed on pFA6a-kanMX6, pFA6a-natMX6 or pFA6a-hphMX6 plasmids. Deletion cassettes were finally used to transform Schizosaccharomyces pombe cells.Primers used for the construction of a strain deleted for swi6 gene: the first 100 bp (capital letters) are complementary to the flanking region of the gene while the last 20 bp (minuscule letters) are complementary to the flanking region of the marker cassette.

  strain Pcf1-YFP: The Kan marker associated with YECitrine was amplified by PCR (plasmid: pFA6a-link-YECitrine-Kan). The PCR product was used to transform a fission yeast wild-type strain. The primers used had the same features mentioned above. strain Pcf2-MYC: The Kan marker associated with 13 Myc repeats was amplified by PCR (plasmid: pFA6a-13Myc-KanMX6). The PCR product was used to transform a fission yeast wild-type strain. Two kinds of transformant were obtained: respectively with 13 Myc repeats and with 4.5 Myc repeats. After western blot analysis, the strain used for the experiments contained 4.5 Myc repeats. The reason was that, in the strain containing the 13 Myc repeats, Pcf2 migrates at the same size of Pcf1-YFP. This could lead to problems in the revelation of the two proteins, by western blot. strain Pcf1-PIP*: To obtain the mutation in the PIP-box, two PCR reactions were performed. For each reaction, a primer containing the desired mutations was used (F-pcf1 PIP* or R-pcf1 PIP*) together with a second primer outside pcf1 ORF (U-pcf1-check or L-pcf1-check). The two PCR products obtained were ligated via fusion PCR, using only the two primers outside pcf1 ORF.

  10 7 cells were plated on YE supplemented with 5fluoroorotic acid (1.6 g/L) (Euromedex). At the same time, 100µl of an appropriate dilution were spread on complete YE, to determine the number of viable cells. Colonies were counted after 4 days at 30°C. The rate of ura4 loss was determined with the method of the median, comparing the number of colonies growing on 5-FOA plates and the viable colonies growing on complete YE plates. Statistical significance was detected using the non-parametric Mann-Whitney test. Colony PCR was performed on 5-FOA resistant colonies to score ura4 deletions and translocations (See 2.1 Material and Methods for colony PCR). The percentage of deletion/translocation events determined by the PCR assay, was used to balance the rate of ura4 loss. Primers used (Sigma-100 μM):

  Chromosome sizes were analyzed by pulse field gel electrophoresis as follows: 30 ml of culture (1.10 7 cells/ml) was washed twice in 30 ml CSE buffer (20 mM citrate/phosphate [pH 5.6], 40 mM EDTA, 1.2 M sorbitol) and then digested in 5 ml CSE containing 1.5 mg/ml Zymolyase 20T (MP biomedicals). Cells pellet was then resuspended in 300 μl of TSE buffer, 45 mM EDTA, 0.9 M sorbitol) and mixed with 400 μl of 1% agarose (Ultra Pure TM Agarose Invitrogen) in TSE and dispensed in 80 μl aliquots to plug molds. Then, cell lysis was performed by incubating agar plugs in 0.25 M EDTA, 50 mM

  40 μg of DNA were resuspendend in 100 μl of sterile water and digested overnight by 10 μl of AseI (10U/μl) or 5 μl of EcoRV (20 U/μl). Both reactions required also 50 μl of Buffer n.3 (10X), 5μl of BSA (100X) and sterile water until a final volume of 500 μl. The digested genomic DNA was precipitated with 50 μl of potassium acetate (5M), 550 μl of ice-cold isopropanol and 1 μl of glycogen (20 mg/ml) and washed once in 70% ethanol. Then, DNA was resuspendend in 25 μl of sterile water and loaded on 0.8% gel. Once the gel was migrated, the genetic material was transferred on a nylon membrane to perform Southern blot. Recombination outcomes were probed with 32 P-ura4 probe (GE healthcare and Applied biosystem), signals were screened with a Typhoon PhosphorImager, and band intensities were quantified with Imagequant software (Amersham Biosciences). Quantification of rearrangements associated with one or two cross-over were performed using ura4 as probe: % of global rearrangements (EcoRV digestion: ura4 inversion + acentric) = intensity of the (inversion+acentric) band/intensity of ((inversion+acentric band) + (RuraR locus band)); % of acentric isochromosome (AseI digestion) = intensity of lower band, corresponding to the acentric/intensity of (acentric band + RuraR locus band) % of ura4 inversion = % of global rearrangements -% of acentric isochromosomes.

To 5 .

 5 10 8 cells add 1/100 of volume of sodium azide 10% (Sigma) and centrifuge. Wash cells in cold water, resuspend pellet in 400 µL EB Buffer (50 mM HEPES High Salt, 50 mM KOAc pH 7.5; 5 mM EGTA, 1% triton X-100; 1 mM PMSF (Sigma) + 1 tablet anti-protease (Roche) and ribolyse cells with glass beads (Sigma-as for TCA extract). Centrifuge and recover surnatant. -Keep 50 µl as INPUT to which add Laemmli 4X and boil 5 min. Keep samples at -20°C. -Keep 300 µl to do IP: add 2 µl of anti-GFP/anti-Myc antibody (Invitrogen) and incubate for one hour, at 4°C. Wash twice 40 µl of Dynabeads Protein-G (Sigma) for sample in 1 ml PBS 1X, 10 min at room temperature and resuspend in 40 µL de EB Buffer. Add the beads to the 300 µL of extract and incubate at 4°C overnight. The day after keep 50 µL of surnatant as UNBOUND FRACTION, then wash beads twice in EB Buffer, 10 min. Resuspend beads in Laemmli (30 µL) and boil for 5 min (IP FRACTION).

  Pcf1-YFP was analyzed in pcf2-d or pcf3-d deleted strains by western-blot (Figure n.28A). As positive control, I used a strain expressing Pcf1-YFP with wild-type pcf2 + and pcf3 + , and as negative control, a strain expressing untagged Pcf1. To quantify the expression level of Pcf1-YFP in these genetic backgrounds, the expression of PCNA was used as loading control. I found that the deletion of pcf2 or pcf3 slightly decrease the expression level of Pcf1 (between 10% and 20%) (Figure n. 28B). This minor effect could be due to the asynchrony of cultures in which different amounts of S-phase cells could account for the observed fluctuation in Pcf1 expression level. Thus, I concluded that deletion of Pcf2 or Pcf3 subunit of CAF-1 has little or no effect on the stability of the largest subunit Pcf1. This does not presume of the functionality of CAF-1 and does not exclude potential functions of

Figure n. 29 :

 29 Figure n. 29: Analysis of growth parameters in strains defective for CAF-1. The growth temperature was 30°C for all experiments. A) Left panel: growth curves in YE (rich media) of indicated strains. Right panel: YE plate with wt, rad22-d, pcf1-d, pcf2-d, pcf3-d and pcf1-d pcf2-d pcf3-d strains. B) Left panel: growth curve in EMM-Glu (minimal media) of indicated strains. Right panel: corresponding plate. The rad22-d strain presents a slow growth phenotype C) Left panel: growth curve in EMM-NH 4 Cl (minimal media) of indicated strains. Right panel: corresponding plate. The initial concentration of cells in the culture was higher (1.10 6 ) than in the previous two media. D) Doubling time of indicated strains and media.

Figure n. 30 :

 30 Figure n. 30: Strains defective for CAF-1 are not sensitive to CPT, HU or UV-C exposure. A) YE plates with different concentrations of CPT, on which serial tenfold-dilutions from indicated strains were spotted. B) YE plates containing different concentrations of HU. C) YE plates exposed to different doses of UV-C. rad3 is the gene coding for the checkpoint kinase Rad3 ATR/Mec1 and the strain deleted for rad3 was used as control for the quality of the plates, because of its hypersensitivity to DNA damaging and replication blocking agents.

Figure n. 31 :

 31 Figure n. 31: Strains defective for CAF-1 are not sensitive to MMC, Cisplatine or MMC exposure A) YE plates containing different concentrations of MMS, on which serial tenfold-dilutions from indicated strains were spotted. B) YE plates containing different concentrations of Cisplatine. C) YE plates containing different concentrations of MMC. The strain deleted for rad3 was used as control for the quality of the plates, because it is hypersensitive to DNA damaging and replication blocking agents.

Figure n. 32 :

 32 Figure n. 32: Strains defective for CAF-1 are not sensitive to TBZ. YE plates containing different concentrations of TBZ, on which serial tenfold-dilutions from indicated strains were spotted. The strains tested were wt, lsd1-W89IA-HA, pcf1-d, pcf2-d and pcf3-d. The strain lsd1-W89IA-HA harbour a mutation of the gene lsd1, whose product is the histone demethylase SWIRM1. The mutation confers a hypersensitivity of this strain to TBZ.

  Figure n. 33). Regarding the hip1-d x pcf1-d cross, it appears that the double mutant hip1-d pcf1-d is unviable because only 2 spores out of 43 hip1-d pcf1-d expected were viable. This low frequency of viable hip1-d pcf1-d spores is not due to a bias in meiotic recombination as the two genes are not genetically linked. The two viable spores could be in fact heterozygous diploid. Regarding the second cross, the double mutant slm9-d pcf1-d seems to be viable because 15 spores out of 36 were viable. The co-lethality of the double mutant pcf1-d hip1-d could be due to redundant functions between CAF-1 and HIRA in maintaining the correct histone pool during the cell cycle, via the selective transcription of core histone genes.

I

  performed preliminary tests with the double mutant pcf1-d slm9-d to test its sensitivity to HU, CPT and UV-C treatment, compared to each single mutant. However, the double mutant does not present any significant increased sensitivity compared to the single mutant slm9-d (data not showed).

Figure n. 33 :

 33 Figure n. 33: Genetic interaction between CAF-1 and HIRA Left panel: tetrads from the cross between pcf1-d and slm9-d strains are represented. Blue squares indicate pcf1-d spores, green squares indicate slm9-d spores and red circles indicate the double mutant pcf1-d slm9-d. Right panel: tetrads from the cross between pcf1-d and hip1-d strains are represented. Blue squares indicate pcf1-d spores, green squares indicate hip1-d spores and red circles indicate the double mutant pcf1-d hip1-d.

Figure n. 34 :

 34 Figure n. 34: CAF-1 is dispensable for recombination between homologous chromosomes and chromosomes loss.A) Scheme of the non-sporulating diploid strain used for the assay and representation of the events (cross over and chromosome loss) that can occur. The resulting phenotypes in term of 5-FOA resistance and growth on low-adenine media are also reported. B) Serial tenfold-dilution from indicated strains, spotted onto YE plate (control plate) and in plate containing low-adenine and 5-FOA. C) Rate of recombination events and rate of chromosome loss in indicated strains. The values correspond to the mean of three independent rates, each one being median of 13 independent cultures. The confidence interval is calculated at 95% (enclosed in bracket) and the fold increase over wt is indicated for each strain.

  (Figure n. 35D). Nonetheless, a similar induction in sisterchromatid recombination by CPT-treatment was observed in pcf1-d cells (Figure n. 35D).

Figure n. 35 :

 35 Figure n. 35: CAF-1 is dispensable for spontaneous and CPT-induced sister-chromatids recombination. A) Scheme of recombination substrate and recombinant products. The black and the gray circles indicate the location of the mutation present in ade6-L469 and ade6-M375 respectively. B) Serial tenfold-dilution from indicated strains, spotted on indicated media. C) Rate of gene conversion and of deletions in indicated strains. The numbers represent the mean of three independent rates, each one being median of 11 independent cultures. The confidence interval is calculated at 95% (enclosed in bracket) and fold increase over wt is indicated for each strain. D) Frequency of gene conversion and deletion in a wt and pcf1-d strain, after 4 hours treatment with 20μM of CPT. DMSO was used as control vehicle. The values reported in the histogram are the mean of two independent experiments and the error bars indicate the standard deviation. The numbers on the top of the bars indicate the induction of gene conversion and deletion by CPT-treatment.

Figure n. 36 :

 36 Figure n. 36: CAF-1 is dispensable for the repair of replicative DSB by sister-chromatid recombination.A) Scheme of a site-specific and strand-specific break at mat1 locus. The white arrow indicates the SSB which is converted in a polar DSB. The black rectangle represents the RTS1 sequence at the centromere-proximal side of mat1 and MPS1 indicates mat1 pause site. The last scheme on the bottom represents the MT switching in a wild-type strain. The white square is the mat2P locus and the grey square is the mat3M. The mat2P and the mat3M provide the intact DNA template, respectively in M and P cells[START_REF] Roseaulin | Mus81 is essential for sister chromatid recombination at broken replication forks[END_REF]. B) Tested strains: PΔ17 pcf1-d is deficient for the SSB (black triangle) but harbours the donors sequences (mat2P and mat3M); M2,3∆ is proficient for SSB (white arrow), but lacks the donors sequences (grey rectangle). Bottom panel: tetrads from the cross of these two strains. Blue squares indicate spores deleted for pcf1 with no SSB and harbouring the donors sequences. Red circles indicate spores deleted for pcf1 experiencing the SSB, but without donors sequences.

  Figure n. 25A). Thus, to control the activity of the RTS1-RFB, the transcription level of rtf1 is regulated via the thiamine repressible promoter nmt41. In the presence of thiamine in the media, the RTS1-RFB is not active (defined as OFF condition) and in the absence of thiamine in the media, the RTS1-RFB is active and the replication fork is arrested at the ura4 locus (defined as ON condition) (see Introduction, Figure n. 25B). The RTS1-RFB was previously shown to behave as hot spot of recombination and chromosomal rearrangements[START_REF] Lambert | Gross chromosomal rearrangements and elevated recombination at an inducible site-specific replication fork barrier[END_REF].It was previously reported that recombination mutants are sensitive to the induction of the RTS1-RFB. Therefore, I first analyzed the sensitivity of cells defective for CAF-1 to the induction of the RTS1-RFB. As strains defective for CAF-1 show a severe slow grow phenotype in EMM-NH 4 Cl, all experiments were performed using EMM-Glu as minimal media. I performed both a serial dilution assay and a survival test in the presence (OFF condition) or in the absence (ON condition) of thiamine in the media. I observed that CAF-1 is not required to promote cell viability, upon induction of the RTS1-RFB. On the contrary, the survival of the rad22 deleted strain was severely decreased when the fork-arrest at ura4 was induced. The reason is the inability of the rad22-d strain to restart the blocked replication fork by recombination (Figure n 37A and B).

Figure n. 37 :

 37 Figure n. 37: CAF-1 is dispensable for cell viability, upon induction of the RTS1-RFB. A) Cell survival of indicated strains experiencing fork arrest at RuraR. Values are the mean of two independent experiments. B) Serial 10-fold dilutions of indicated strains, spotted onto EMM-Glu containing thiamine (pause OFF) or not (pause ON).

  -FOA R cells). Analysis of chromosomal rearrangements by PCR showed that ectopic recombination induced by fork-arrest results in translocations and genomic deletions (Figure n. 38A; Iraqui, Chekkal et al. 2012 in press). Exploiting this assay, my aim was to investigate the role of CAF-1 in ectopic recombination induced by fork-arrest. Induction of the RTS1-RFB resulted in 11 fold increased in the rate of ura4 loss. As previously reported, the rate of ura4 loss was not increased in rad22-d strain in response to fork-arrest (Figure n. 38B). If compared to the wt strain, rad22-d strain exhibits a 8 fold decrease in the rate of ura4 loss upon fork arrest, thus confirming that loss of this marker is mediated by homologous recombination (Figure n. 38B). Strains deleted for pcf1, pcf2 or pcf3 showed a similar and significant 3 to 5 fold decrease in the rate of ura4 loss, compared to wt strain, showing that CAF-1 promotes genetic instability induced by the RTS1-RFB (Figure n. 38 C and D).Then, I analyzed by PCR the nature of chromosomal rearrangements mediated by ectopic recombination (Figure n. 39A). In the wt strain, the RTS1-RFB induced genomic deletion by 19.4 fold and translocations by 29.3 fold, over the OFF condition. In contrast, strains deleted for pcf1-d, pcf2-d or pcf3-d showed a 2.3 to 5.5 fold reduction in genomic deletion and a 3.5 to 9.9 fold reduction in translocation events, compared to wt strain (Figure n. 39B and C).

1

  implies the three subunits, suggesting that the histone-chaperone function is involved in the regulation of recombination. My data suggest that CAF-1 could be a new regulator of recombination outcomes, especially by acting in non-allelic recombination.

Figure n. 38 :

 38 Figure n. 38: Assay to analyze fork-arrest-induced rearrangements between dispersed repeated sequences. A) Induction of the RTS1-RFB in RuraR cells leads to loss of ura4 marker, as assayed by 5-FOA resistance. Blue arrows represent RTS1 sequences and its polarity at ura4 locus (chr. III) and at mat locus (chr. II). The ura4 gene is represented in red. The black and orange arrows indicate the primers TLIII and TLII, to analyze translocation. Black and green circles represent the centromeres of chromosome III and chromosome II, respectively. B) Rate of ura4 loss assayed by 5-FOA resistance. 20-30 cultures were analyzed by fluctuation analysis for wild-type and rad22-d strains. The rate of ura4 loss was determined using the median method. C) Rate of ura4 loss assayed by 5-FOA resistance in indicated strains. The values on the histogram correspond to the mean of at least three independent median rates (11 cultures for each experiment). The error bars represent the standard deviation and the statistical significance was calculated using the non-parametric Mann Whitney test. D) Table indicating the induction of ura4 loss by the RTS1-RFB in indicated strains (rate of ura4 loss in ON conditions/rate of ura4 loss in OFF conditions), and the decrease over wt.

Figure n. 39 :

 39 Figure n. 39: CAF-1 promotes ectopic recombination upon fork-arrest induction. A) Examples of colony PCR analysis of 5-FOA R colonies obtained with rtf1 induction (ON conditions) in wt, pcf1-d and rad22-d strains. TLII and TLIII primers flank RTS1 at 120 bp and 160 bp on chr. II and III respectively and amplify a product of 1140 bp (black and orange arrows indicate the primers TLIII and TLII in the Figure38A). Rng3 primers amplify rng3 gene, which is an essential gene located between RuraR and the telomere, giving a PCR product of 647 bp. Ura4 primers flank ura4 locus and amplify a product of 960 bp. B) The % of deletion events, as determined by the PCR assay, was used to balance the rate of ura4 loss. The values reported in the histogram correspond to the mean of 3 independent median rates. Error bars indicate the standard deviation. Right table: induction of genomic deletion by the RTS1-RFB (rate of genomic deletion in ON conditions/rate of genomic deletion in OFF conditions) and the relative decrease over wild-type. C) The % of translocation events, as determined by the PCR assay, was used to balance the rate of ura4 loss. The values reported are means of 3 independent median rates. Error bars correspond to the standard deviation. Right table: induction of genomic translocations by the RTS1-RFB (rate of genomic translocations in ON conditions/rate of genomic translocations in OFF conditions) and the relative decrease over wild-type.

  recombination proteins are recruited (see Introduction, Figure n. 26A). Once the initial replication-block is removed, fork-restart occurs without changing template. However, stalled nascent strand can occasionally switch template and anneal with the second RTS1 sequence, in inverted orientation. This template exchange reaction results in joint-molecules formation (JMs) that are visualized through the analysis of replication intermediates by 2-dimensional gel electrophoresis (2DGE). Two kinds of JMs formed between RTS1 repeats were reported: JM-A that is proposed to correspond to an extended D-loop (after initiation of DNA synthesis on the non-contiguous template) and JM-B that contains Holliday junction-like structures (HJs-like) formed between the two RTS1 repeats (see Introduction, Figure n. 26C). D-loop intermediates are thought to be precursors of HJs-like structures. Both JMs are dependent on Rad22, while JM-A depends on both Rhp51 and Rad22. The resolution of HJs-like structures gives rise to three kinds of chromosomal rearrangements: the formation of acentric and dicentric iso-chromosomes (resolution associated to one cross-over) and the switch of ura4 orientation (resolution associated to two cross-overs) (see Introduction, Figure n. 27A).

  first studied the role of CAF-1 in the efficiency of replication-induced recombination by analyzing JMs by 2DGE (Figure n. 40A). As previously reported, JMs were efficiently formed in the wt RuraR strain upon induction of the RTS1-RFB (Figures n. 40B and C). Interestingly, strains defective for CAF-1 (pcf1-d, pcf2-d or pcf3-d strains) showed a similar 3 to 4 fold reduction in JM-A intensity and a 8 to 11 fold reduction in JM-B intensity, compared

Figure n. 40 :

 40 Figure n. 40: CAF-1 defects result in a decreased amount of JMs upon fork-arrest at RuraR. A) Diagrams of replication intermediates (RI) within AseI restriction fragment analyzed by 2DGE (top) and their structures (bottom: black arrowheads indicate RTS1 orientation). Left: RIs observed when ura4 locus is replicated passively (no fork arrest at RTS1 barrier). Right: RIs observed in RuraR cells upon fork arrest. Expected mass of arrested forks and calculated mass of JMs are given. JM-A and JM-B indicate D-loop and HJs like structures, respectively. B) 2DGE analysis of RIs from indicated RuraR strains grown for 24 hours with (fork arrest OFF) or without thiamine (fork arrest ON). Numbers indicate the percentage of fork arrested by the RTS1-RFB (± standard deviation). C) Quantification of B. The values correspond to the mean of three independent experiments and the error bars correspond to the standard deviation..

Figure n. 41 :

 41 Figure n. 41: hypotheses drawn for CAF-1 dependent phenotype.

  . Compared to the wt, a significant reduction (a 2-3 fold decreased, p<0.03) in accumulation of both acentric and switch of ura4 orientation was observed in each single mutant (pcf1-d pcf2-d or pcf3-d) (Figures n. 42A and B).

Figure n. 42 :

 42 Figure n. 42: Decreased level of products of JMs resolution in the absence of CAF-1. A) RFLA of indicated RuraR strains grown in the presence of thiamine (OFF condition) and for 24h and 48h after thiamine removal. B) Kinetics of rearrangements formation according to the number of generation experiencing fork arrest at RuraR. The values correspond to the mean of three independent experiments and the error bars indicate the standard error of the mean.

  , the acentric chromosome accumulated in the wt RuraR strain, upon fork arrest at the RTS1-RFB (Figures n. 43A and C). In agreement with the data shown in Figure n. 42, I observed a 2-3 fold (p<0.0003) reduction in the accumulation of acentric chromosomes in each single mutant, compared to the wt strain (Figures n. 43A and C). As this phenotype is common to each single mutated strain, this suggests that the histone chaperone function of CAF-1 is involved in this phenotype. To reinforce this hypothesis, I analyzed the pcf1-d pcf2-d pcf3-d triple mutant, in which the three genes encoding for CAF-1 subunits were inactivated. The triple mutant showed a reduction in the accumulation of acentric chromosome to the same extends than each single mutant, demonstrating that deletions of genes encoded CAF-1 subunits are epistatic for replication-induced recombination (Figure n. 43A and C).

  induced recombination (Figure n. 43). Therefore, I verified that the residual level of recombination products remains dependent on the main recombination factor, Rad22. I created two strains, pcf1-d rad22-d and pcf2-d rad22-d and analyzed the accumulation of acentric chromosomes by PFGE. The acentric chromosomes were not detected in pcf1-d rad22-d RuraR, pcf2-d rad22-d RuraR and rad22-d RuraR strains (Figure n. 43B), thus demonstrating that the remaining level of chromosomal rearrangements induced by the RTS1-RFB is indeed dependent on Rad22. Surprisingly, disomic chromosomes III were observed in the pcf2-d rad22-d, but not in pcf1-d rad22-d strain, independently of the induction of the RTS1-RFB. The presence of two chromosomes III could be due to gross chromosomal rearrangements, leading to the production of an abnormal chromosome III (maybe fusion with another one) or to the breakage of chromosome III. The difference between pcf1-d rad22-d and pcf2-d rad22-d strains could be related to a Pcf2 function that is not shared by the other subunits of the complex. Nonetheless, these data highlight of a potential interplay between Rad22 and Pcf2 in the maintenance of genome stability.

Figure n. 43 :

 43 Figure n. 43: Analysis of acentric chromosome formation by PFGE. A) PFGE analysis of wt, pcf1d, pcf2-d, pcf3-d and pcf1-d pcf2-d pcf3-d strains in OFF condition and in ON condition (24h and 48h after thiamine removal). The size of the chromosome III is 3.5 Mb and the size of the acentric chromosome is 1.2-1.5 Mb. B) PFGE analysis of wt, rad22-d, pcf1-d, pcf1-d rad22-d and pcf2-d rad22-d in OFF condition and in ON condition (24h and 48h after thiamine removal). The size of the chromosome III is 3.5 Mb and the size of the acentric chromosome is 1.2-1.5 Mb. C) Kinetic of acentric chromosome accumulation after thiamine removal in indicated RuraR strains. The values correspond to the mean of at least three independent experiments and error bars indicate the standard error of the mean.

  GFP. The expression level ofRad22-GFP was not affected by the deletion of pcf1-d, pcf2-d or pcf3-d, as verified by western-blot (Figure n. 44A).

Figure n. 44 :

 44 Figure n. 44: Recombination centres and Rad22 recruitment at arrested forks are not affected by the absence of CAF-1. A) Total protein extracts from indicated strains were analyzed by westernblot. Rad22-GFP (68.8 kDa) was detected using an anti-GFP antibody and PCNA (28.9 kDa) was detected using an anti-human PCNA antibody. B) Analysis of spontaneous Rad22-GFP foci in indicated strains. Differential Interference Contrast (DIC-grey) and GFP fluorescent signal (green) in wild-type (top panels) or pcf1-d cells (bottom panels), carrying rad22-gfp. Red arrows indicate the presence of Rad22-GFP foci. C) Quantification of B in indicated strains. Values are means of at three independent experiments (for wt and pcf1) or two (pcf2-d and pcf3-d). Errors bars correspond to standard deviation. At least 300 nuclei were analyzed per strain and experiment. D) Graphic representing the level of Rad22 recruitment at the Rura4R locus in indicated strains, grown for 24 hours in the presence (fork-arrest OFF) or the absence of thiamine (fork-arrest ON). Values correspond to the mean of three independent experiments and error bars correspond to the standard error of the mean.

RTS1

  sequence at ura4 (uraR). In this case, template exchange cannot occur because of the absence of the second RTS1 repeat; thus arrested forks are restarted exclusively on the initial template. Previous studies have shown that, in the absence of Rad22 or Rhp51, termination signal visualized by 2DGE accumulates at uraR, showing that the RTS1-RFB behaves as a hot spot of replication termination in the absence of recombination (see Introduction, Figure n. 25D). Therefore, termination signals are used as sign of impaired fork recovery. Analysis of replication intermediates by 2DGE showed that the accumulation of termination signals and the level of arrested forks in uraR strains defective for CAF-1 are at the same level than in the wt strain (Figures n.

Figure n. 45 :

 45 Figure n. 45: Arrested forks are stable and prone to recombination-dependent restart in the absence of CAF-1. A) Diagrams of replication intermediates (RI) within the AseI restriction fragment analyzed by 2DGE (top) and their structures (bottom: black arrowheads indicate RTS1 orientation. Left: RIs observed when the ura4 locus is replicated passively (no fork arrest at the RTS1 barrier). Right: RIs observed in uraR cells, upon fork arrest. B) 2DGE analysis of RIs from indicated strains harbouring the uraR system, grown for 24 hours in the presence of thiamine (pause OFF) or not (pause ON). Numbers indicate the percentage of fork arrested at uraR ± standard deviation. C) Quantification of B. The values correspond to the mean of three independent experiments and the error bars correspond to the standard error of the mean.

Figure n. 46

 46 Figure n. 46 CAF-1 acts in the Rhp51-dependent pathway. A) Scheme representing the two pathways of replication-induced recombination: Rad22-and Rhp51-dependent (left panel) or Rad22dependent and Rhp51-independent (right panel). Examples of RIs analysis by 2DGE in wt and rhp51d strains are shown on the bottom part. Note that, in the absence of Rhp51, D-loop intermediates are not present while HJs-like structures are still detectable. B) PFGE analysis of indicated strains in the presence of thiamine (OFF condition) or 24h and 48h after thiamine removal (ON conditions). The size of the chromosome III is of 3.5 Mb and the size of the acentric chromosome is of 1.2-1.5 Mb. C) Kinetic of acentric chromosome accumulation after thiamine removal in indicated RuraR strains. The values correspond to the mean of at least three independent experiments and error bars indicate the standard error of the mean.

Figure n. 47 :

 47 Figure n. 47: CAF-1 and Srs2 act in a same pathway of replication-induced recombination. A) PFGE analysis in indicated strains in OFF condition and in ON condition (48h after thiamine removal). The size of the chromosome III is of 3.5 Mb and the size of the acentric chromosome is of 1.2-1.5 Mb. B) Kinetic of acentric chromosome accumulation after thiamine removal in indicated RuraR strains. The values correspond to the mean of at least three independent experiments and error bars indicate the standard error of the mean.

Figure n. 48 :

 48 Figure n. 48: Genetic interaction between genes encoded CAF-1 subunits and rqh1. A) PFGE analysis of indicated strains in OFF condition and in ON condition (24h and 48h after thiamine removal) The size of the chromosome III is of 3.5 Mb and the size of the acentric chromosome is of 1.2-1.5 Mb. B) Kinetic of acentric chromosome accumulation after thiamine removal in indicated RuraR strains. The values correspond to the mean of at least three independent experiments and error bars indicate the standard error of the mean.

Figure n. 49 :

 49 Figure n. 49: CAF-1 stabilizes D-loop intermediates by counteracting the activity of Rqh1. A) Analysis of RIs by 2DGE analysis in indicated RuraR strains grown for 24 hours in the presence of thiamine (pause OFF) or not (pause ON). The red star indicates cross-linked DNA samples. C) Quantification of A. The values correspond to the mean of two independent experiments.

Figure n. 50

 50 Figure n. 50 CAF-1 promotes ectopic recombination induced by fork-arrest by counteracting the anti-recombinase Rqh1. A) Top panel: rate of ura4 loss assayed by 5-FOA resistance in indicated strains and conditions.Values correspond to the mean of three independent median rates (each one determined from 9 independent cultures using the median method). The error bars represent the standard deviation and the statistical significance was calculated using the non-parametric Mann Whitney test. Bottom panel: table representing the induction of ura4 loss by the RTS1-RFB in

Figure n. 51 :

 51 Figure n. 51: Absence of gene silencing after short induction of fork arrest by the RTS1-RFB. A) Serial ten-fold dilutions of indicated strains, previously grown in presence of thiamine (OFF condition), spotted onto complete YE and YE containing 5FOA plates. B) Serial ten-fold dilutions of indicated strains, previously grown in the absence of thiamine for 72 hours (ON condition), spotted onto complete YE and YE containing 5FOA plates. The liquid cultures in OFF and ON conditions, from which dilutions were obtained, were diluted every 24 hours to avoid cell saturation.

Figure n. 52 :

 52 Figure n. 52: Contribution of Swi6 to allelic homologous recombination A) Serial ten-fold dilutions of indicated strains, spotted on YE plates and exposed to increased doses of UV-C. B) Analysis of SCE: the rate of gene conversion and the rate of deletion is reported for indicated strains.The values represent the mean of three independent rates, each one being median of 11 independent cultures. The confidence interval is calculated at 95% (enclosed in bracket). Fold increase over wt is indicated for each strain. C) Analysis of recombination between homologous chromosomes: the rate of recombination events and rate of chromosome loss is reported for indicated strains. The values correspond to the mean of three independent rates, each one being median of 13 independent cultures. The confidence interval is calculated at 95% (enclosed in bracket) and the fold increase over wt is indicated for each strain.

  Finally, I analyzed the level of acentric chromosome in swi6-d RuraR cells, upon induction of the RTS1-RFB. In contrast to pcf1-d strain, swi6-d cells accumulated acentric chromosome to the same extent as the wt cells (Figure n. 53A and B). Therefore the deletion of swi6 gene does not mimic CAF-1 defects in replication-induced recombination, strongly suggesting that CAF-1 function in heterochromatin maintenance is not required to stabilize D-loop intermediates.

Figure

  Figure n. 53: The deletion of swi6 gene does not phenocopy the deletion of pcf1 gene in replication-induced recombination. A) PFGE analysis of wild-type, swi6-d and pcf1-d strains in OFF condition and in ON condition (after 24h and 48h of forkarrest induction). The chromosome III and the acentric chromosome were visualized using the rng3 probe. The size of the chromosome III is 3.5 Mb and the size of the acentric chromosome is 1.2-1.5 Mb. B) Kinetic of acentric chromosome accumulation after thaimine removal in indicated RuraR strains. The values represent the mean of the % of acentric chromosome, obtained in three independent experiments and error bars represent the standard error of the mean.

Figure n. 55 :

 55 Figure n. 55: Mutations in the PIP-box of Pcf1 abolished its interaction with PCNA, but not with Pcf2. A) Left panel: alignment of human p150, budding yeast Cac1 and fission yeast Pcf1. Conserved domains are represented in grey (PEST, E/D) and black (KER). Stars indicate PCNA binding site (PIP box). Right panel: PIP-box consensus sequence (in green) and PIP-box sequence in pcf1 + (in red) and its mutation in pcf1-PIP*.In green, hydrophobic "h" and aromatic "A" amino acids are defined. B) Immunoprecipitation of Pcf1-YFP, harbouring or not the mutation in the PIP-box sequence, was performed using an anti-GFP antibody. C) Immunoprecipitation of Pcf2-MYC, harbouring or not the mutation in the PIP-box sequence of Pcf1, was performed using an anti-MYC antibody. The Input for α-MYC is absent because the concentration of the Pcf2-MYC was not enough to be detected by western blot. D) Immunoprecipitation of Pcf2-MYC, harbouring or not the mutation in the PIP-box sequence of Pcf1, was performed using an anti-MYC antibody. The Input for α-MYC is absent because the concentration of the Pcf2-MYC was not enough to be detected by western blot. Subsequent western blot revelation of Pcf1, Pcf2 and PCNA was achieved via anti-GFP, anti-MYC, and anti-human PCNA antibodies, respectively.

Figure n. 56 :

 56 Figure n. 56: The interaction between PCNA and Pcf1 is required to promote replicationinduced recombination. A) PFGE analysis of the wild-type, pcf1-PIP* and pcf1-d strains in OFF condition and in ON condition (48h after thiamine removal). The chromosome III and the acentric chromosome were visualized using the rng3 probe. The size of the chromosome III is 3.5 Mb while the size of the acentric chromosome is 1.2-1.5 Mb. B) Kinetic of acentric chromosome accumulation after thiamine removal in indicated strains. The values represent the mean of the % of acentric chromosome, obtained from at least three independent experiments and the error bars indicate the standard error of the mean. C) 2DGE analysis of replication intermediates within the AseI-restriction fragment in indicated wt RuraR and pcf1-PIP* RuraR strains, upon induction of fork-arrest. All DNA samples were cross-linked.

Figure n. 57 :

 57 Figure n. 57: Pcf1 promotes ectopic recombination induced by fork-arrest via its interaction with PCNA. A) Top panel: Rate of ura4 loss assayed by 5-FOA resistance in wild-type, pcf1-d and pcf1-PIP*. The rate of ura4 loss was determined doing the mean of the media obtained from at least three different experiments (9 cultures were analyzed by fluctuation analysis for each experiment). Error bars define standard deviation and statistical significance was calculated using the nonparametric Mann Whitney test. Bottom panel: table reporting the induction of ura4 loss by the RTS1-RFB (rate of ura4 loss in ON conditions/ rate of ura4 loss in OFF conditions) in indicated strains. Decrease over the wild-type is reported. B) Examples of PCR analysis of 5-FOA R colonies obtained followed rtf1 induction (ON conditions) in pcf1-PIP* strains. The primers used are: TLII and TLIII, Rng3 and Ura4.

  Rhp51-dependent pathway and by counteracting the anti-recombinase activity of Rqh1, I tested if inactivation of CAF-1 subunits could also rescue the very slow growth phenotype of srs2-d rqh1-d. Therefore, triple mutant srs2-d rqh1-d pcf1-d and srs2-d rqh1-d pcf2-d were created and their ability to support cell growth was analyzed. As expected, both pcf1 and pcf2 deletions suppress the slow growth phenotype of srs2-d rqh1-d, but not to the same extent as the deletion of rhp51 (Figures n. 58A-D). Indeed, the generation time of srs2-d rqh1-d pcf1-d or srs2-d rqh1-d pcf2-d was of 6.7 hours, compared to 5.7 hours for srs2-d rqh1-d rph51-d.These data provide further strong evidences that CAF-1 complex acts in the same pathway than Rhp51 and that CAF-1 affects the stability of joint molecules produced by Rhp51.

Figure n. 58 :

 58 Figure n. 58: The deletions of pcf1 or pcf2 rescue the slow growth phenotype of srs2-d rqh1-d. A) Genetic interaction with pcf1. Indicated strains were streaked on plates containing EMM-Glu media. B) Genetic interaction with pcf2. Indicated strains were streaked on plates containing EMM-Glu media. C-D) Deletion of pcf1 or pcf2 does not rescue the slow growth phenotype of srs2-d rqh1-d, to the same extent as the deletion of rhp51. D) Generation time in hours of indicated strains.

Figure n. 59 :

 59 Figure n. 59: Two hypothesis to explain CAF-1 role in promoting fork arrest-induced chromosomal rearrangements. A) CAF-1 might increase fork restart on the initial template to the detriment of template exchange. B) CAF-1 might have a direct role in promoting template exchange of nascent strands.

  3. a direct role for CAF-1 in the stabilization of JMs. The data I obtained during the investigation of the three hypotheses mentioned above, lead to propose that CAF-1 has a direct role in homologous recombination mechanisms: CAF-1 promotes homologous recombination by counteracting the dissolution of D-loop intermediates by Rqh1 (Figure n. 60). Indeed, CAF-1 could be recruited via its interaction with PCNA, to promote histones deposition during extension of the D-loop by DNA synthesis. Histones deposition might then stabilize D-loop intermediates by preventing their dissolution by the helicase Rqh1. Thus, by counteracting Rqh1 function, CAF-1 might be part of an equilibrium regulating stabilization/dissociation of early recombination intermediates.

Figure n. 60 :

 60 Figure n. 60: A model for D-loops stabilization by CAF-1 dependent nucleosome assembly. CAF-1 is recruited via the interaction between Pcf1 and PCNA during D-loops extension, to promote the chromatinization of early recombination intermediates (D-loops). Doing so, CAF-1 counteracts Rqh1 helicase activity and prevents the dissolution of D-loop intermediates.

  . It has also been demonstrated that the double mutant srs2-d rqh1-d presents a very slow growth phenotype, probably due to the accumulation of toxic recombination intermediates. Indeed, this phenotype is rescued by the deletion of rhp51, rhp55 or rhp57 preventing the formation of the D-loop (Doe and Whitby 2004-Figure n. 58C). Deletion of pcf1 or pcf2 rescues this slow growth phenotype as well (Figure n. 58C). These data support the view that CAF-1 acts in the same pathway than Rhp51, stabilizing the D-loop and promoting the formation of recombination intermediates that cannot be dissolved by helicases in the srs2-d rqh1-d strain.

  possible explanation for MMS resistance of pcf1-d or pcf2-d or pcf3-d strains could be that Rad51-dependent joint molecules are not stable in the absence of CAF-1, leading to a decrease in the amount of toxic recombination intermediates. These toxic recombination intermediates are normally dissolved by Rqh1. An experiment to demonstrate that the resistance of CAF-1 defective strains to MMS is dependent on Rqh1 activity will be to test the the sensitivity to MMS of double mutants pcf1-d rqh1-d and pcf2-d rqh1-d, A loss of resistance to MMS in these double mutants will provide further evidences of the interplay between Rqh1 and CAF-1.

  exploited by McIIwraith and colleagues in 2005, to study DNA polymerases involved in DNA synthesis from strand invasion intermediates(McIIwraith, Vaisman et al. 2005).To score JMs and the product of their resolution, I exploited the conditional fork arrest substrate RuraR at ura4 locus. To widen the knowledge horizons of CAF-1 involvement in Dloop stabilization, it will be of interest to create various RuraR constructs, in which the distance between RTS1 repeats will be decreased, to determine how much of DNA synthesis is needed to observe the stabilizer function of CAF-1. The length of the DNA wrapped around the nucleosome is about 147 bp and the length of the DNA linker between nucleosomes can be very different from region to region in the genome. Considering that the actual size between RTS1 repeats is 1.7 Kb, the maximal number of nucleosomes can be deposit on the D-loop is of 11. Modifying the distance between RTS1 repeats might be useful also to investigate if the ability of Rqh1 to dissociate D-loop is sensitive to the number of nucleosomes that are loaded, or if just one nucleosome might be enough to block its dissociative function.CAF-1 deposits preferentially newly synthesized histones on DNA and the mark of such histones is the acetylation of H3 on lysine 56 (H3K56Ac). Considering the importance of this mark during DNA replication and repair(Introduction 3.2.4 and 4.2), further perspectives could be to investigate the function of H3K56Ac in D-loop stabilization by CAF-1, using a strain expressing histones H3 that cannot be acetylated on lysine 56. Dr. Xemalce provided me of a strain in which H3.3 and H4.3 encoding genes are deleted by arg3, H3.1 and H4.1 encoding genes are deleted by his3 and the lysine 56 of histone H3.2 is mutated in arginine (H3.1/H4.

  which a variety of recombination mediators (Rad22, Rhp54, Rhp55-Rhp57, Rlp1-Rdlp1-Sws1) and negative regulators (Srs2, Fml1, Rqh1) act in a synergistic or antagonistic manner, to process Rad51 filament and D-loop intermediates, displacing the equilibrium of the balance toward different possible recombination pathways, such as DSBR and SDSA.Once the D-loop is formed, DNA polymerases delta and eta initiate DNA synthesis to elongate this early recombination intermediate and different mechanisms are proposed for the stabilization of the extended D-loop. During allelic recombination, the long sequence homology, allows CAF-1 independent stabilization mechanisms to occur, such as Rad51dependent branch migration[START_REF] Murayama | Formation and branch migration of Holliday junctions mediated by eukaryotic recombinases[END_REF]. Indeed, my data support a dispensable function for CAF-1 in supporting allelic recombination. The rate of recombination between homologous chromosomes and chromosome loss in strains defective for CAF-1 are the same than in the wild-type strain. Considering also the experiment to score spontaneous sister chromatid exchanges, all my data are coherent with the fact that CAF-1 is dispensable for allelic recombination, in S. pombe.The other side of the coin consists of the involvement of CAF-1 in ectopic (non-allelic) recombination. Non-allelic recombination occurs between dispersed repeated sequences in the genome, leading to aberrant outcomes threatening genome stability (Introduction 2.5.1).During ectopic recombination, the shorter length of homology between donor and acceptor DNA molecules renders Rad51-dependent branch migration impossible and the role of CAF-1 in D-loop stabilization might be crucial. These innovative data reveal a novel regulation level of homologous recombination mechanisms and outcomes (allelic versus non-allelic recombination), by CAF-1 dependent chromatin assembly, affirming the importance of CAF-1 in genome stability maintenance. Indeed, the role of CAF-1 in D-loop stabilization implies a channelling of the recombination pathways toward the DSBR model and double HJs formation, to the detriment of the synthesis-dependent strand annealing (SDSA) pathway. To confirm this speculation, it is envisageable to use a plasmid gap repair assay, which measures the repair of a linearized plasmid through recombination with homologous DNA on the chromosome. Exploiting this assay, it will be possible to evaluate how CAF-1 influences the rate of mitotic crossing-overs[START_REF] Orr-Weaver | Yeast recombination: the association between double-strand gap repair and crossing-over[END_REF] Sun, Nandy et al. 2008).Another implication of my model is that, during the switch of nascent strands from one template to another one, invoked for example during Micro-homology Mediate BIR (MMBIR -see Introduction 2.5.2), the deposition of nucleosomes onto newly synthesized strand, could constitute a physical barrier to the re-annealing of the filament with the initial template.It would have been interesting to dissect the role of CAF-1 during homologous recombination also in meiosis and not only in vegetative growing cells. Unfortunately S. pombe diploids, homozygous for pcf1-d or pcf2-d or pcf3-d present defects in meiosis leading to abnormal ascus morphology, abnormal number of spores containing in the ascus and severely reduced spore viability (data not shown;[START_REF] Dohke | Fission yeast chromatin assembly factor 1 assists in the replication-coupled maintenance of heterochromatin[END_REF]. Thus, I concentrated on mitotic recombination in vegetative cells, where the deletion of pcf1 or pcf2 or pcf3 genes do not present any significant growth phenotype, except for their sensitivity to the nitrogen source.

Figure 1 .

 1 Figure 1. Conditional replication fork-arrest assays. A. Diagrams of fork-arrest constructs. Centromere-proximal and telomere-proximal regions are represented in black and grey, respectively. Strong or putative replication origins (ori) and the centromere are indicated by yellow, green and black circles, respectively. Blues arrows indicate the polarity of the RTS1-RFB. The ura4 + gene is indicated in red and the arrow indicates its direction of transcription.Representations of the primary arrested fork structure are given for each construct. The name of each fork-arrest construct is given using the following nomenclature: ''t'' and ''ori'' refer to the telomere and the replication origin 3006/7, respectively; '','' and ''.'' indicate the RTS1barrier and its polarity (, blocks replication forks moving from the ori3006/7 towards the telomere, and . blocks replication forks moving from the telomere towards the origin 3006/7. B. Diagrams of replication intermediates (RIs) within the AseI fragment analysed by 2DGE (top panel). Representative RIs analysed by 2DGE in indicated strains in OFF (Rtf1 being repressed) and ON (Rtf1 being expressed) conditions. Signal corresponding to arrested forks, joints-molecules (JMs) and termination structures are indicated by black, red and green arrows, respectively. Note that the t.ura4-ori construct does not result in a strong fork arrest as the RTS1-RFB is not orientated in the main direction of replication (see text for details). doi:10.1371/journal.pgen.1002976.g001

Figure 2 .

 2 Figure 2. A single fork-arrest induces GCRs that are stimulated by inverted repeats near the site of fork arrest. A. Diagrams of chromosome II containing or not the RTS1 sequence (blue arrow or RTS1-d) and of chromosome III containing ura4 + alone or associated with RTS1-RFB constructs. The RTS1 sequence maps near the mat1 locus where it helps to ensure unidirectional replication [62]. Primers used for amplifying the 1 Kb ura4 fragment or the 650 bp rng3 fragment are depicted in red and grey, respectively. Primers used to amplify the translocation junction (1.2 kb) are represented in orange on chromosome II (TLII) and in black on chromosome III (TLIII). B. Representative PCR-amplifications from 5-FOA R colonies of the indicated strains; ON and OFF refers to the RTS1-RFB being active or not, respectively. PCR products and their sizes are indicated on the figure. C. Effect of intra-and inter-chromosomal recombination between RTS1 repeats on fork-arrest-induced genomic deletion. RTS1-RFB activity and ura4 location with respect to the RFB are given for each construct. The % of deletion events, as determined by the PCR assay, was used to balance the rate of ura4 loss. Then, the RFB-induced deletion rate was calculated by subtracting the rate obtained in the presence of thiamine (Rtf1 being repressed) from the rate obtained in the absence of thiamine (Rtf1 being expressed). The values reported are means of at least 3 independent median rates. Error bars correspond to the standard error (SE). D. Effect of Rqh1 on RFB-induced deletions (left) and translocations (right), as described for panel C. Error bars indicate SE. Statistically significant fold differences between the rqh1-d and the wild-type strains are indicated with an *. E. Representative PCR amplifications from 5-FOA R colonies of the rqh1-d t-ura4,ori strain, as described for panel B. (Refer to Figure S1 for corresponding rates of deletion and translocation when Rtf1 is expressed or not). doi:10.1371/journal.pgen.1002976.g002

Figure 3 .

 3 Figure3. Fork-arrest induces mutations. A. Effect of intra-and inter-chromosomal recombination between RTS1 repeats on fork-arrest-induced mutation rates (base-substitutions, frame-shifts and small insertions or deletions between short tandem repeats). RTS1-RFB activity and ura4 location with respect to the RFB are given for each construct. The % of mutation events, as determined by the PCR assay and sequencing, was used to balance the rate of ura4 loss. Then, the RFB-induced mutation rate was calculated by subtracting the rate obtained in the presence of thiamine (Rtf1 being repressed) from the rate obtained in the absence of thiamine (Rtf1 being expressed). The values reported are means of at least 3 independent median rates. Error bars correspond to SE. (Refer to FigureS2for corresponding rates of mutation when Rtf1 is expressed or not). B. Rate of mutation for indicated strains; ON and OFF refers to the RTS1-RFB being active or not, respectively. The % of mutation events, as determined by the PCR assay and sequencing, was used to balance the rate of ura4 loss. The values reported are means of at least 3 independent median rates. Error bars correspond to SE. Statistically significant fold differences in mutation rates between the ''OFF'' and ''ON'' conditions are indicated with an *. C. Spectra of mutation events in indicated strains upon RFB induction (refer to Table2for exact numbers and to FigureS3for mapping of deletions/duplications and their features). D. Strains harbouring the ura4 alleles with a single base-substitution or frame-shift or duplication of 20 or 22 nt, together with the RTS1-RFB in the t-ura4,ori configuration were streaked onto the indicated media after cell growth with (RFB ''OFF'') or without (RFB ''ON'') thiamine. The bottom diagram indicates strain positions and the mutation events required to obtain Ura + revertants. E. PCR analysis of Ura + revertants isolated from the t-ura4-dup20,ori strain (duplication of 20 nt in ura4) after RFB induction. With the primers used, a 106 bp fragment is amplified from the ura4 + strain and a 126 bp fragment is amplified from the t-ura4-dup20,ori strain. F. Sequence alignments of ura4-dup22, ura4-dup20, ura4 + alleles and corresponding Ura + revertants. Micro-homologies are indicated in grey and duplicated sequences are underlined in black. The phenotype of each allele is indicated on the figure. G. Map of deletion and duplication events within the ura4 ORF. doi:10.1371/journal.pgen.1002976.g003

Figure 4 .

 4 Figure 4. Fork recovery by homologous recombination results in replication slippage. A. Serial tenfold-dilutions from indicated strains (t-ura4-dup20-ori associated or not with the RTS1-RFB) were spotted onto the indicated media after cell growth with (Rtf1 -, repressed) or without (Rtf1 +, expressed) thiamine. RTS1-RFB activity is given for each construct and condition. B. Frequency of Ura + revertants in indicated strains after cell growth with (Rtf1 repressed) or without (Rtf1 expressed) thiamine. The RTS1-RFB activity is given for each construct and condition. Values correspond to the mean of at least three independent experiments and error bars correspond to the standard error of the mean (SEM). C. Rate of replication slippage in the indicated strains and conditions. The rate of Ura + revertants was calculated using the method of the median from at least 11 independent cultures. Values in brackets indicate the 95% confidence interval. Statistical significance was detected using the nonparametric Mann-Whitney U test. D. Serial tenfold-dilutions from the strains indicated spotted onto the media indicated after cell growth without thiamine. RTS1-RFB activity ''-'' refers to the strain t-ura4-dup20-ori and ''+'' refers to the strain t-ura4-dup20,ori. E. Kinetics of Ura + revertants frequency for the strains indicated as a function of the number of generations after thiamine removal. RTS1-RFB activity ''-'' refers to the strain t-ura4-dup20-ori and ''+'' refers to the strain t-ura4-dup20,ori. The values reported are the means of two experiments. F. The rate of replication slippage/generation for the strains indicated with (t-ura4-dup20,ori) or without (t-ura4-dup20-ori) the active RTS1-RFB. The rate was calculated from the slope of the curves presented in panel F. The values reported are means of three independent experiments and error bars correspond to SE. doi:10.1371/journal.pgen.1002976.g004

Figure 5 .

 5 Figure 5. Collapsed forks, but not stalled forks, induce replication slippage. A. Left panel: the frequency of Ura + revertants as a function of time-contact with indicated drugs for the indicated ura4 alleles (single base-substitution, frame-shift, duplication of 20 nt). Right panel: the frequency of Ura + revertants in response to UV-C irradiation as a function of dose for the indicated ura4 alleles. The values reported are means of two independent experiments. Numbers indicate fold difference in the frequency of Ura + revertants between the treated and untreated control samples. For ura4 alleles containing base-substitutions or frame-shifts, the mutation event required to obtain Ura + revertants is indicated on the figure. B. Serial tenfold-dilutions from ura4-dup20 strain spotted onto the media indicated after treatment with MMC or CPT as indicated. C. Frequency of Ura + revertants after the indicated treatments in the ura4-dup20 strain. DMSO (the vehicle) was used as control for CPT treatment. The values reported are means of at least three independent experiments. Error bars correspond to SEM. doi:10.1371/journal.pgen.1002976.g005

Figure 6 .

 6 Figure 6. Model of replication-stress-induced genetic instability at collapsed forks. Collapsed forks might arise from torsional stress, fork breakage (i.e. at nick, ICLs), or proteins tightly-bound to DNA. Replisome disassembly at collapsed forks may favour the unwinding of the nascent strand on which Rad51 nucleates. At this initial stage of fork resumption by recombination, homology-driven template exchange can promote intraor inter-recombination resulting in GCRs. Fork recovery by recombination overcomes the initial replication block and allows an inaccurate replisome to form (see text for details). doi:10.1371/journal.pgen.1002976.g006

Table n .4 -List of strains used in this study

 n

	VP111 VP338		h-pcf3::Kan, rtf1nmt41::sup35, ade6-704, leu 1-32, RTS1b:ura4+ h-, pcf1-PIPboxMut, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+	This study This study
	VP114 VP340		h-pcf2::Kan, rtf1nmt41::sup35, ade6-704, leu 1-32, RTS1b:ura4+ P Δ17::leu2, ura4-D18, leu1-32	This study Arcangioli
	VP120 VP346		h-smt0, pcf1::Kan, pcf2::Kan, pcf3::Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ M 2,3 Δ::leu2, ade6 M-210, ura4-D18, leu1-32	This study Arcangioli
	VP133 VP354		h-smt0, pcf2::Kan, rhp51::Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-, pcf1::Kan, rqh1::Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+	This study This study
	SL 917 VP358		h-smt0, ura4-D18, ade6 M-375 int:puc8/ura4+/ade64-69 h -, pcf1::ura4+, slm9::Kan, leu1-32, ura4-D18	Hartsuiker This study
	VP152 VP385		h-smt0, pcf1::Kan, ura4-D18, ade6 M-375 int:puc8/ura4+/ade64-69 pcf1::Kan, P Δ17::leu2, ura4-D18, leu1-32, ade6 M-216	This study This study
	VP155 VP388	Legend: RTS1b -> RTS1 sequences were integrated on both sides of ura4 locus h-smt0, pcf2::Kan, ura4-D18 ade6 M-375 int:puc8/ura4+/ade64-69 h-smt0, pcf1-PIPboxMut:YFP-Kan, ade6-704, leu1-32, ura4-D18	This study This study
	VP158 VP409		RTS1C -> RTS1 sequence was integrated at the centromeric side of ura4 locus h-smt0, pcf3::Kan, ura4-D18 ade6 M-375 int:puc8/ura4+/ade64-69 h-smto, pcf2-Myc (4.5rep)-KanR ade6-704, leu 1-32, ura 4-D18	This study This study
	Strain name VP161 VP394	Genotype h-smt0, swi6::Nat, ura4-D18 ade 6M-375 int:puc8/ura4+/ade64-69 h-smt0, ade6-704, leu1-32, ura4-D18, pcf1:YFP-Kan	Notes This study This study
	VP001 VP186 VP425		h-smt0, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-smt0, ade6 M-210, ura4-D18/ade6 M-216 h-smto, pcf2-Myc(4.5rep)-KanR, pcf1PIPbox-YFP-KanR, ade 6-704, leu 1-32, ura 4-D18	Lambert Hartsuiker This study
	VP002 VP190 VP426		h-smt0, rad22::Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-smt0, pcf1::Kan, ade6 M-210, ura4-D18/h-smt0, pcf1::Kan, ade6 M-216 h-smto, pcf2-Myc(4.5rep)-KanR, pcf1-YFP-KanR, ade 6-704, leu 1-32, ura 4-D18	Lambert This study This study
	VP003 VP198 SL80		h+, pcf1::Kan, ura4-D18, leu1-32, ade6 M-216 h-smt0, pcf2::Kan, ade6 M-210, ura4-D18/h-smt0, pcf2::Kan, ade6 M-216 h+ rqh1::kan ade6-704 leu1-32 ura4D18	Francesconi This study Lambert
	VP005 VP203 SL279		h+, pcf2::Kan, ura4-D18, leu1-32, ade6 M-216 h-smt0, pcf3::Kan, ade6 M-210, ura4-D18/h-smt0, pcf3::Kan, ade6 M-216 h-smt0 rhp51::Kan rtf1nmt41::sup35 ade6-704 leu1-32 RTS1b::ura4 +	Francesconi This study Lambert
	VP007 VP208 SL294		h+, pcf3::Kan, ura4-D18, leu1-32, ade6 M-216 h-smt0, swi6::Nat, ade6 M-210, ura4-D18/h-smt0, swi6::Nat, ade6 M-216 h-rad22GFP::Kan rtf1 nmt41::sup35 ade6-704 leu1-32 RTS1b::ura4 +	Francesconi This study Lambert
	VP009 VP217 SL511		h-smt0, pcf2::Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-, ura4:pECFP-pcna, pcf1:YFP-Kan, ura4-D18 h-smt0 rqh1::Kan rtf1nmt41::sup35 ade6-704 leu1-32 RTS1b::ura4 +	This study This study Lambert
	VP015 VP 219 SL350		h-smt0, pcf1::Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-smt0, pcf2::Kan, rad22:GFP-Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-rtf1nmt41::sup35 ade6-704 leu1-32 RTS1C::ura4 +	This study This study Lambert
	VP016 VP229		h-smt0, pcf3::Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-smt0, pcf1::Kan, rad22:GFP-Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+	This study This study
	VP018 VP232		h-, ade6-704, leu1-32, ura4-D18 h+, pcf1:YFP-Kan, leu1-32, ura4-D18, ade6 M-216	Lambert Meister
	VP020 VP236		h+, rad3::ura4+ , ade6-704, leu1-32, ura4-D18 h-smt0, pcf1::Kan, rqh1::Kan, rtf1nmt41::sup35, ade6-704, leu 1-32, RTS1b:ura4+	Lambert This study
	VP021 VP241		h+, rad22:GFP-Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-smt0, pcf2::Kan, rqh1::Kan, rtf1nmt41::sup35, ade6-704, leu 1-32, RTS1b:ura4+	Lambert This study
	VP023 VP244		h-smt0, lsd1-W89IA-HA::Kan, ade6-704, leu1-32, ura4-D18 h-, hip::Kan, leu1-32, ura 4 D-18, lys1	Arcangioli Saitoh
	VP024 VP245		h-mgs1::ura4+, leu1-32, ade6-704, ura4-D18 h+, pcf1:YFP-Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+	Lambert This study
	VP036 VP256		h-smt0 swi6::Nat, ade6-704, leu1-32, ura4-D18 h-smt0, pcf1:YFP-Kan, pcf2::ura4+, leu1-32, ade6-704	This study This study
	VP045 VP259		h-smt0, pcf3::Kan, rad22:GFP-Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-smt0, pcf1:YFP-Kan, pcf3::Kan, leu1-32, ade6-704	This study This study
	VP050 VP266		h-smt0, pcf1::Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1C:ura4+ h-smt0, srs2::Nat, pcf1::ura4+, ade6-704, leu 1-32, ura4-D18	This study This study
	VP052 VP270		h-smt0, pcf2::Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1C:ura4+ h-smt0, srs2::Nat, pcf2::ura4+, ade6-704, leu1-32, ura4-D18	This study This study
	VP056 VP272		h-smt0, pcf3::Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1C:ura4+ h-, slm9::Kan, leu1-32, ura4-D18, lys1	This study Saitoh
	VP069 VP290		h-smt0, swi6::Nat, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-smt0, rqh1::Kan, pcf2::ura4+, ade6-704, leu1-32, ura4-D18	This study This study
	VP077 VP294		h-smt0, pcf2::Kan, srs2::Nat, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-smt0, srs2::Nat, rqh1::Kan, ade 6-704, leu1-32, ura4-D18	This study This study
	VP087 VP300		h-smt0, pcf1::Kan, srs2::Nat, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-smt0, srs2::Nat, rqh1::Kan, pcf1::ura4+, ade6-704, leu1-32, ura4-D18	This study This study
	VP094 VP307		h-smt0, pcf1::Kan, rad22::Nat, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-smt0, pcf1-PIPboxMut, rtf1nmt41::sup35, ade6-704, leu 1-32, RTS1b:ura4+	This study This study
	VP098 VP313		h-smt0, pcf2::Kan, rad22::Nat, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+ h-smt0, pcf1::Kan, rhp51::Kan, rtf1nmt41::sup35, ade6-704, leu1-32, RTS1b:ura4+	This study This study
	VP103 VP316		h-smt0, pcf1::Kan, rtf1nmt41::sup35, ade6-704, leu 1-32, RTS1b:ura4+ h-smt0, rqh1::Kan, pcf1::ura4+, ade6-704, leu1-32, ura4-D18	This study This study
	VP109 VP 320		h-pcf1::Kan, rtf1nmt41::sup35, ade6-704, leu 1-32, RTS1b:ura4+ h+ srs2::Nat, rqh1::Kan, pcf2::ura4+, ade6-704, leu1-32, ura4-D18.	This study This study
			100 101

Table 1 .

 1 Rates of ura4 loss (including genomic deletion, translocation, and mutation events), calculated using the method of the median. the following nomenclature is used: ''t'' and ''ori'' refer to the telomere and the replication origin 3006/7, respectively; '','' and ''.'' indicate the RTS1-RFB and its polarity (, blocks replication forks moving from the ori3006/7 towards the telomere, and . blocks replication forks moving from the telomere towards the origin 3006/ 7). b event/cell/division 610 28 6 standard error. Values are means of at least 3 independent rates.

				Fold induction	Fold induction by
	strains a	Rate of ura4 loss b		by Rtf1 expression (2/+ thiamine) c	the RFB (relative to the t-ura4-ori construct)
		+ thiamine	2 thiamine
		(Rtf1 repressed)	(Rtf1 expressed)
	t-ura4-ori	4.661	4.361.8	0.9
	t-ura4,ori	4.160.9	13.962.8	3.4 (p = 0.002)	3.2
	t-ura4,ori RTS1-d	4.961.4	11.464.1	2.3 (p = 0.03)	2.6
	t,ura4-ori	3.961.3	4.862.5	1.2	1.1
	t.ura4,ori	5.862.1	97.4651.5	16.8 (p = 0.004)	22.6
	t.ura4,ori RTS1-d	4.962.2	19.563.3	4 (p = 0.006)	4.5
	t.ura4-ori	3.261.7	3.661.8	1.1	0.8
	t,ura4.ori	6.661.7	50.3614.2	7.6 (p = 0.014)	11.7

a c statistical significance was determined using the nonparametric Mann-Whitney U test. doi

:10.1371/journal.pgen.1002976.t001

Table 2 .

 2 Mutations spectra in the indicated strains.

		t-ura4-ori a		t.ura4-ori		t-ura4,ori		t.ura4,ori		t,ura4-ori		t-ura4,ori rad22-d
	Rtf1 expression	2	+	2	+	2	+	2	+	2	+	2	+
	Fork arrest at ura4	2	2	2	2	2	+	2	+	2	+	2	+
	Transitions b
	GCRAT	5			1	7	14	6	2		5	5	5
	ATRG C	3		1	2	4	9	1	4	4	7	4	6
	Transversions b
	GCRT A	3	5	1		4 5	2 0	5	2	4	2	3	4
	GCRC G	1	3	4	2	2	3	2			1	8	5
	ATRC G	3	3	1	1	6	4	2	2		1	4
	ATRT A		1	5	3	4	2	4	3	3	1	2	3
	Rate of base	4.661.1	3.761.5	3.361.6	4.261.3	3.460.9	6.461.2 d 2.661.1	6.561.5 d 2.561.3	3.361.7	115634	152633
	substitutions c
	+ Frame-shift b :
	in homo-nt		1							1
	in mono-nt		1	2	1	2	1	1		1		1
	-Frame-shift b :
	in homo-nt					1	4		1			1	2
	in mono-nt					1	3			1			1
	Rate of frame-shift c		0.660.25	0.560.3	0.560.1	0.260.06	160.2 d	0.160.06	0.560.1	0.760.4		8.762.6	20.464.4
	Del/Dupl b :
	Deletions					9	27		4		1		2
	Duplications					5	12
	Rate of Del/Dupl c		,0.3		,0.5	0.760.2	4.961	,0.1	2.160.5	,0.2	0.260.1	,3.7	1362.8
	Other (complexe)					1
	Total	15	14	14	10	87	99	21	18	14	18	28	28

PLOS Genetics | www.plosgenetics.org

October 2012 | Volume 8 | Issue 10 | e1002976

ACKNOWLEDGMENTS

Acknowledgments

We thank members of Lambert's lab, Roland Chanet, Tony Carr, and Ted Weinert for helpful discussions and comments on the manuscript. We thank Sylvain Martineau, Joe ¨l Blaisonneau, and Elodie Olivier for technical assistance.

This work was supported by the Institut Curie, the CNRS, ANR grants ANR-Piribio09-44854 and ANRJCJC10-1203 01, and La Ligue Contre le Cancer (comite ´Essonne) to SAEL. VP was funded by the ITN ''Image DDR'' PITN-GA-2008-215148. II was funded by the Fondation pour la Recherche Me ´dicale (FRM) and AC by l'Association pour la Recherche sur le Cancer (ARC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

incubation for 12-16 hours in thiamine-free media. By analyzing replication intermediates by native 2-dimensional gel electrophoresis (2DGE), it was reported that more than 95 % of forks moving from ori 3006/7 toward the telomere (tel) are efficiently blocked by the RTS1-RFB at the uraR locus. Arrested forks were not detected without Rtf1 induction . each subunit, independently of CAF-1. Therefore, further experiments were performed using the three single deleted strains: pcf1-d, pcf2-d or pcf3-d. Total protein extracts from strains pcf1 + :YFP, pcf1 + , pcf1 + :YFP pcf2-d and pcf1 + :YFP pcf3-d were analyzed by western-blot. Pcf1-YFP (89.2 kDa) was detected using an anti-GFP antibody and PCNA (28.9 kDa) was detected using an anti-human PCNA antibody. B) The expression level of Pcf1-YFP and PCNA were estimated using ImageJ software. The ratio between Pcf1-YFP and PCNA expression level in pcf1 + :YFP was considered as 1 and the expression of Pcf1-YFP in pcf1 + :YFP pcf2-d and pcf1 + :YFP pcf3-d was quantified relative to that of pcf1 + :YFP.

Strains defective for CAF-1 are sensitive to the nitrogen source

To investigate the role of CAF-1 on cell growth and fitness, I studied the ability of strains defective for CAF-1 to grow on different culture media. I compared the growth parameters of single pcf1-d, pcf2-d or pcf3-d null mutant with a wild-type (wt) strain.

I tested both liquid and solid rich media (YE) and two minimal media containing different nitrogen sources: ammonium chloride (EMM-NH 4 Cl) and glutamate (EMM-Glu). These media were selected as they are commonly used to grow the yeast S. pombe, but also because my further experiments have required the use of conditional addition of particular substances, such as thiamine, which is already present in rich media. I observed that, in both YE and EMM-Glu, the doubling time for each strain defective for CAF-1 is comparable to the doubling time of the wt strain (around 3 hours) (Figure n. 29A,B and D). In contrast, the three strains (pcf1-d, pcf2-d and pcf3-d) exhibited a very slow growth phenotype on agar plate containing EMM-NH 4 Cl and were unable to divide on liquid media containing EMM-NH 4 Cl (Figure 29C). These data showed that the absence of CAF-1 does not affect cell cycle length, but renders cells sensitive to the nitrogen source. I first supposed that the absence of CAF-1 might lead to a defect in nitrogen metabolism and/or in the transporters used to import ammonium chloride. This might be linked to a role of CAF-1, directly or indirectly, in transcription processes [START_REF] Kim | Potential role of the histone chaperone, CAF-1, in transcription[END_REF][START_REF] Heyd | The p150 subunit of the histone chaperone Caf-1 interacts with the transcriptional repressor Gfi1[END_REF] to wt strain. These data show that CAF-1 affects JMs formed between RTS1 sequences, upon fork arrest, either by promoting their formation or by promoting their stability. Furthermore, as the strains deleted for each of the three subunits exhibit the same phenotype, then the role of CAF-1 in promoting or stabilizing JMs is likely related to its histone chaperone function.

CAF-1 stabilizes Rhp51-dependent Joint-molecules (D-loop)

In the RuraR system, template exchange occurs by two pathways:

-A Rad22-and Rhp51-dependent pathway, resulting in D-loop and HJs-like structures formation, -A Rad22-dependent but Rhp51-independent pathway, resulting in HJs-like structures without D-loop formation [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF].

Indeed, the rhp51-d strain does not exhibit the signal corresponding to JM-A (D-loop), while

CAF-1 stabilizes D-loop structures by counteracting their dissociation by Rqh1.

Knowing that CAF-1 affects replication-induced recombination by acting on the Rhp51 pathway, I hypothesized that CAF-1 might regulate the stability of D-loop structures by preventing their dissolution by DNA helicases, such as Srs2 or Rqh1. If my hypothesis was correct, deletion of helicase genes could rescue the instability of JMs observed in the absence of CAF-1. Both Srs2 and Rqh1 are DNA helicases proposed to act during early steps of homologous recombination, by either dismantling the Rad51 filament or by dissolving D-loop intermediates, thus promoting SDSA pathway [START_REF] Fabre | Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication[END_REF][START_REF] Aylon | Molecular dissection of mitotic recombination in the yeast Saccharomyces cerevisiae[END_REF][START_REF] Ira | Srs2 and Sgs1-Top3 suppress crossovers during doublestrand break repair in yeast[END_REF][START_REF] Robert | Mrc1 and Srs2 are major actors in the regulation of spontaneous crossover[END_REF][START_REF] Oh | BLM ortholog, Sgs1, prevents aberrant crossing-over by suppressing formation of multichromatid joint molecules[END_REF][START_REF] Oh | RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, resolve aberrant joint molecules during meiotic recombination[END_REF][START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF]see Introduction 2.3.2). In addition, Rqh1 plays a crucial role in preventing mitotic cross-overs by promoting the dissolution of double Hollidayjunctions [START_REF] Doe | Partial suppression of the fission yeast rqh1(-) phenotype by expression of a bacterial Holliday junction resolvase[END_REF].

To investigate genetic interactions between srs2 and pcf1 or pcf2, I created pcf1-d srs2-d and To further support this conclusion, I analyzed the genetic interaction between pcf1 and rqh1 in ectopic recombination induced by the RTS1-RFB at the RuraR locus. As previously reported [START_REF] Lambert | Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange[END_REF], the induction of ura4 loss by fork-arrest was 3 times higher in Nonetheless, deletion of pcf1 did not fully abolish the high level of ura4 loss observed in rqh1-d strain, especially regarding the level of genomic deletion. Therefore, Rqh1 has additional functions in preventing genome instability at arrested forks, not counteracted by CAF-1.

The interaction between PCNA and Pcf1 is required to stabilize D-loop intermediates

During DNA replication, CAF-1 is targeted to replication foci through the interaction between the largest subunit of CAF-1 with PCNA. Moreover, the histone deposition function of CAF-1 requires its recruitment at the replication fork, through its interaction with PCNA [START_REF] Moggs | A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage[END_REF][START_REF] Krawitz | Chromatin assembly factor I mutants defective for PCNA binding require Asf1/Hir proteins for silencing[END_REF][START_REF] Ben-Shahar | Two fundamentally distinct PCNA interaction peptides contribute to chromatin assembly factor 1 function[END_REF]). The inability of CAF-1 to interact with PCNA results in an inability to promote nucleosomal assembly in vitro [START_REF] Krawitz | Chromatin assembly factor I mutants defective for PCNA binding require Asf1/Hir proteins for silencing[END_REF].

Firstly, I confirmed that CAF-1 is targeted to replication foci in S. pombe (Figure n. 54A and B). For this purpose, I used a strain in which PCNA was already tagged with a cyan fluorescent protein (PCNA-CFP) [START_REF] Meister | Nuclear factories for signalling and repairing DNA double strand breaks in living fission yeast[END_REF] p150 could switch its function toward an active histone chaperone mode, together with the other subunits of CAF-1 complex.

In an effort to demonstrate that Pcf1 is recruited on D-loop intermediates, I investigated the recruitment of Pcf1 at the RuraR locus, by performing ChIP analysis using a strain expressing Pcf1-YFP. Unfortunately, I was not able to achieve my purpose maybe because the recruitment of Pcf1 at a specific locus (RuraR), in specific conditions (fork arrest system ON) and in an asynchronous population, is below the resolution limit of the ChIP technique.

Therefore, I focused on the possibility to detect a co-localization between the fluorescently tagged RuraR locus and Pcf1-YFP. Indeed, Dr. Costes, developed strains in which the RuraR locus is fluorescently tagged: LacO sequences were integrated 7.9 Kb away from ura4 locus. Despite that D-loop stabilization by CAF-1 requires its interaction with PCNA, this does not provide evidences that histones are indeed deposited onto extended D-loop. A caveat of my work was the difficulty to create a strain in which CAF-1 does not interact with histones. Indeed, CAF-1 interacts with H3-H4 dimers via the three subunits and there are no point mutations, described in the literature, that allow abolishing CAF-1 interaction with H3/H4. Furthermore, there are not clear mapped region of interaction between the different CAF-1 subunits and the dimer H3-H4. To investigate nucleosome assembly on D-loop intermediates, a possible option is a biochemical approach: synthetic D-loop substrates could be incubated

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Italian -Mother tongue.

English -Optimal level for spoken and written English. -Training in using different kinds of microscope for in vivo imaging and techniques of deconvolution and de-noising. -Training in practical DNA combing and interpretation of results. -Theorical lessons on the mathematical basis of microscopy and on how to build a microscope.

October -July 2009:

Stage at Pr. Russo's laboratory on "Characterization of replication pattern at the frataxin region (9q13)" at the University of Padua (Italy).

-Use of molecular combing to study the DNA replication at 9q13 in lymphoid cells, to analyse particular features that could lead to the development of Friedreich Ataxia.

May -June 2007:

Stage at Pr. Rigoni's laboratory on " Heterologue expression, purification and activity essay of a Fe-Hydrogenase: Hyd-A" at the University of Padua (Italy).

Author Summary

The appropriate transmission of genetic material during successive cell divisions requires the accurate duplication and segregation of parental DNA. The semi-conservative replication of chromosomes during S-phase is highly accurate and prevents accumulation of deleterious mutations. However, during each round of duplication, there are many impediments to the replication fork machinery that may hinder faithful chromosome duplication. Homologous recombination is a universal mechanism involved in the rescue of replication forks by rebuilding a replication apparatus at the fork (by mechanisms that are not yet understood). However, recombination can jeopardize genome stability because it allows genetic exchanges between homologous repeated sequences dispersed through the genome. In this study, we employ a fission yeast-based arrest of a single replication fork to investigate the consequences of replication fork arrest for genome stability. We report that a single blocked fork favours genomic deletions, translocations, and mutations; and this instability occurs during fork recovery by recombination.

We also report that a single arrested fork that resumes its progression by recombination is prone to causing replication slippage mediated by micro-homology. We propose that deletions/duplications observed in human cancer cells suffering from replication stress can be viewed as scars left by error-prone replication forks restarted by recombination.

Supporting Information

Figure S1 Fork-arrest results in GCRs in a recombinationdependent manner. A. The rate of deletion for indicated strains, in the presence (Rtf1 repressed) and in the absence (Rtf1 expressed) of thiamine. Numbers of RTS1 repeats present in the S. pombe genome and the presence of a visible fork arrest (based on 2DGE presented on Figure 1B) are given for each strain. The % of deletion events, as determined by the PCR assay, was used to balance the rate of ura4 loss. The values reported are means of at least 3 independent median rates. Error bars correspond to the standard error (SE). Statistically significant fold differences in the rate of deletion events between the Rtf1 ''repressed'' and ''expressed'' conditions are indicated with an *. B and C. Rate of deletion (B) and translocation (C) for the strains indicated; ON and OFF refers to the RTS1-RFB being active or not, respectively. The % of deletion and translocation events, as determined by the PCR assay, was used to balance the rate of ura4 loss. The values reported are means at least 3 independent median rates. Error bars correspond to SE. Statistically significant fold differences in the rates of deletion or translocation events between the ''OFF'' and ''ON'' conditions are indicated with an *.

Abstract

The replication of chromosomes can be challenged by endogenous and environmental factors, interfering with the progression of replication forks. Therefore, cells have to coordinate DNA synthesis with mechanisms ensuring the stability and the recovery of halted forks. Homologous recombination (HR) is a universal mechanism that supports DNA repair and the robustness of DNA replication. Nonetheless, mechanisms regulating HR pathways, such as ectopic versus allelic recombination, remain poorly understood. Another essential pathway for genome stability is the wrapping of newly replicated DNA around nucleosomes, leading to the constitution of a chromatin fibre, which allows the structural organization of the genetic material. In Saccharomyces cerevisiae, deficiencies in chromatin assembly pathways lead to replication forks instability and consequent increase in the rate of HR. Histone chaperones play a crucial role during chromatin assembly, thus I decided to focus on the H3-H4 histone chaperone Chromatin Assembly Factor 1 (CAF-1), to study its role in HR processes in Schizosaccharomyces pombe. Indeed, HR includes a DNA synthesis step and little is known about the associated chromatin assembly. My data excluded a role for CAF-1 in allelic recombination and in the maintenance of forks stability. However, CAF-1 was found to play an important role during ectopic recombination, in promoting chromosomal rearrangements induced by halted replication forks. My data support a model according to which CAF-1 allows the stabilization of early recombination intermediates (D-loop), via nucleosome deposition during the elongation of these intermediates. Doing so, CAF-1 counteracts the dissociation of early recombination intermediates by the helicase Rqh1. Therefore, CAF-1 appears to be part of an equilibrium that regulates stability/dissociation of early steps of recombination events. Importantly, I found that the role of CAF-1 in this equilibrium is of particular importance during non-allelic recombination, revealing a novel regulation level of HR mechanisms and outcomes by chromatin assembly.

Key words: homologous recombination, Chromatin Assembly Factor 1 (CAF-1), chromatin assembly, D-loop stabilization, ectopic recombination.