Sharon Begley

Delphine Benarroch-Popivker

Sabrina Pisano

Aaron Mendez-Bermudez

Liudmyla Lototska

Parminder Kaur

Serge Bauwens

Nadir Djerbi

Chrysa M Latrick

Vincent Fraisier

Bei Pei

Alexandre Gay

Emilie Jaune

Kevin Foucher

Julien Cherfils-Vicini

Eric Aeby

Simona Miron

Arturo London ˜ O-Vallejo

Jing Ye

Marie-He´ Le` Ne Le Du

Hong Wang

Eric Gilson

Marie-Jose ` Phe Giraud-Panis

Replication protein A ssDNA single-strand DNA SSA single-strand annealing TIN2 TERF1-interacting nuclear factor

Keywords: Télomères, RAP1, TRF2, fusions inter-chromosomiques, recombinaison nonhomologue (NHEJ), sénescence réplicative x x telomeres, RAP1, TRF2, chromosome fusions, NHEJ, replicative senescence. xi

Les télomères sont des séquences d'ADN, généralement répétées en tandem, localisées à l'extrémité des chromosomes linéaires. Une des fonctions principales des télomères est de différencier l'extrémité des chromosomes des cassures double-brin, et ainsi de prévenir l'activation des voies de réparation de l'ADN. Chez les mammifères, cette fonction est plus spécifiquement assurée par le complexe shelterin. Il s'agit d'un complexe hétérogène composé de six protéines distinctes: TRF1, TRF2, POT1, RAP1, TPP1 et TIN2, qui interagit spécifiquement avec l'ADN télomérique. Au sein de ce complexe, les protéines RAP1 et TRF2 coopèrent afin d'empêcher l'extrémité des chromosomes d'être perçue comme un dommage de l'ADN, ce qui autrement aboutirait à des fusions inter-chromosomiques suite au processus de réparation. La protéine TRF2 se lie directement à la molécule d'ADN dans laquelle elle s'enroule de façon spécifique. Cette propriété est primordiale pour générer une structure d'ADN en forme de boucle, appelée t-loop, et dont le bon fonctionnement des télomères dépend.Les travaux effectués au cours de cette thèse ont mis en évidence deux scenari indépendants dans lesquels la protéine RAP1 assure un rôle critique dans la stabilité des télomères. Premièrement, RAP1 peut prévenir les fusions inter-chromosomiques dans des cellules exprimant une forme altérée de TRF2 incapable de former des t-loops. Deuxièmement, l'inhibition de RAP1 dans des cellules en sénescence réplicative conduit à l'activation des voies de réparation de l'ADN et à la formation de fusions inter-chromosomiques. Ces observations font écho à des résultats précédents obtenus dans des cellules HeLa traitées avec l'inhibiteur de la télomérase BIBR1532, et dont l'expression de la protéine RAP1 était abolie par shRNA. De plus, j'ai montré que les fusions interchromosomiques engendrées par la perte de RAP1 sont dépendantes de la ligase IV, qui est un acteur principal de la voie de réparation de l'ADN par recombinaison non-homologue (NHEJ). Dans l'ensemble, ces travaux démontrent l'importance de la protéine RAP1 dans la stabilité des télomères lorsque la protéine TRF2 est non fonctionnelle, mais aussi dans des situations physiologiques telles que la sénescence réplicative.

First of all, I would like to thank the members of my Thesis committee, Dr. Corine Bertolotto, Dr. Paula Martinez and Dr. Stéphane Marcand, for their courage to accept my invitation and willingness to evaluate my thesis thoroughly. I greatly appreciate your efforts and time spent on my work.

Then I would like to extend my gratitude to my PhD supervisor Pr. Eric Gilson. It was a rare luck and privilege to hit the jackpot twice: being granted a Signalife PhD fellowship and being accepted into your team after project rotations. Thank you very much for allowing me to work on RAP1 and senescence and even more, thank you for believing in my work despite all the project ups and downs we faced together throughout the PhD.

My very special thanks go to my co-supervisor Dr. Aaron Mendez-Bermudez. I thank you enormously for being always by my side during this extreme but surely unforgettable PhD journey. I always admire your optimism and hard work, and I owe you a lot in terms of professional and personal skills I acquired while working with you. I must admit I always felt very privileged and happy to work with you. Thank you for all the amazing discussions and team work, and I'm looking forward to seeing our work published any time soon.

Many many thanks to my lab colleagues, past and present, for welcoming me in the team and providing unbelievable care and support during my PhD. Specifically, I thank Rita for sharing so many memorable moments together, for your helping hand with literally everything, for being my elder sister even though I don't have a real one. Sabrina, my dear "mommy" friend, thank you for taking care of me in very difficult times and for your very big heart. Delphine, thanks a lot for always supporting and encouraging me, especially during my last year. Marie-Jo, I thank you for teaching me a hardcore biochemistry, such as EMSA and overhang assay. Nadir, thank you for being very patient with my orders and thanks a lot for always willing to help. Julien, I would like to thank you for being always a witty and positive person and I learned a lot from you about cancer and immunology. Alex, thanks for reminding me to buy some groceries and for your fantastic lemon cakes, which I will definitely miss in the future. Many thanks to our PhD crew, Alice, Martin, Sol, Charlene, for many great and sometimes crazy moments in the lab and not only. Alice, thanks for your interesting talks about corals, Martin, I learned from you a few cool methods to work with neurons, Sol, you're my autophagy expert, Charlene, you are the one xii who knows all the challenges to work with senescent cells. I also thank Claire, Melanie, Serge and many others for contributing to a good atmosphere in the team. I would like to mention also the contribution of Mounir and Marta during my first year of PhD.

I would like to express my gratitude also to our "floor-mates", teams Liti and Cristofari. Thank you for being amazing neighbours and for sharing with me reagents when I needed the most. Special thanks go to my "stay-late-in-the-lab" mates, Arpita and Ben, for their presence during sometimes late working hours. Arpita, I will never forget our iconic trip to Berlin together with Rita. Ben, I am very grateful for your kind help with a lot of issues I had, no matter whether it's personal or professional. Thank you also for providing me with an excellent French language expertise.

Many thanks go also to Ludo from the cytometry platform and Sabine for taking care of my radioactivity orders and being so supporting and cheerful person.

I would like to thank my high school teachers, Kavetska Tamara Borysivna and Vasyliuk Tetiana Ivanivna for always believing in me and being my real motivation to study biology and always stay curious. Thanks a lot to all my NaUKMA University teachers, for absolutely intense and fruitful 6 years of studies. Many thanks to Doan Svitlana Ivanivna and Kondratov Oleksandr, my Bachelor and Master thesis supervisors, who motivated me to pursue scientific career.

I absolutely thankful to all my friends who supported me during my PhD and not only.

Special thanks to Natalia and Maksym, as well as Daria. I will never forget our trips and the 10th Nice-Cannes marathon in which we participated, they are among the best memories I had during my PhD. Many thanks to Sergii and Tania, for their enthusiasm in cheering me up always when I needed the most.

I am grateful to all the Signalife Labex community that became my second family. Among many people I met thanks to this amazing PhD Program, I would like to acknowledge Dr. Konstanze Beck and Anna Bliznyuk. I greatly appreciate your help with all the administrative procedures and for making me integrate into new culture and country smoothly. Many thanks to Lenka, Ramona and Nikita for many wonderful moments we shared and for your presence in the most difficult times. I feel very blessed to have you as my friends. My very warm wishes and special thanks go also to my Signalife buddy and flatmate Denisa. Words are not enough to express my gratitude to you. Hope we will all cross our paths in the future.

xii Oleksiy, among many things I am grateful to you, I would like to thank you especially for always believing in me and being my motivation to apply for PhD grants. I wouldn't be here writing these acknowledgements without your love and support. I admire your scientific approach in our daily life, and I am very grateful to all the wonderful moments we spent together and looking forward to many more to come.

Last, but not least, I would like to thank my family. Specifically, my mom and dad, I absolutely adore you for being so loving and caring parents, for so much support I got from you during all my life and especially during my PhD.

Дякую усім, хто підтримував мене протягом моєї роботи над дисертацією, і особлива подяка моїй сім'ї, батькам, за любов і підтримку.

Introduction

Over the course of evolution, mammalian telomeres developed the shelterin complex, a very elegant way to protect chromosome extremities from various DNA damaging insults. This complex is comprised of six proteins that can employ and control different interactors with one final goal: keep the telomeres functional.

One of highly dangerous events in the life of telomeres is chromosome fusion.

Chromosome fusions usually occur when two chromosomes fuse end-to-end forming dicentric or ring chromosomes. Fusion can be executed by several different DNA repair pathways with or without telomere loss as an outcome. Non-homologous end-joining repair (NHEJ) and homologous recombination repair (HRR) are two common DNA repair pathways that promote telomere fusions.

In human, there are two key telomeric proteins that inhibit chromosome fusions: TRF2 and RAP1. TRF2 recruits RAP1 to telomeres and thus is considered as the master regulator of telomere protection, whereas the role of RAP1 at mammalian telomeres has been debatable for a long time.

Therefore, this Thesis manuscript sheds light on human RAP1 and its role in telomere protection.

In Chapter 1, the literature overview is a resume of the most recent and relevant findings in the field of DNA repair and DNA damage in connection to telomere fusion.

Chapter 2 represents the actual results of the thesis project. It is grouped into two articles. Article 1 is the published manuscript, which focuses mainly on how TRF2 protects telomeres in the context of DNA topology. We demonstrate that TRF2 can wrap DNA around its homodimerization domain (TRFH). The TRFH-dysfunctional mutant of TRF2 (called Topless) is not able to wrap telomeric DNA, has decreased ability to promote t-loop formation and does not protect against DNA damage response (DDR), whereas it was able to rescue chromosome fusions. As part of the PhD project, we show that Top-less was not able to protect chromosomes from NHEJ upon RAP1 dysfunction. The latter result was an inspiration for Article 2, where we focus solely on the RAP1 role in telomere protection in the context of replicative senescence. Specifically, we found that in senescent cells RAP1 becomes essential to protect telomeres from DDR checkpoint and NHEJ repair. In Article 2 (the manuscript in preparation for publication) the main findings of my PhD work are described.

Chapter 3 is the final chapter of this dissertation. It discusses the main findings and suggests future work that would be important to better understand the mechanism of RAP1dependent telomere protection in senescent cells.

During my PhD training, I have contributed to another research project on the role of TRF2 in pericentromere function. The results were recently published. Since this is not my main research project, the results are not discussed in this manuscript, but the article is attached in Appendix I. Article 3.

Chapter 1

Telomere fusion: control, mechanisms and consequences in Drosophila melanogaster upon ionizing radiation. He was the first to introduce the name "telomere", which originates from the Greek words telos (end) and meros (part), and he used this term to describe the end parts of chromosomes [Muller HJ., 1938]. Simultaneously, McClintock highlighted the importance of telomeres during her studies of plant chromosomes in corn cells. She noted that the loss of natural chromosome ends (telomeres) destabilizes cellular genomes, causing chromosomes to become "sticky" and undergo adhesion and fusion at their ends, with consequent formation of dicentric chromosomes. She also demonstrated that the ends could be restored if chromosomes acquired a new telomere [START_REF] Mcclintock | The Behavior in Successive Nuclear Divisions of a Chromosome Broken at Meiosis[END_REF][START_REF] Mcclintock | The Stability of Broken Ends of Chromosomes in Zea Mays[END_REF].

When the Watson-Crick double helical structure of DNA was resolved in 1953, it immediately suggested a mechanism of its replication -each strand in the duplex acts as a template to guide the synthesis of its complement. However, understanding the mechanism of the semi-conservative DNA replication [Meselson M. and Stahl FW., 1958] identified the "end replication problem", consisting of the inability of cells to completely replicate the linear ends of DNA [START_REF] Gilson | The telomere story or the triumph of an openminded research[END_REF]. The first formulation of the end replication problem was focused on the lagging strand synthesis process where the gap generated by removal of the RNA primer at the 5'-end cannot be filled at the end of the chromosomal DNA, resulting in shortening of the newly synthesized strands with each round of DNA replication. This lagging strand problem was revisited later on by Cech and colleagues on the basis of the structure of the parental telomere extremity that corresponds to a 3'-overhang: the lagging chromatid is expected to somehow reproduce the 3' overhang while the leading chromatid ends as a blunt DNA if the DNA polymerase synthesizes until the last nucleotide and by a 5'-overhang if the polymerase stop before, but in any case the genetic information of the 3' overhang of the parental DNA is lost. Thus, it was postulated that the end replication is more a leading strand than a lagging strand problem [START_REF] Lingner | Telomerase and DNA end replication: no longer a lagging strand problem?[END_REF]. Watson also predicted the existence of a protective mechanism to prevent the chromosomal shortening [Watson JD. et al., 1972]. For Olovnikov, the terminal replication problem was the cause of a progressive telomere shortening, which also acted as an internal clock to determine the number of divisions that a cell can undergo during its lifespan. Therefore, telomere shortening could not only control the process of ageing but also acts as a molecular clock that counts the number of cycles that the cell can support [Olovnikov AM., 1973]. This is also consistent with the "Hayflick limit", an observation made in the early sixties showing that cultured primary fibroblasts have a limited number of divisions [START_REF] Hayflick | T he limited in vitro lifetime of human diploid cell strains[END_REF].

Structure and length of telomeres vary greatly among different species. The basic units of telomeres are tandem repeats, for instance, T2AG3 in mammals. Telomeric DNA is doublestranded with a short 3 '-tail (150-300 kb) in the very end of the chromosome. However, plants can have blunt-ended telomeres [START_REF] Kazda | Chromosome end protection by blunt-ended telomeres[END_REF]. In species with relatively long telomeres the 3'-overhang can fold backwards and invade the double-stranded telomere DNA forming t-loops (Figure 1). Interestingly, t-loops have been discovered among different species. For example, Trypanosoma form very tiny t-loops, less than 1 kb in length, whereas field pea harbours extremely large t-loops, up to 50 kb in size [de Lange T., 2004].

T-loops are considered as structural barriers that protect telomeric DNA from being recognized by DDR machinery [START_REF] Van Ly | Telomere Loop Dynamics in Chromosome End Protection[END_REF].

Both G-rich and C-rich telomeric strands may form additional complex DNA structures.

For example, the G-rich strand can adopt a four-stranded G-quadruplex structure involving planar G-tetrads of guanine, while the C-rich strand can form the so-called i-motif with intercalated C•C+ base pairs (Figure 1). Different G-quadruplex structures exist, and they may be important to protect 3'-tails [START_REF] Phan | Human telomeric DNA: G-quadruplex, i-motif and Watson-Crick double helix[END_REF].

Chromosomal DNA extremities can be recognized as accidental double strand breaks (DSBs) and treated as such by the cell leading to cell cycle arrest (DDR checkpoint) and recombination (DDR repair) [Shay JW., 2004]. Therefore, the natural ends of chromosome must be protected both from DDR checkpoint and repair.

Altogether, telomeres have to deal with two major problems: end replication and end protection. It turns out, they can do so with a help from different proteins that are described in the next section. A. G-quadruplex. B. t-loop. C. i-motif. Illustration from [Giraud-Panis MJ. et al., 2013].

Factors that maintain and protect telomeres

To overcome the end protection problem, cells developed a few strategies to keep the equilibrium of the telomere length, such as telomerase and alternative lengthening. At the same time, end protection problem can be effectively solved by means of the capping proteins.

Telomerase was discovered in Tetrahymena thermophila by Greider and Blackburn [Greider CW. and Blackburn EH., 1985]. This protein is composed of two essential components: TERT (telomerase reverse transcriptase) and TERC (telomerase RNA component). TERC binds to the 3'-tail and serves as a template for TERT, which elongates telomeres (Figure 2) [Schmidt JC. and Cech TR., 2015]. Interestingly, in human and several other vertebrate species, but not all, TERT expression and telomerase activity are severely shut down in somatic tissues at the end of embryogenesis with the exception of progenitor or stem cells but to a level insufficient to fully replenish telomeric DNA ends at each round of cell division [Cong YS. et al., 2002].

An alternative way to counteract telomere attrition is based on homologous recombination and is called alternative lengthening of telomeres (ALT) pathway. Whereas approximately 85-90% of tumours utilize telomerase to elongate their telomeres [Kim NW. et al., 1994], some cancers (notably tumours of mesenchymal origin) use the ALT pathway that relies on homologous recombination [Apte MS. and Cooper JP., 2017].

Several telomere capping proteins, protecting telomeres from unwanted DDR activation, exist among different organisms. The prototypes of telomere capping protein complexes were identified in budding yeast, consisting mainly in two complexes: shelterin and CST (Cdc13-Stn1-Ten1). Shelterin is restricted to the RAP1 protein, which specifically binds telomeric DNA repeats to protect telomere DNA from fusion, while CST plays a key role against telomeric DNA degradation, checkpoint activation and telomere replication [Giraud-Panis MJ. et al., 2010].

The equivalents of the shelterin and CST complexes are found in many (if not all) eukaryotic organisms but with a great diversity of protein composition (Figure 3) [Giraud-Panis MJ. et al., 2013]. In mammals, shelterin is comprised of six proteins: TRF1 and TRF2 that through a Myb-like domain named Telobox bind to double-stranded DNA (dsDNA), TPP1 binding to POT1, which binds to the 3'-overhangs, TIN2 making a protein bridge between TRF1/TRF2 and TPP1 and finally RAP1 that, in contrast to budding yeast, binds indirectly to telomeric DNA via a direct interaction with TRF2 (Figure 3) [de Lange [START_REF] De Lange | Shelterin: the protein complex that shapes and safeguards human telomeres[END_REF]Giraud-Panis MJ. et al., 2013].

Chromosome fusions and DNA repair

3.1. The "good" and the "bad" of fused chromosomes: lessons from evolution Over the years, eukaryotic chromosomes acquired certain differences in their shape, size, composition, and number. These features made species distinguishable among each other, therefore, they appear to be important targets of evolution.

Simply, two ways of chromosome number evolution exist: fusion and fission, which lead to two different consequences for the genome: either reduction or amplification in the number of the existing genetic material [START_REF] Schubert | Chromosome evolution[END_REF].

In terms of evolution, there is a large body of evidence that end-to-end fusions lead to reduction in the total number of chromosomes. For example, fusion of two ancestral primate chromosomes created human chromosome 2 [START_REF] Ijdo | Origin of human chromosome 2: an ancestral telomere-telomere fusion[END_REF] Mycetophylax conformis and Mycetophylax morschi [Cardoso DC. et al., 2014], and plant Arabidopsis thaliana [Lysak MA. et al., 2006]. Evolution of the budding yeast genome is characterized by the whole-genome duplication (from n=8 to n=16 chromosomes). However, it has been observed that in some other yeast species, such as Zygosaccharomyces, Kluyveromyces, Lachancea, and Ashbya, the number of chromosomes varies from 6 to 8. The most common event in reducing chromosome number among those yeast is telomere end-to-end fusions [Gordon JL. et al., 2011]. Recently, two independent groups created Saccharomyces cerevisiae strains with dramatically reduced number of chromosomes [reviewed in [START_REF] Liti | Yeast chromosome numbers minimized using genome editing[END_REF]. Luo et al. engineered yeast with n=2 chromosomes, and Shao and colleagues fused all the chromosomes into a single chromosome in a functional yeast [START_REF] Shao | Creating a functional single-chromosome yeast[END_REF][START_REF] Luo | Karyotype engineering by chromosome fusion leads to reproductive isolation in yeast[END_REF]. Both studies concluded that reduced chromosome number causes no major growth defects when cells are grown under various conditions and stresses. The groups showed that the n = 1 and n = 2 strains can undergo sexual reproduction, albeit with reduced efficiency compared with wild-type yeast, and produce spores that are slightly less viable. Therefore, these engineered yeast strains constitute powerful resources for studying fundamental concepts in chromosome biology [START_REF] Liti | Yeast chromosome numbers minimized using genome editing[END_REF]. Probably the most impressive example of natural chromosome reduction is the Indian muntjac (Muntiacus muntjak), whose females only have 6 chromosomes, and its males only 7 [Wurster DH. and Benirschke K., 1970]. By means of comparative mapping and sequencing approach, Tsipouri and colleagues characterized the sites of ancestral chromosomal fusions in the Indian muntjac genome [START_REF] Tsipouri | Comparative sequence analyses reveal sites of ancestral chromosomal fusions in the Indian muntjac genome[END_REF]. Specifically, they screened an Indian muntjac bacterial artificial chromosome library with a telomere repeat-specific probe. They found that all seven Indian muntjac sequences, that were analyzed, contained centromeric satellite I repeat sequences immediately adjacent to the telomeric-repeat block [START_REF] Tsipouri | Comparative sequence analyses reveal sites of ancestral chromosomal fusions in the Indian muntjac genome[END_REF]. Furthermore, high frequency of tandem fusions, which arise from telomere and centromere repetitive elements, has been proposed as the main mechanism of stasipatric (rapid) speciation that is common among muntjacs [START_REF] Wang | Rapid and parallel chromosomal number reductions in muntjac deer inferred from mitochondrial DNA phylogeny[END_REF].

Presence of telomere and centromere or pericentromere repeats and their duplication at the fusion site is a proof of DNA damage repair by non-homologous end-joining. This type of repair is usually error-prone and can give rise to certain types of genetic instability through initiation of 'breakage-fusion-bridge' (BFB) cycles, first discovered by McClintock in Zea mays [START_REF] Mcclintock | The Behavior in Successive Nuclear Divisions of a Chromosome Broken at Meiosis[END_REF][START_REF] Mcclintock | The Stability of Broken Ends of Chromosomes in Zea Mays[END_REF]. Such cycles start with the loss of telomeres at the ends of the chromosomes (Figure 4). Then DNA is replicated, and sister chromatids with fused ends are formed. During anaphase, centromeres of those sister chromatids are pulled in the opposite directions forming bridges as the ends are fused. While pulling centromeres apart from each other, a break of the bridge occurs at any point in a way that a daughter cell receives an uneven chromosome without telomeres. Telomeres can be restored by telomerase, but if the chromosome still lacks telomeres at the ends, the BFB cycle will continue during the next cell division.

Figure 4. Breakage-fusion-bridge cycles.

BFB cycles cause duplications, deletions, inversions as secondary rearrangements in the chromosomes. Genetic instability that occurs becomes a driving force of evolution. As described above, it can lead to appearance of new species (which can be considered as "good"). On the other hand, it can be a cause of establishing and promoting different malignancies (which for a normal cell and the whole organism is usually considered as "bad") [START_REF] Selvarajah | The breakage-fusion-bridge (BFB) cycle as a mechanism for generating genetic heterogeneity in osteosarcoma[END_REF]Kwei KA. et al., 2010;Martínez P. and Blasco MA., 2017;[START_REF] Maciejowski | Telomeres in cancer: tumour suppression and genome instability[END_REF]. Therefore, detailed studies of the mechanisms that lay behind genetic instability are needed to better understand how the switch between "good" and "bad" occurs.

The next part of this manuscript is focused on DNA repair mechanisms that are in connection to telomere fusion.

The DNA double-strand break repair mechanisms promoting chromosome fusions

In normally functioning cells, chromosome fusion must be prevented in order to maintain genome stability. In this regard, cells developed several mechanisms that inhibit fusion at natural chromosome ends. This is one of the main function accomplished by telomeres. Among the telomere strategies to prevent fusion, one can cite peculiar DNA structures (t-loops, 3'-overhangs), shelterin and other associated telomere factors.

In the absence of a proper anti-fusion activity, chromosome extremities can fuse by different recombinational repair mechanisms: homologous recombination repair as well as classical and alternative non-homologous end-joining (Figure 5).

In the next section of this manuscript, the mentioned pathways will be presented in detail with a focus on their relationship to chromosome fusions.

Homologous recombination repair. Homologous recombination repair (HRR) or

homology-directed repair (HDR) is a high-fidelity pathway of DSB repair. Although many different proteins and some of the non-coding RNAs are implicated in HRR, and several different mechanisms exist, the basic principles are conserved among prokaryotes and eukaryotes. This type of repair relies on homologous recombination, where a homologous DNA template is used to repair and restore the sequence around the break. DSB repair by HRR in mitotic cells favours the use of the sister chromosome over the homologous chromosome as a template donor [Kadyk LC. and Hartwell LH., 1992]. Notably, repair of DSBs by means of HRR can lead to two different consequences: crossover and non-crossover.

For example, crossover occurs during meiosis and can be also used to generate genetic diversity [START_REF] Baudat | Regulating double-stranded DNA break repair towards crossover or non-crossover during mammalian meiosis[END_REF]Heyer WD. et al., 2010]. However, the primary mechanism of HRR, gene conversion, does not result in the crossover, which makes it a faithful DNA repair process. Also, synthesis-dependent strand annealing pathway (SDSA) does not result in crossovers and is important to preserve genomic integrity [Verma P. and Greenberg RA., 2016]. When DSBs cannot be processed by the conventional mechanism of HRR, cells decide between SDSA, double-strand break repair (DSBR), break-induced replication (BIR), or single-strand annealing (SSA) (Figure 6) [Chapman JR. et al., 2012;Verma P. and Greenberg RA., 2016;Wright WD. et al., 2018].

Regardless of which choice has been made, initial steps of HRR share the same principles. First, after a DSB occurs, broken DNA ends undergo nucleolytic end resection to generate 3'-ssDNA overhangs. Generation of 3'-overhangs can be characterised by a two-step mechanism. First, in higher eukaryotes, an immediate recruitment of Mre11-Rad50-Nbs1 (MRN) along with CtIP complex occurs at the sites of DSB [Lamarche BJ. et al., 2010;[START_REF] Langerak | Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks[END_REF]. MRN-CtIP removes small oligonucleotides to generate a short protruding end [START_REF] Muraki | The DNA damage response at dysfunctional telomeres, and at interstitial and subtelomeric DNA double-strand breaks[END_REF]. Next, several other enzymes are recruited to produce long single-stranded overhangs by resection, for instance, Exonuclease 1 (EXO1), DNA replication helicase/nuclease 2 (DNA2) [Mimitou EP. and Symington LS., 2009;[START_REF] Jasin | Repair of strand breaks by homologous recombination[END_REF]. Furthermore, Bloom helicase (BLM) can be important for longrange resection of DNA ends [Nimonkar AV. et al., 2011], as well as Werner helicase (WRN).

The latter can substitute BLM in DNA2-mediated resection [START_REF] Sturzenegger | DNA2 cooperates with the WRN and BLM RecQ helicases to mediate longrange DNA end resection in human cells[END_REF].

Therefore, BLM and WRN act epistatically and ensure the single-strand 3'-overhang formation on both strands of the break.

Figure 6. Different pathways of homologous recombination repair in human.

Modified from [Heyer WD. et al., 2010].

Moreover, breast cancer suppressor BRCA1 can also take part in the initial steps of HRR, since it has been shown to interact with MRN [START_REF] Zhong | Association of BRCA1 with the hRad50-hMre11-p95 complex and the DNA damage response[END_REF] and CtIP [START_REF] Yu | The C-terminal (BRCT) domains of BRCA1 interact in vivo with CtIP, a protein implicated in the CtBP pathway of transcriptional repression[END_REF]] and promote HRR [Moynahan ME. et al. 1999;Stark JM. et al. 2004], as does CtIP [Sartori AA. et al. 2007;[START_REF] Bennardo | Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair[END_REF]]. Interestingly, BRCA1 may control the CtIP-dependent recruitment of DNA2 to DNA damage sites for subsequent DSB resection [Hoa NN. et al., 2015]. It has been demonstrated that BRCA1-A complex comprised of ubiquitin interacting motif (UIM) containing protein RAP80, adapter protein Abraxas, MERIT40 (mediator of RAP80 interactions and targeting 40 kDa, also known as NBA1), BRCC45, and deubiquitinylating enzyme BRCC36 guides BRCA1 to the sites of DSB through interaction with UIMs of RAP80 [reviewed in Greenberg RA., 2008;Daley JM. et al., 2014;[START_REF] Her | Factors forming the BRCA1-A complex orchestrate BRCA1 recruitment to the sites of DNA damage[END_REF]. Also, a proper recruitment of BRCA1 to the DSB sites is controlled by lncRNA DDSR1 [START_REF] Sharma | A BRCA1-interacting lncRNA regulates homologous recombination[END_REF].

After 3'-overhangs are generated, they are immediately covered by ssDNA-binding replication protein A (RPA). This binding prevents the formation of unwanted secondary structures on ssDNA [START_REF] Chen | RPA coordinates DNA end resection and prevents formation of DNA hairpins[END_REF]. The next step is to load Rad51 on 3'-overhangs.

In humans, several proteins can be important to replace RPA with Rad51. Among them, Rad52 appears to be essential to physically replace RPA with Rad51 and promote, therefore, formation of the nucleoprotein filament [Sugiyama T. and Kowalczykowski SC., 2002;[START_REF] Plate | Interaction with RPA is necessary for Rad52 repair center formation and for its mediator activity[END_REF]. Notably, it has been also shown that BRCA2 interacts directly with Rad51 and recruits it to the RPA-coated ssDNA at the DSB site [START_REF] Her | Factors forming the BRCA1-A complex orchestrate BRCA1 recruitment to the sites of DNA damage[END_REF]. Therefore, several mechanisms exist for the proper functioning of initial steps of HRR.

When the filament is formed, Rad51 initiates the search for a homologous template followed by the donor DNA strand invasion, formation of a D-loop and subsequent DNA synthesis mediated mainly by DNA polymerase δ in eukaryotes [START_REF] Maloisel | DNA polymerase delta is preferentially recruited during homologous recombination to promote heteroduplex DNA extension[END_REF][START_REF] Mcvey | Eukaryotic DNA Polymerases in Homologous Recombination[END_REF]. Strand invasion and formation of the D-loop is mediated by Rad54 (a protein that belongs to the SNF2/SW12 family in humans), which removes Rad51 from the filament [START_REF] Kanaar | Human and mouse homologs of the Saccharomyces cerevisiae RAD54 DNA repair gene: evidence for functional conservation[END_REF]Li X. and Heyer WD., 2009;Mazin AV. et al., 2010].

To complete HRR, three different scenarios are possible (Figure 6). First, if the second DNA end is present, mitotic cells mainly follow the SDSA pathway [Andersen SL. and Sekelsky J., 2016]. Therefore, either of 3'-overhangs or even both of them can invade the donor template. The invading strand is further displaced during the D-loop migration and the newly formed DNA strand anneals back to the ssDNA overhang of the second end, resulting in a non-crossover product [Heyer WD. et al., 2010]. However, a second possibility is the creation of a double Holiday junction (dHJ), which can result either in the crossover or non-crossover outcome depending on the proteins involved in the processing. For example, BLM together with topoisomerase 3 alpha (Top3A) process dHJs in a way that crossover does not occur [Wu L. and Hickson ID., 2003].

BIR takes place when there is only one accessible DNA end. The available 3'-overhang invades the homologous DNA and then extends to the end of the chromosome. In higher eukaryotes, BIR is an important mechanism to repair and restart broken replication forks, as well as it can contribute to the alternative lengthening of telomeres [reviewed in Verma P. and Greenberg RA., 2016].

SSA is another type of DSB repair, which can be considered as an alternative pathway of HDR [Verma P. and Greenberg RA., 2016]. SSA is initiated when DSB occurs between homologous direct repeats. These repeats first are resected bidirectionally, then nucleases cleave off unpaired 3'-overhangs. The final step is annealing and ligation of the DSB.

Remarkably, SSA does not require Rad51 filament, therefore, is Rad51-independent. Because the nuclease cleavage can result in deletion of repeats, SSA is a mutagenic process [Verma P. and Greenberg RA., 2016;[START_REF] Bhargava | Regulation of Single-Strand Annealing and its Role in Genome Maintenance[END_REF].

In summary, homologous recombination repair is represented by several pathways. All these pathways are conserved among different organisms, and some of them are redundant.

However, given how complex is the interaction among different proteins within one pathway, new approaches emerge in order to better dissect the mechanism of HRR. One of these approaches relies on super-resolution microscopy methods to study the process at singlemolecule resolution [START_REF] Kaniecki | A change of view: homologous recombination at single-molecule resolution[END_REF].

If HRR is not properly executed, this can lead to rapid telomere resection and loss followed by appearance of telomere-free ends and massive telomere-free chromosome fusions. Since HDR relies on the presence of homologous DNA template, it favours formation of sister chromatid fusions and can promote unequal sister chromatid exchange that will create fragile chromosomes [Rudd MK. et al., 2007].

Non-homologous end-joining repair.

Non-homologous end-joining is a second type of repair that cells employ on a regular basis. Described as a "willy-nilly" end-joining [Deriano L. and Roth DB., 2013], it relies on joining damaged DNA strands together. It can be either very robust and precise if the ends do no miss nucleotides or do not require further processing; otherwise, it can lead to certain genetic instability or diversity [Lieber MR., 2010;Chang HHY. et al., 2017]. For example, V(D)J recombination in immune cells absolutely requires NHEJ and is considered as a normal physiological process [START_REF] Malu | Role of non-homologous end joining in V(D)J recombination[END_REF], whereas incongruous NHEJ may promote cancer formation [Sishc BL. and Davis AJ., 2017]. The latter is due to formation of dicentric chromosomes that initiate BFB cycles or chromotrypsis [Maciejowski J. and de Lange T., 2017]. If NHEJ acts between two telomeres, it fuses chromosomes as an immediate outcome [START_REF] Marcand | How do telomeres and NHEJ coexist?[END_REF]. Therefore, NHEJ is the prime mechanism to create both intra-and inter-chromosome fusions.

Nowadays many different proteins involved in classical or canonical NHEJ have been characterized (which is often referred to as c-NHEJ), however, the basic principles on how this pathway is executed are the same among various species. In general, the c-NHEJ can be divided into three very general steps: DSB recognition, processing, and ligation (Figure 7) [Lieber MR., 2010;[START_REF] Yang | Non-homologous end joining: advances and frontiers[END_REF].

Figure 7. Non-homologous end-joining repair in human.

There are two essential components to make NHEJ work: DNA-PK and the ligase IV complex [Waters CA. et al., 2016]. However, presence of those two complexes can be enough only if the DSB forms blunt ends and do not require further processing. If the DSB is followed by incompatible DNA ends, direct ligation cannot be performed, and therefore several endprocessing and annealing proteins are on call before the ligation can occur [Chang HH. et al. 2016;Chang HHY. et al., 2017].

DSB recognition. The first protein, which is recruited within seconds to the sites of DSB, is Ku. In human, Ku is very abundant (500 000 molecules per cell) and demonstrates the strong affinity for DNA binding [Fell VL. and Shild-Poulter C., 2015]. Nevertheless, both in vitro and in vivo studies show that just two molecules of Ku are enough to cover the sites in the vicinity of the damaged DNA, presumably, each one covering the broken ends [Roberts SA. and Ramsden DA., 2007;[START_REF] Britton | A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair[END_REF]. In eukaryotes, Ku is present as a heterodimer, which is called Ku70/80. After being recruited to the sites of damage, Ku70/80 promotes sequestration of several other NHEJ factors for the appropriate repair (nucleases, polymerases, ligases), thus, Ku acts as a hub or scaffold protein [Fell VL. and Shild-Poulter C., 2015]. In yeast, there are Yku70/80 orthologs for mammalian Ku proteins. Strikingly, Yku is not an essential protein in yeast, whereas loss of human Ku86 leads to massive telomere loss and cell death [START_REF] Wang | Ku86 represses lethal telomere deletion events in human somatic cells[END_REF].

Ku forms the DNA-PK complex together with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [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]. DNA-PKcs has been discovered only in higher eukaryotes so far. Recently, the cryo-EM structure of human DNA-PK has been solved. Two research groups independently demonstrated that DNA-PKcs and Ku70/80 together form a DNA-binding bridge or tunnel. DNA-PKcs is relatively proximal, and Ku70/80 is distal, to the free DNA end. DNA-PKcs and Ku70/80 both wrap around one and a half turn of the DNA duplex with the blocked DNA end flanking outside of the complex [START_REF] Yin | Cryo-EM structure of human DNA-PK holoenzyme[END_REF][START_REF] Sharif | Cryo-EM structure of the DNA-PK holoenzyme[END_REF]. Notably, DNA-PKcs alone barely binds to DNA but strongly binds to DNA in the presence of Ku70/80 [START_REF] Yin | Cryo-EM structure of human DNA-PK holoenzyme[END_REF]. DNA-PKcs can be autophosphorylated or trans-phosphorylated by ATM. These two states of phosphorylation regulate the switch between recruitment of Artemis or ligase reaction [START_REF] Uematsu | Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks[END_REF][START_REF] Jiang | Differential phosphorylation of DNA-PKcs regulates the interplay between end-processing and end-ligation during nonhomologous endjoining[END_REF].

DSB processing.

Components of the DNA-PK complex can recruit to the sites of DSB DNA end-processing factors such as Artemis [START_REF] Riballo | A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci[END_REF], Werner [START_REF] Chen | WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repair[END_REF]Shamanna RA. et al., 2016], polynucleotide kinase-phosphatase (PNKP) [START_REF] Shimada | Polynucleotide kinase-phosphatase enables neurogenesis via multiple DNA repair pathways to maintain genome stability[END_REF], APTX-polynucleotide kinase-phosphatase-like factor 1 (APLF) [Macrae CJ. et al., 2008;Grundy GJ. et al., 2013], DNA polymerases Pol λ and Pol μ [Capp JP et al., 2006;[START_REF] Chayot | DNA polymerase μ is a global player in the repair of non-homologous end-joining substrates[END_REF], terminal deoxynucleotidyl transferase (TdT) [START_REF] Boubakour-Azzouz | Terminal deoxynucleotidyl transferase requires KU80 and XRCC4 to promote N-addition at non-V(D)J chromosomal breaks in non-lymphoid cells[END_REF]. Depending on how complex is the DSB, the mentioned factors can be required for the accurate DNA end cleavage and annealing in order to facilitate further ligation [START_REF] Yang | Non-homologous end joining: advances and frontiers[END_REF]. Interestingly, mammalian DNA-PKcs and the rest of the mentioned processing factors (except DNA polymerases) do not have orthologs in budding yeast S.cerevisiae [START_REF] Dudásová | Non-homologous end-joining factors of Saccharomyces cerevisiae[END_REF]. Instead, MRX complex becomes of outstanding importance to execute NHEJ [Emerson CH. and Bertuch AA., 2016]. Regarding the DNA polymerases in yeast, Pol4 is a Pol X family polymerase (related to mammalian polymerases λ and μ). Moreover, yeast employs also Pol3 (mammalian Polδ) [START_REF] Ramsden | Polymerases in nonhomologous end joining: building a bridge over broken chromosomes[END_REF].

DSB ligation. Importantly, c-NHEJ is distinct in this regard, because it relies on the ligase IV function [START_REF] Wang | Genetic evidence for the involvement of DNA ligase IV in the DNA-PK-dependent pathway of non-homologous end joining in mammalian cells[END_REF]. In mammals, ligase IV forms a complex with XRCC4

and XLF [START_REF] Ahnesorg | XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining[END_REF]. XRCC4 and XLF are particularly important for bridging DNA molecules and therefore promoting ligase IV activity [Andres SN. et al., 2012]. PAXX (XRCC4 paralogs) is a regulator of XRCC4 [START_REF] Xing | Interactome analysis identifies a new paralogue of XRCC4 in non-homologous end joining DNA repair pathway[END_REF]. Besides XRCC4ligase IV complex, it interacts with Ku70 directly and promotes Ku accumulation at the break [START_REF] Ochi | DNA repair. PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair[END_REF][START_REF] Liu | PAXX promotes KU accumulation at DNA breaks and is essential for end-joining in XLF-deficient mice[END_REF]]. An emerging view is that PAXX is an additional protein recruited to hard-to-repair DSBs [Tadi SK. et al., 2016], where it can promote DNA polymerase λ activity [START_REF] Craxton | PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair[END_REF].

In yeast, ligation occurs due to the activity of DNA ligase IV or Dnl4 in S. cerevisiae.

Dnl4 is strongly associated with Lif1. If Lif1 is dysfunctional, Dnl4 becomes unstable [START_REF] Herrmann | Saccharomyces cerevisiae LIF1: a function involved in DNA double-strand break repair related to mammalian XRCC4[END_REF]]. Mrx and Yku were reported to promote association of the Dnl4-Lif1 complex to the DSB, as well as Nej [Emerson CH. and Bertuch AA., 2016].

Alternative non-homologous end-joining pathway.

It has been reported that critically short telomeres tend to fuse end-to-end via non-canonical end-joining that requires microhomology [Letsolo BT. et al., 2010].

Alternative NHEJ (a-NHEJ) in certain literature reviews can also be referred to as alternative end-joining (a-EJ). An early evidence for the existence of alternative end-joining pathways came from studies in Ku-deficient budding yeast [Boulton SJ. and Jackson SP., 1996]. In mammals, similar observations were made in p53 knockout mice lacking the components of the NHEJ machinery, but yet supporting insertions, deletions and microhomology [START_REF] Zhu | Unrepaired DNA breaks in p53-deficient cells lead to oncogenic gene amplification subsequent to translocations[END_REF].

A-NHEJ is distinct from c-NHEJ and HRR on several counts. First, it does not necessarily require homology to function as HRR does. However, certain types of fusions that occur through a-NHEJ can use microhomology, which makes it similar to the SSA pathway [START_REF] Verma | Noncanonical views of homology-directed DNA repair[END_REF]Sallmyr A. and Tomkinson AE., 2018]. The latter pathway is called MMEJ or MHEJ (microhomology-mediated repair). In contrast to SSA, MMEJ relies on very short homologies, less than 20 bp [Pannunzio NR. et al., 2014;[START_REF] Mladenov | DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferation-dependent regulation[END_REF]. The final step in the repair is ligation of DNA, but in comparison with c-NHEJ it is ligase IV independent process, which is executed via either ligase III or I [START_REF] Wang | DNA ligase III as a candidate component of backup pathways of nonhomologous end joining[END_REF][START_REF] Simsek | DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation[END_REF][START_REF] Masani | Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination[END_REF].

The distinct characteristics of a-NHEJ are the key players involved in the pathway: poly(ADP-ribose) polymerase1 (PARP1), DNA polymerase θ (POLQ), and Ligase III/I [Chang HHY. et al., 2017] (Figure 8). The main role of PARP1 is to catalyse the polymerization of ADP-ribose units -derived from the ADP donor NAD+ -resulting in the attachment of either linear or branched PAR polymers to itself or other target proteins. PARP1 is therefore believed to be a sensor of DNA damage [START_REF] Ray Chaudhuri | The multifaceted roles of PARP1 in DNA repair and chromatin remodelling[END_REF]. By means of several biochemical and super-resolution microscopy approaches, it has been demonstrated that PARP1 competes with KU for DNA DSB repair. KU can be removed from the sites of DSBs by PARylation that is performed by PARP1 [START_REF] Wang | PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways[END_REF][START_REF] Yang | Super-resolution imaging identifies PARP1 and the Ku complex acting as DNA doublestrand break sensors[END_REF]. Furthermore, PARP1 recruits MRN complex to the repair centre (Figure 8) [Haince JF. et al., 2008]. In analogy to HRR, MRN together with CtIP may be necessary for end processing and removal/recruitment of other proteins [Lamarche BJ. et al., 2010]. Contrary to HRR, MMEJ does not require γ-H2AX, neither BLM nor EXO1 for end processing [Truong LN. et al., 2013].

After DNA is recognized and processed, DNA polymerase θ is needed for a stable annealing of DNA strands. Polθ uses short microhomology (2-6 bp) for annealing. Notably, if this microhomology is not present, due to its transferase activity, Polθ can add several nucleotides to create microhomology at the site of the break [START_REF] Kent | Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ[END_REF].

Remarkably, polymerase θ was found to promote a-NHEJ at dysfunctional telomeres in cooperation with PARP1 [Mateos-Gomez PA. et al., 2015]. Recently, another DNA polymerase β was reported to participate in a-NHEJ [START_REF] Ray | DNA polymerase beta participates in DNA End-joining[END_REF].

Figure 8. Alternative NHEJ in human.

The final step of a-NHEJ is ligation of the annealed DNA. End ligation is ligase IVindependent and relies on the activity of either ligase III or I. Ligase III seems more efficient in comparison to Ligase I [START_REF] Lu | Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining[END_REF]. Ligase III can form a complex with XRCC1, which was found to co-exist with MRN in a-NHEJ [Caldecott KW. et al., 1994;[START_REF] Della-Maria | Human Mre11/human Rad50/Nbs1 and DNA ligase IIIalpha/XRCC1 protein complexes act together in an alternative nonhomologous end joining pathway[END_REF].

Whether this pathway is a backup of the main c-NHEJ or acts independently, there is a body of evidence that a-NHEJ is employed by the cell to create genetic diversity [START_REF] Ottaviani | The role of microhomology in genomic structural variation[END_REF]. Nevertheless, there are still many outstanding questions that have to be explored for better understanding how a-NHEJ works and what is the prime importance of this pathway.

DNA damage response at the sites of double-strand break

In eukaryotes, DNA damage response (DDR) is a cascade of signalling events within the cell as a response to DNA damage. Like a classical signal transduction pathway, it is comprised of signal sensors, transducers, mediators and effectors. One of the peculiarities of this signalling pathway is that instead of ligand-receptor interactions, DDR machinery upstream events rely on the direct recognition and further processing of damaged DNA molecules. The sensors of this pathway are the proteins that directly recognize damaged DNA and activate upstream DDR kinases. Then the signal is amplified through the activation of different mediator kinases, and the final effectors spread the signal that will determine the fate of the cell.

DNA repair and DDR pathway are tightly connected with each other. MRN complex is essential in this regard because it appears to be in the front line, where different repair choices and DNA damage sensing merge [Williams RS. et al., 2007]. In the context of DDR, MRN is believed to play a role as a sensor of the damage. It is activated immediately at the damaged sites and directly binds dsDNA. Also, MRN acts as the main factor required for the rapid localization of ATM (ataxia-telangiectasia mutated) to DSBs [Lee JH. and Paull TT., 2005]. It is important to mention that ATM and ATR (ATM-and Rad3-Related) pathways are d i s t i n c t f r o m e a c h o t h e r. AT M r e s p o n d s t o D S B s , w h e r e a s AT R -t o both ssDSBs and dsDSBs with a greater preference to ssDSBs, and is particularly important to repair the DNA lesions that occur during replication [START_REF] Maréchal | DNA damage sensing by the ATM and ATR kinases[END_REF].

At the sites of DSBs, ATM is a transducer protein kinase, which becomes activated by phosphorylation of its serine residue Ser1981 in human [Bakkenist CJ. and Kastan MB., 2003]. It has been shown that ATM activation is impaired in cells with MRN deficiencies [START_REF] Uziel | Requirement of the MRN complex for ATM activation by DNA damage[END_REF]. Furthermore, the carboxyl terminus of Nbs1 (a protein of the MRN complex) is known to interact with ATM [START_REF] Falck | Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage[END_REF]. Moreover, recently it was demonstrated that several proteins can enhance ATM signalling via direct interaction with the MRN complex. For instance, a signalling mediator, p53-binding protein 1 (53BP1) through it BRCT domain binds to MRN complex directly and regulate ATM phosphorylation of its substrates [Lee JH. et al, 2010]. Rad17, a replication checkpoint protein, also binds directly to MRN and is required for the early recruitment of the MRN complex to the DSB site, and it contributes to ATM activation [START_REF] Wang | Rad17 recruits the MRE11-RAD50-NBS1 complex to regulate the cellular response to DNA double-strand breaks[END_REF]. Smad7 interacts with Nbs1 and enhances the interaction between ATM and Nbs1 upon DNA damage response, leading to phosphorylation of downstream substrates [START_REF] Park | Smad7 enhances ATM activity by facilitating the interaction between ATM and Mre11-Rad50-Nbs1 complex in DNA double-strand break repair[END_REF].

Among substrates that are phosphorylated by ATM are BRCA1, Chk2, p53, H2AX, MDC1. The latter two act in cooperation. It has been described that H2AX phosphorylation is performed by ATM as one of the upstream events of DDR activation. The phosphorylated histone is called γH2AX. γH2AX, in turn, acts as a hub for nuclear foci formation, the DDR centres where many DNA repair proteins and chromatin remodelling factors are accumulated [START_REF] Iijima | Dancing on damaged chromatin: functions of ATM and the RAD50/MRE11/NBS1 complex in cellular responses to DNA damage[END_REF][START_REF] Clouaire | Taming Tricky DSBs: ATM on duty[END_REF][START_REF] Podhorecka | H2AX Phosphorylation: Its Role in DNA Damage Response and Cancer Therapy[END_REF].

Formation of γH2AX foci is one of the key steps in DDR signalling and repair in the context of chromatin. MDC1 was found to directly interact with γH2AX and therefore contribute to the γH2AX foci formation. At the same time, MDC1 interacts with ATM. Thus, it acts as a mediator between ATM and γH2AX and helps spread phosphorylation of γH2AX by ATM over long chromosome distances [Stewart GS. et al., 2003;Lee JH. et al., 2005;[START_REF] Stucki | MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks[END_REF].

Although there is no doubt that phosphorylation of H2AX is essential for the DDR pathway, it has been documented that many other chromatin modifications occur, such as DNA methylation, different histone modifications etc., which require specific chromatin remodelling factors [reviewed in Polo SE. and Jackson SP., 2011]. Notably, γH2AX triggers cascades that rely on ubiquitylation and SUMOylation in order to recruit BRCA1 and 53BP1 to the damaged sites [reviewed in Daley JM. and Sung P., 2014;Muraki K. and Murnane JP., 2017].

In heterochromatin repair, ATM through its substrate Chk2 phosphorylates KAP1 and also stimulates further dissociation of heterochromatin protein HP1-β from H3K9me3 around DSBs [Goodarzi AA. et al., 2008;[START_REF] Bolderson | Kruppel-associated Box (KRAB)associated co-repressor (KAP-1) Ser-473 phosphorylation regulates heterochromatin protein 1β (HP1-β) mobilization and DNA repair in heterochromatin[END_REF]. Also, cells that do not form 53BP1 foci, fail to form phosphorylated KAP1 foci [START_REF] Noon | 53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair[END_REF]. Interestingly, changes in the chromatin structure upon DDR activation have been reported to increase chromosome mobility [reviewed in Hauer MH. and Gasser SM., 2017;Smith MJ. and Rothstein R., 2017;[START_REF] Marnef | Organizing DNA repair in the nucleus: DSBs hit the road[END_REF]. This phenomenon is believed to be common in yeast, where damaged DNA becomes highly mobile and moves within the nucleus to the repair centres [START_REF] Lisby | Colocalization of multiple DNA doublestrand breaks at a single Rad52 repair centre[END_REF]. Notably, DSBs that are unable to be repaired move to the yeast nuclear periphery [START_REF] Nagai | Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase[END_REF]. In higher eukaryotes, chromosome mobility is relatively weaker compared to yeast, however, it does occur. For example, increased chromosome movement of uncapped telomeres in mouse cells has recently been associated with the 53BP1 repair protein and LINC-domain complex [START_REF] Dimitrova | 53BP1 promotes nonhomologous end joining of telomeres by increasing chromatin mobility[END_REF][START_REF] Lottersberger | 53BP1 and the LINC Complex Promote Microtubule-Dependent DSB Mobility and DNA Repair[END_REF].

As mentioned before, a second transducer kinase pathway can be activated as a response to DNA damage. This pathway relies on ATR. In the DSB repair, ATR is activated when the resection of DNA ends takes place, and therefore ssDNA overhangs of certain length are present [START_REF] Shiotani | Single-stranded DNA orchestrates an ATM-to-ATR switch at DNA breaks[END_REF]. In this process, RPA that coats ssDNA, is required for the recruitment of the ATR-ATRIP complex to the sites of DNA damage [Zou L. and Elledge SJ., 2003]. In order to be activated at the site of ssDNA, ATR-ATRIP interacts with several other proteins. For example, TopBP1 is one of the best characterized proteins that contains an ATR-activation domain to promote ATR kinase activity through interaction with both ATR and ATRIP [START_REF] Kumagai | TopBP1 activates the ATR-ATRIP complex[END_REF]Mordes DA. et al., 2008].

Interestingly, TopBP1 can be activated through phosphorylation by ATM [Yoo HY. et al., 2007]. Apart from that, ATM may also promote the recruitment of of TopBP1 to sites of DNA damage through γH2AX and Mdc1 [START_REF] Wang | MDC1 collaborates with TopBP1 in DNA replication checkpoint control[END_REF].

It turned out that the MRN complex (through its subunit Nbs1) is important for activation of ATR [START_REF] Shiotani | Two distinct modes of ATR activation orchestrated by Rad17 and Nbs1[END_REF]. In line with this, MRN can also recruit TopBP1 to ssDNA-to-dsDNA junctions [Duursma AM et al., 2013]. Recently another TopBP1independent way to activate ATR was described. Human RPA-binding protein ETAA1 can directly bind to RPA and propagate ATR signalling [START_REF] Haahr | Activation of the ATR kinase by the RPA-binding protein ETAA1[END_REF]Lee YC. et al., 2016]. Last but not least, ATR can be activated via autophosphorylation [START_REF] Liu | ATR autophosphorylation as a molecular switch for checkpoint activation[END_REF].

Altogether, activation and recruitment of ATR-ATRIP complex to the sites of DSBs involves several factors and yet more to be discovered. The key substrate in the ATR pathway is Chk1. Activation of Chk1 triggers important pathways in cell homeostasis, such as response to replication stress, apoptosis and many others [Flynn RL. and Zou L., 2011;Blackford AN. and Jackson SP., 2017].

Anti-fusion mechanisms at telomeres

3.4.1. Telomere factors controlling fusions. In budding yeast, several different mechanisms to prevent fusions have been described. One of them relies on the protein Rap1 (Repressor Activator Protein 1). In 1985, this protein was initially identified as a DNA binding factor which interacts specifically with the 5'-upstream region of three yeast genes, TEF1, TEF2 and RP51A, whose products are part of the translation apparatus [START_REF] Huet | A general upstream binding factor for genes of the yeast translational apparatus[END_REF]. At that time, this DNA binding factor was temporarily called TUF, for translational upstream factor [START_REF] Huet | A general upstream binding factor for genes of the yeast translational apparatus[END_REF]. Although there was no known connection at that time, another study identified this factor to bind telomeric repeats directly [START_REF] Berman | Identification of a telomere-binding activity from yeast[END_REF]. The link between the two has been established later when Shore and Nasmyth purified the same protein than TUF and described it as a transcriptional regulator that can play a role in either repression or activation of transcription, and therefore dubbed it Rap1 [START_REF] Shore | Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements[END_REF]. Important discoveries were then to show that Rap1 is localized on telomeric DNA in budding yeasts [Conrad MN. et al., 1990;[START_REF] Klein | Localization of RAP1 and topoisomerase II in nuclei and meiotic chromosomes of yeast[END_REF], covers the entire length of telomeric DNA [START_REF] Gilson | Distortion of the DNA double helix by RAP1 at silencers and multiple telomeric binding sites[END_REF] and regulates telomere length [Lustig AJ. et al., 1990]. Many more outstanding findings were observed later on, which broaden the spectrum of yeast Rap1 functions in heterochromatin formation, telomerase regulation and senescence [START_REF] Moretti | Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1[END_REF][START_REF] Hecht | Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: a molecular model for the formation of heterochromatin in yeast[END_REF][START_REF] Marcand | Silencing of genes at nontelomeric sites in yeast is controlled by sequestration of silencing factors at telomeres by Rap 1 protein[END_REF][START_REF] Maillet | Evidence for silencing compartments within the yeast nucleus: a role for telomere proximity and Sir protein concentration in silencer-mediated repression[END_REF][START_REF] Marcand | A protein-counting mechanism for telomere length regulation in yeast[END_REF]Platt JM. et al., 2013].

Rap1 is a key protein to protect against c-NHEJ in yeast [Pardo B. and Marcand S., 2005]. It can do so either directly through its RCT domain or via recruitment of two other proteins, Sir4 and Rif1 [START_REF] Marcand | Multiple pathways inhibit NHEJ at telomeres[END_REF]. In addition to Rap1, Nej1 in a complex with Lif1 and Dnl4 prevent telomere fusions due to telomerase dysfunction [Liti G. and Louis EJ, 2003]. Notably, yeast Ku heterodimer (Yku) rapidly associates with the DNA at the damaged sites and prevents resection of DNA through inhibition of MRX complex, which as a consequence prevents fusions [Bertuch AA. and Lundblad V., 2003;Celli GB. et al., 2006].

Finally, higher order telomeric chromatin conformation could play a role in budding yeast to prevent fusion. Although it was not possible to detect conventional t-loops, it was reported that yeast telomeres can form fold-back structures through Rif2-mediated Rpd3L recruitment to telomeres [START_REF] Poschke | Rif2 promotes a telomere fold-back structure through Rpd3L recruitment in budding yeast[END_REF].

The identification of human RAP1 was obtained thanks to a yeast two-hybrid screen of HeLa cells with TRF2 as a bait [START_REF] Li | Identification of human Rap1: implications for telomere evolution[END_REF]. Comparison of RAP1 structure within different species (H.sapiens, S.cerevisiae, K.lactis) revealed a high degree of domain conservation; however, the sequence similarities are surprisingly low [START_REF] Li | Identification of human Rap1: implications for telomere evolution[END_REF]. Importantly, in contrast to budding yeast, mammalian RAP1 does not bind telomeric DNA directly but through its direct interaction with TRF2 [START_REF] Li | Identification of human Rap1: implications for telomere evolution[END_REF]. Some of the yeast Rap1 functions were confirmed in mice and humans. For instance, it was demonstrated that both mouse and human RAP1 binds to telomeric and extra-telomeric sites and regulates the transcription of its target genes, specifically those involved in the metabolism control [Martinez P. et al., 2010;[START_REF] Yang | Human telomeric proteins occupy selective interstitial sites[END_REF][START_REF] Yeung | Nontelomeric role for Rap1 in regulating metabolism and protecting against obesity[END_REF]Martinez P. et al., 2013].

Interestingly, a cytoplasmic fraction of RAP1 was found to regulate NF-κB signalling pathway [START_REF] Teo | Telomere-independent Rap1 is an IKK adaptor and regulates NF-kappaB-dependent gene expression[END_REF]. Some early studies also reported that RAP1 can negatively regulate the telomere length [START_REF] Li | Rap1 affects the length and heterogeneity of human telomeres[END_REF]O'Connor MS. et al., 2004], although, this was not confirmed by means of TALEN RAP1 knockout [Kabir S. et al., 2014]. Since different cell lines were used to measure the length of telomeres upon RAP1 downregulation, this may suggest that RAP1 controls the length in cell type-dependent fashion.

Although yeast RAP1 is a key anti-fusion protein, conflicting results regarding its role as an anti-fusion factor in mammals were reported. Indeed, mouse telomeres lacking RAP1 did not develop DNA damage response activation [Sfeir A. et al., 2010;Kabir S. et al., 2014] but can lead to telomere recombination by HDR [Sfeir A. et al., 2010]. As an outcome, this can trigger telomere resection and fusion [Rai R. et al., 2016]. In vitro, human RAP1 has been shown to protect against NHEJ either in cooperation with TRF2 or upon tethering to the telomeric DNA when TRF2 is removed [Bae NS. and Baumann P., 2007;Sarthy J. et al., 2009;Bombarde O. t al., 2010]. However, none of the studies in mice revealed RAP1 role as anti-NHEJ factor [Martinez P. et al., 2010;Sfeir A. et al., 2010] except one observation where upon telomerase dysfunction, RAP1-defficient mice are characterized by progressive telomere shortening, telomere end-to-end fusions and telomere loss [Martinez P. et al., 2016].

In this regard, Rai and co-workers identified that BRCT and Myb domains of RAP1 are important to prevent telomere-free fusions and signal-free ends [Rai R. et al., 2016]. They showed that RAP1 in cooperation with TRF2 are required to fully repress PARP1 and SLX4 localization at telomeres and further t-loop resolution and telomere loss due to circlemediated excision [Rai R. et al., 2016].

Importantly, the anti-fusion properties of yeast Rap1 are expected to depend on its interacting partner TRF2. Interestingly, in addition to be the RAP1 recruiter at telomeres, TRF2 exhibits potent anti-fusion activities independently of RAP1. TRF2 dysfunction leads to massive end-to-end-fusions, which are ligase IV-dependent [van Steensel B. et al., 1998;[START_REF] Smogorzewska | DNA ligase IV-dependent NHEJ of deprotected mammalian telomeres in G1 and G2[END_REF].

What is the mechanism of telomere protection that depends on TRF2?

One mechanism relies on t-loops, which are the terminal loops that results from invasion of the 3' overhang into the duplex part of telomeric DNA forming a lasso-like structure [Doksani Y. et al., 2013;Benarroch-Popivker D. et al., 2016]. TRF2 promotes the formation and stabilization of t-loops and protects them from cleavage by resolvases [Poulet A. et al., 2009;Doksani Y. et al., 2013;[START_REF] Schmutz | TRF2 binds branched DNA to safeguard telomere integrity[END_REF]. It does so through either basic N-terminal domain [Saint-Leger A. et al., 2014] or by means of homodimerization domain (TRFH) [Benarroch-Popivker D. et al., 2016, presented in this manuscript].

TRF2 also interacts with other proteins to prevent NHEJ, like Ku, in order to repress initial steps of NHEJ [START_REF] Ribes-Zamora | TRF2 interaction with Ku heterotetramerization interface gives insight into c-NHEJ prevention at human telomeres[END_REF]. TRF2 also cooperates with Apollo to protect from fusions and aberrant telomere recombination [START_REF] Lenain | The Apollo 5' exonuclease functions together with TRF2 to protect telomeres from DNA repair[END_REF][START_REF] Van Overbeek | Apollo, an Artemis-related nuclease, interacts with TRF2 and protects human telomeres in S phase[END_REF]Lam YC. et al., 2010]. Apollo is a Artemis-like nuclease that has 5ʹ to 3ʹ exonuclease activity, which can be regulated by TRF2 [START_REF] Ye | TRF2 and apollo cooperate with topoisomerase 2alpha to protect human telomeres from replicative damage[END_REF].

Topoisomerase III alpha was shown to influence chromosome stability in cooperation with BLM and TRF2 because its dysfunction results in formation of anaphase bridges and degradation of the 3'-overhangs [START_REF] Temime-Smaali | Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres[END_REF]. Additionally, ERCC1/XPF complex interacts with TRF2 and is important for the maintenance of the 3'-overhang, which per se is sufficient to prevent telomere fusion, even when TRF2 is inhibited [Zhu XD. et al., 2003].

Outside mammals, Taz1, a functional homolog of TRF2 in fission yeast [START_REF] Deng | Fission yeast telomere-binding protein Taz1 is a functional but not a structural counterpart of human TRF1 and TRF2[END_REF], interact with a RAP1 homolog to prevent telomere fusion [Miller KM. et al., 2005].

It has been reported that a-NHEJ is activated in cells lacking Ku and is enhanced by further TPP1-POT1 and TRF2 removal [START_REF] Sfeir | Removal of shelterin reveals the telomere end-protection problem[END_REF]. In addition, DNA-PK inhibits a-NHEJ in vitro [START_REF] Bombarde | TRF2/RAP1 and DNA-PK mediate a double protection against joining at telomeric ends[END_REF].

How cell cycle controls telomere fusion.

DDR at telomeres is controlled by several shelterin factors. TRF2 has been shown to prevent ATM activation, whereas TPP1-POT and TRF1 -ATR [START_REF] Guo | Dysfunctional telomeres activate an ATM-ATR-dependent DNA damage response to suppress tumorigenesis[END_REF]Denchi EL. and de Lange T., 2007;[START_REF] Sfeir | Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication[END_REF].

Besides excessive DNA damage, TRF1 dysfunction is characterized by multiple telomere signals that lead to chromosome fragility and sister fusions as a result of aberrant telomere recombination [Martinez P. et al., 2009;[START_REF] Sfeir | Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication[END_REF].

It is noteworthy that DDR at telomeres is coupled with cell cycle and number of divisions. Thus, a body of evidence indicates that different DDR proteins accumulate at telomeres in the cell cycle-dependent manner [Verdun RE. et al., 2005]. Numerous studies show that DDR and DNA repair proteins compete with each other and with shelterin for binding to telomeric DNA, and this guides the choice of DNA repair pathway [START_REF] Deng | Multiple roles for MRE11 at uncapped telomeres[END_REF][START_REF] Dimitrova | Cell cycle-dependent role of MRN at dysfunctional telomeres: ATM signaling-dependent induction of nonhomologous end joining (NHEJ) in G1 and resection-mediated inhibition of NHEJ in G2[END_REF][START_REF] Rai | NBS1 Phosphorylation Status Dictates Repair Choice of Dysfunctional Telomeres[END_REF]Muraki K. and Murnane JP., 2018]. In line with this, the main competition occurs between HRR and c-NHEJ (Figure 9). It is now known that c-NHEJ, as well as a-NHEJ, can be active throughout the cell cycle.

However, HRR outcompetes c-NHEJ in S/G2 phases, whereas NHEJ is a preferred choice in G1, and both HRR and c-NHEJ inhibit a-NHEJ pathway throughout the cell cycle [Daley JM. and Sung P., 2014]. Interestingly, during mitosis cells tend to keep any repair activities shut down, and therefore repair the accumulated damage later in interphase because repair during mitosis can be highly deleterious [Orthwein A. et al., 2015].

Figure 9. Cell-cycle control of DSB repair.

The choice between NHEJ and HRR depends on competition between 53BP1 and MRN/ BRCA1 (Figure 10) [START_REF] Dimitrova | Cell cycle-dependent role of MRN at dysfunctional telomeres: ATM signaling-dependent induction of nonhomologous end joining (NHEJ) in G1 and resection-mediated inhibition of NHEJ in G2[END_REF][START_REF] Zimmermann | 53BP1: pro choice in DNA repair[END_REF]. c-NHEJ at telomeres depends on 53BP1. At functional telomeres, TRF2 keeps the ends blocked for DDR, whereas removal of TRF2 leads to the occupation of telomeres by phosphorylated 53BP1. In G1, Rif1 and PTIP are recruited to 53BP1 to mediate exclusion of BRCA1 and MRN/CtIP and prevent therefore 5'-resection (Figure 10). In S/G2, CtIP becomes phosphorylated by CDK and forms a complex with MRN and BRCA1. This complex replaces 53BP1 at telomeres and initiates resection [START_REF] Zimmermann | 53BP1: pro choice in DNA repair[END_REF]Daley JM. and Sung P., 2014]. Recent work also revealed that phosphorylation/dephosphorylation of Nbs1 (MRN subunit) and its association with TRF2 acts as a switch between all three repair pathways [START_REF] Rai | NBS1 Phosphorylation Status Dictates Repair Choice of Dysfunctional Telomeres[END_REF]. Moreover, through modulation of de-ubiquitination, TRF2 can also control the recruitment of RNF168, which in turn recruits 53BP1 [START_REF] Okamoto | A two-step mechanism for TRF2-mediated chromosome-end protection[END_REF].

Figure 10. DSB occupancy in G1 and S/G2 phases of cell cycle.

In primary cells, accumulation of DDR at telomeres increases with the number of cell divisions and is passed through generations [Cesare AJ. et al., 2013]. Therefore, telomere becomes a target of persistent and irreparable damage [START_REF] Hewitt | Telomeres are favoured targets of a persistent DNA damage response in ageing and stress-induced senescence[END_REF]Suzuki M. et al., 2012;Fumagalli M. et al., 2014]. Together with telomere shortening that occurs through divisions, DDR may lead to ceased divisions and replicative senescence or apoptosis if the cell cannot survive damage anymore [START_REF] Arnoult | Complex interactions between the DNA-damage response and mammalian telomeres[END_REF]. Interestingly, only five dysfunctional telomeres are enough to trigger senescence [Kaul Z. et al., 2011].

Many other proteins and protein modifications that are not described here, has been shown to control DDR and DNA damage at telomeres and therefore, control chromosome fusions. A recent work shows evidence that in mammals besides huge protein network, DDR at deprotected telomeres is mediated by telomeric lncRNAs, which are called DDRNAs [START_REF] Rossiello | DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs[END_REF].

Altogether, telomere fusions can occur through different DSB repair pathways which are controlled by comprehensive protein and RNA networks.

34

Chapter 2

Research project

Objectives of the study

In the begin of the research project (2015), there was no clear vision about RAP1 role in telomere protection. The RAP1 paradox emerged based on several observations. In yeast, it has been shown to be among the main factors to protect telomeres from fusions [Pardo B. and Marcand S., 2005;[START_REF] Marcand | How do telomeres and NHEJ coexist?[END_REF]. However, in mammals its role at telomeres was questionable due to the fact that mouse and human RAP1 knockout cells do not display telomere-dysfunction phenotypes, and RAP1-deficient mice are alive and fertile with no chromosome fusions over generations [Sfeir A. et al., 2010;Martinez P. et al., 2010;Kabir S. et al., 2014]. On the other hand, in vitro artificially tethered to telomeres, RAP1 was able to rescue fusions upon TRF2 dysfunction [Sarthy J. et al., 2009]. Thus, it was tempting to speculate that RAP1 role in telomere protection could be masked by the immense effect of TRF2 at telomeres. Therefore, we aimed to decipher how RAP1 controls NHEJ in human. For this reason, we set the next objectives:

1. To study the contribution of RAP1 in the control of telomere fusions upon TRF2 dysfunction. To do so, we used a topology-deficient mutant of TRF2 (Top-less). The results are discussed in section 2. Article1. 2. To reveal whether RAP1 has any role in telomere protection of replicative senescent cells. The results of this part are discussed in section 3. Article 2. The shelterin proteins protect telomeres against activation of the DNA damage checkpoints and recombinational repair. We show here that a dimer 0 of the shelterin subunit TRF2 wraps 90 bp of DNA through several lysine and arginine residues localized around its homodimerization domain. The expression of a wrapping-deficient TRF2 mutant, named Top-less, alters telomeric DNA topology, decreases the number of terminal loops (t-loops), and triggers the ATM checkpoint, while still protecting telomeres against non-homologous end joining (NHEJ). In Top-less cells, the protection against NHEJ is alleviated if the expression of the TRF2-interacting protein RAP1 is reduced. We conclude that a distinctive topological state of telomeric DNA, controlled by the TRF2-dependent DNA wrapping and linked to t-loop formation, inhibits both ATM activation and NHEJ. The presence of RAP1 at telomeres appears as a backup mechanism to prevent NHEJ when topology-mediated telomere protection is impaired. A p value < 0.05 was calculated for the difference between the medians of the 2 volume distributions, attesting that, as for TRF2, bigger complexes have smaller CL and vice-versa.

(H) 2D-probability density map of contour length (CL) and circumference obtained for the TRF2/DNA complexes representing the probability to find a protein/DNA complex with a given DNA contour length and the corresponding circumference. Note the slope close to 1 of the linear fit.

(I) Distribution of the calculated circumference for TRFH/DNA complexes obtained from the deconvoluted AFM data set shown in Figure 1B. (D) Positions of the protected lysines in the TRFH domain infer chirality in the interaction, thus forcing strands to cross. From earlier work [START_REF] Amiard | A topological mechanism for TRF2-enhanced strand invasion[END_REF] we know that TRF2 introduces positive supercoils in a relaxed circular substrate. Two models can be drawn:

In I, DNA strands are crossing at the top of the TRFH structure giving a right handed wrapping.

This would explain the positive supercoils caused by TRF2 in DNA.

In II, DNA strands are crossing at the bottom of the TRFH structure. In this case the wrapping is left handed. This does not fit with the positive supercoils reported. (G) Same experiment as above using an anti-TIN2 antibody.

Material and Methods

AFM imaging

Complexes deposition:

10 µl of a solution of DNA and proteins in 5 mM HEPES pH 7.4, 150 mM KCl and 1 mM MgCl 2 was incubated 20 min at 25°C. The protein/DNA molar ratios used were the following:

(2.5/10) nM for TRF2, (1100/7) nM for TRFH, (5/10) nM for Top-less. After incubation, samples were crosslinked with glutaraldehyde (0.1% final concentration) for 30 min on ice. Before applying the sample on freshly cleaved mica, the concentration of MgCl 2 was increased to 10 mM. After 2 min on mica the sample was washed with 1 ml of deionized water and dried under a gentle N 2 flow. Imaging was performed on a Multimode 8 equipped with E-scanner controlled by a Nanoscope V (Bruker AXS, Santa. Barbara, CA), in air under Tapping Mode using silicon tips (RTESP, 300kHz). Images were recorded at 1.5-2.0 Hz over 1 μm wide scan area (512×512 pixels). Raw images were flattened using the manufacturer's software (Nanoscope Analysis 1.40)

and converted into TIF files.

Contour Length and volume measurements:

Contour lengths (CLs) for each molecule were manually traced and measured using Image J software (http://imagej.nih.gov/ij/). For DNA-protein complexes the read-through DNA length method was used. Measurements of the naked DNA were performed using the naked molecules found in the images corresponding to the different binding experiments.

Although, the expected contour length for a B-DNA molecule of 650 bp is 221 nm (650 bp* 0.34 nm/bp), the measured mean values obtained for each naked DNA is shorter (192 ± 11 nm, 189 ± 9 nm and 188 ± 9 nm for TRF2, TRFH and Top-less binding experiment respectively). This discrepancy is related to a DNA shortening possibly due to a partial B-to A-form transition induced by the drying step [START_REF] Rivetti | Accurate length determination of DNA molecules visualized by atomic force microscopy: evidence for a partial B-to A-form transition on mica[END_REF]. The mean helical rise corresponding to the three different naked DNA mean CLs is then 0.29 nm/bp, that gives rise to 93 bp of DNA wrapping (27nm/0.29 nm/bp).

Volumes were calculated as ellipsoids using the formula:

V=4/3 *π *(D/2*d/2*h)
where D, d and h correspond to major diameter, minor diameter and height respectively.

These parameters were measured using Image SXM software (www.liv.ac.uk/~sdb/ImageSXM).

At least 130 objects were scored for each condition.

Volume deconvolution:

The dimensions of an object imaged by AFM are affected by the broadening effect due to the tipsample convolution radius. The relationship between the experimental width of the sample in the image, W, the radius of curvature of the tip, Rc, and the radius of curvature of the sample, Rm, is

given by the equation [START_REF] Bustamante | Scanning force microscopy of nucleic acids and nucleoprotein assemblies[END_REF]:

W=4Rc Rm 1/2
If two objects are measured with the same probe the ratio between them is the following:

W1/W2 = (R1/R2) 1/2
The double-stranded DNA width (2 nm), involved in the protein complex, can be used as an internal reference for size. This allows us to obtain the real diameters for the protein complex [START_REF] Nettikadan | Quantitative analysis of the transcription factor AP2 binding to DNA by atomic force microscopy[END_REF].

R1= (W1/W2) 2 * R2
Where R1 and R2 are the real dimensions of the protein complex and the DNA respectively, while W1 and W2 are their measured dimensions.

Using the deconvoluted values corresponding to the protein diameters, it is possible to calculate the deconvoluted volumes.

Circumference estimation :

Once the deconvoluted values for the minor d and major D diameters are obtained, using the Ramanujan approximation it is possible to calculate the deconvoluted circumference of the ellipsoid using the following formula:

C ≈ π *(3*(d+D)-((3d+D)*(d+3D)) 1/2 )
Plots and statistics :

All the histograms represent the distribution of a measured or calculated parameter expressed in percentage of events. To obtain the mean value corresponding to each subpopulation emerging from multimodal distributions, a multi-Gaussian fitting has been applied using the QtiPlot data analysis and scientific visualization (http://soft.proindependent.com/qtiplot.html).

All the parameters obtained by the Gaussian fitting are expressed in the text as mean ± FWHM

(Full width at half maximum). The 2D-probability density map of contour length (CL) and circumference measured by AFM for the complexes TRF2/DNA is obtained using R open source software (http://www.R-project.org). The software was used to calculate the bivariate kernel density estimation. The resulting 2D map represents the probability to find a protein/DNA complex with a given DNA contour length and the corresponding protein circumference. The darker the region in which the data fall, the higher is their probability density.

The linear fit applied to the scatter plot corresponding to the correlation graph of CLs as a function of circumferences for TRF2/DNA complexes was performed imposing a y-intercept of 192 nm (mean value of the corresponding naked DNA) and calculated using the QtiPlot software.

The analysis of the TRFH/DNA volume distributions as a function of TRFH/DNA CL distribution was performed using GraphPad Prism v 5.03. The results are shown as a box and whiskers plot. To the two volume populations the Wilcoxon matched-pairs signed rank test was applied giving a p value < 0.05.

Strand invasion assay, topology assays, EMSAs, and Holliday junction migration assays

Strand invasion assays were performed as described previously [START_REF] Poulet | The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA[END_REF].

Topology assays were also performed as described previously [START_REF] Amiard | A topological mechanism for TRF2-enhanced strand invasion[END_REF][START_REF] Poulet | The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA[END_REF], but using pLTelo, a pLEU500-Tc [START_REF] Chen | Activity of a plasmid-borne leu-500 promoter depends on the transcription and translation of an adjacent gene[END_REF] -based plasmid containing 650 bp of human telomeric repeats between BstAPI and BamHI sites. EMSAs were performed using a 106bp DNA probe containing 16 TTAGGG repeats flanked by a 5-bp (CAGCC) sequence at the 5' and a 5 bp (CCTTG) sequence at the 3' end. A total of 5 nM of 5' labeled probe was incubated in a total volume of 10 µl in 20 mM HEPES pH 8, 100 mM NaCl, and 500 ng/µl of acetylated BSA on ice for 15 min. Ficoll was added to a final concentration of 3% and the samples loaded on a 1% agarose gel with 0.5 TBE under 7 V/cm. Migration was performed at the same voltage for 30 min. The gels were then dried and analyzed using phosphorimager screens. Analysis was performed on a Typhoon FLA 9500 (GE Healthcare) using the Image Quant software (GE Healthcare). Holliday junction migration assays were performed as described previously (Poulet et al., 2009).

Circular dichroism (CD)

Far-UV CD spectra (between 195 nm and 260 nm) were recorded using a Jasco J-815 spectropolarimeter equipped with a Peltier temperature control unit. The spectra were acquired as an average of five scans with a scan speed of 100 nm/min and a response time of 2 s. CD measurements were performed at 20°C, using 1-mm quartz cells. TRF2 and Top-less samples were at 4.6 µM in 10 mM Tris buffer, pH 8.0, 60 mM NaCl, 10% glycerol, and 0.2 mM DTT.

Pull-down assay

A total of 10 µg of purified His-fusion TRF2 or His-fusion Top-less proteins were incubated with cobalt-based magnetic beads (Dynabeads, Lifetechnologies) at 4°C for 30 min in 50 mM sodium phosphate pH 8, 300 mM NaCl, and 0.01% Tween 20. After two washes using the same buffer, 15 µg of purified Rap1 were added at 4°C for 90 min. The supernatant (unbound fraction, UB) was precipitated with cold acetone and resuspended in Laemmli loading buffer.

After two washes, the magnetic beads containing the His-tagged proteins and associated Rap1 protein (bound fraction, B) were resuspended in Laemmli loading buffer and analyzed by SDS-PAGE.

Direct Stochastic Optical Reconstruction Microscopy (STORM) experiments

Preparation of nuclei, psoralen crosslinking and chromatin spreading :

Samples were prepared using the protocol described in Doksani et al (Doksani et al., 2013) with minor modification: 5 × 10 6 nuclei (HT1080 super-telomerase cells with down-regulation of endogenous TERF2 by siRNA and ectopic expression of TRF2 or Top-less) were isolated as described in [START_REF] Pipkin | A reliable method to display authentic DNase I hypersensitive sites at long-ranges in single-copy genes from large genomes[END_REF][START_REF] Pipkin | A reliable method to display authentic DNase I hypersensitive sites at long-ranges in single-copy genes from large genomes[END_REF][START_REF] Pipkin | A reliable method to display authentic DNase I hypersensitive sites at long-ranges in single-copy genes from large genomes[END_REF], resuspended in 1 ml of NWB (10 mM Tris-HCl pH 7.4, 15 mM NaCl, 60 mM KCl, 5 mM EDTA, 300 mM sucrose), and incubated in a 3.5 cm dish, on ice, in the dark, while stirring for 5 min with 100 μg/ml Trioxsalen (SIGMA). Nuclei were exposed to 365 nm UV light at 2 cm from the light source (model UVL-56, UVP) for 30 min, while stirring on ice. After crosslinking, nuclei were collected, washed once with ice-cold NWB, and resuspended in 250 µl of NWB. For spreading, nuclei were diluted 1:10 in spreading buffer (10 mM Tris-HCl 7.4, 10 mM EDTA, 0.05% SDS, 1 M NaCl, pre-warmed at 37°C) and 100 μl of the suspension was immediately spread on a 18 mm diameter 1.5H coverslip (Marienfeld) using a Shandon Cytospin 3 (600 rpm, 1 min, medium acceleration). Samples were fixed in methanol at -20°C for 10 min followed by 1 min in acetone at -20°C. The coverslips were washed in PBS 1x and dehydrated through a 70%, 95%, 100% ethanol series before performing FISH.

Fluorescence in situ hybridization

The PNA probe [CCCTAA]3, conjugated with Alexa Fluor 647 fluorophore (PNA Bio INC.), was resuspended in water at a stock concentration of 20 µM and diluted 1:100 in the hybridization buffer solution (70% formamide, 10 mM Tris-HCl pH 7.2, 1:10 blocking buffer) before FISH labeling. 10 µl of this solution was put on a glass slide and ethanol-dried samples on coverslips were then put on top of the drop. The slide-coverslip "sandwich" was placed at 80ºC for 10 min on heat block, with the slide-side facing the block, to allow DNA denaturation.

Then the samples were put overnight in the dark at room temperature in a humidified box in order to let the hybridation reaction to occur. The coverslip was then removed from the slide and washed twice for 15 min with 70% formamide; 10 mM Tris-HCl pH 7. , 1997-2014) taking into account only the objects having a length ≥ 1500 μm (corresponding to 5000 bp). Molecules having gaps longer than 0.5 μm and kinked, knobbed-like or branched molecules were not scored as in Doksani et al. (Doksani et al., 2013).

Western blots

A total of 30 µg of total extract was loaded on a 4-20% acrylamide gradient SDS gel in Laemmli buffer. After separation, proteins were transferred on an Immobilon-FL PVDF membrane (Millipore) and TRF2 was revealed using an anti-TRF2 primary antibody from mouse (Imgenex IMG-124A) and an IRdye-labeled goat anti-mouse antibody (Li-Cor) under the conditions recommended by the supplier. Bands were revealed using the Odyssey apparatus and corresponding software (Li-Cor). For shelterin proteins, the following antibodies were used:

Rabbit Polyclonal anti-TRF1 (Santa Cruz Biotechnology, Inc., sc-6165-R); Rabbit Polyclonal anti-POT1 (Novus Biologicals, NB100-56429); Rabbit Polyclonal anti-TPP1 (Bethyl Laboratories, Inc., A303-069A): Rabbit Polyclonal anti-TIN2 (Abcam, ab64386); Rabbit Polyclonal anti-RAP1 (Bethyl Laboratories, Inc., A300-306A); Rabbit Monoclonal anti-CHK2 (phospho T68), Abcam ab32148); Mouse Monoclonal anti-CHK2 (BD Biosciences, 611571).

Chromatin immunoprecipitation

Anti-Myc ChIP was performed as described previously [START_REF] Simonet | The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats[END_REF] with minor modifications. Briefly, HeLa cells were cross-linked for 12 min with 1% formaldehyde and washed with cold PBS. Cells were centrifuged and the pellet was resuspended in cell lysis buffer

(5 mM PIPES pH8, 85 mM KCl, 0.5% NP40 and protease inhibitors). The cells were disrupted with a dounce homogenizer and centrifuged at 4°C. The pellet was resuspended in nucleus lysis buffer (50 mM Tris-HCL pH 8, 10 mM EDTA, 1% SDS, protease inhibitors) and cells were sonicated using a Bioruptor to obtain an average fragment size of 400 bp. IPs were set up with 40 µg of DNA, and Myc-Tag (9B11 Cell Signaling, mouse) and H3 (1791 abcam, rabbit polyclonal) antibodies were incubated overnight. Magnetic beads (Dynabeads, Life Technologies) were added for 2 hours. The beads were washed with a low salt buffer (150 mM NaCl, 1% Triton X-100, 0.1% SDS) and a high salt buffer (500 mM NaCl, 1% Triton X-100, 0.1% SDS), followed by a lithium salt buffer (0.25 M LiCl, 1% NP40, 1% deoxycholic acid). Chromatin was eluted with 1% SDS and 0.1 M NaHCO 3 solution, and the cross-link was reversed at 65°C overnight.

The DNA was treated with RNase for 20 min, proteinase K for 1 hour at 50°C, prior to phenolchloroform purification, and ethanol precipitation. DNA samples were dissolved in TE buffer, blotted onto a N+ Hybond membrane (GE Healthcare) using a slot blot apparatus, and hybridized with the same probe as used for the trioxsalen experiments. Membranes analysis was performed as described for trioxsalen experiments. For TRF2, RAP1 and TIN2 ChIP the following antibodies were used: Rabbit polyclonal anti-RAP1 from Bethyl (A300-306A); Rabbit polyclonal anti-TIN2 from Abcam (ab64386); Rabbit polyclonal anti-TRF2 from Novus Biologicals (NB110-57130).

TERRA slot blot

RNA was extracted from 5 million HeLa cells treated (or not) with doxycycline as described above and transduced by the Empty, TRF2, or Top-less expressing vectors (see below for transduction conditions) using the RNAeasy kit from Qiagen. RNA (20 µg) from each condition was digested with 2 units of RNase free DNaseI (New englend Biolabs) at 37°C for 10 min and heated at 75°C for 10 min. From these, 10 µg were digested with 1 µg of RNase (Life Technologies) at 37°C for 10 min. A total of 5 µl of 5 loading buffer (80 mM MOPS, 6 mM EDTA, 2.6% formaldehyde, 30% formamide, 20 mM sodium acetate) was added and the samples were heated at 75°C for 10 min before slot blotting using a N+ Hybond membrane (GE Healthcare). Before and after slot blotting, wells were washed with 200 µl of 10x SSC. After UV crosslinking of the membrane and baking at 80°C during 15 min, bands were revealed by sequential hybridization in Church buffer with a telomeric probe (the same used for Trioxsalen experiments) and a probe obtained from a 500-bp fragment corresponding to the sequence of the human 26S RNA (precursor of 18S RNA [START_REF] Vincent | S26 ribosomal protein RNA: an invariant control for gene regulation experiments in eucaryotic cells and tissues[END_REF]). Membranes analysis was performed as described for trioxsalen experiments.

Immunofluorescence detection of telomere dysfunction-induced foci

Slides were fixed with 4% formaldehyde in PBS at room temperature for 10 min, and then incubated for 90 min with blocking buffer (PBS, 1% Triton X-100, 1% BSA and 5% Donkey serum), followed by incubation overnight at 4°C with anti-TRF1 (sc-6165; Santa Cruz Biotechnology) and anti-γH2AX (05-636; Upstate) antibodies. Cells were then washed with PBS and incubated with anti-rabbit Alexa488 (A21206; Molecular probes) and anti-mouse Alexa555 (A31570; Molecular probes) antibodies. After washing with PBS, the nucleus was labeled with DAPI (VECTASHIELD mounting medium with DAPI, Vector Laboratories). For IF-PNA FISH labelling, slides were first treated as above using a rabbit anti-53BP1 antibody (NB100-305; Novus Biological) followed by a goat anti-rabbit Alexa 488 antibody (111-545-144; Jackson ImmunoResearch) then fixed again with 4% formaldehyde in PBS at room temperature for 2 min, de-hydrated by successive incubation in 50%, 75% and 100% ethanol for three min.

Hybridization was performed at 80°C in70% Formamide, 10 mM Tris pH 7.2 for three min followed by an incubation overnight at room temperature. Slides were washed first in the Formamide, Tris solution above, then in a 150 mM NaCl, 50 mM Tris pH 7.5 solution and finally PBS. Mounting was performed as above.

IF images were produced using a Zeiss LSM 5 Exciter confocal laser scanning microscope (Zeiss, Jena, Germany) and analyzed using the ZEN software. PNAFISH/IF images were obtained on a DeltaVision Elite microscope (GE Healthcare).

Metaphase spreads analysis

For chromosome analysis, cells were arrested in metaphase for 3 hours at 37ºC with 50 ng/ml of colcemid (KaryoMAX, Invitrogen). Cells were incubated for 15 min at 37ºC in hypotonic solution (75 mM KCl), fixed in methanol:acetic acid (3:1), and spread on cold, wet, ethanol-cleaned slides. Slides were fixed in 4% formaldehyde in PBS for 2 min, washed in PBS, digested with pepsin (0.5 mg/ml, 0.01 N HCl) for 10 min at 37ºC, washed in PBS, fixed in 4% formaldehyde in PBS for 2 min, washed in PBS, dehydrated in increasing concentrations of ethanol, and air-dried. Hybridization was then performed using FITC-conjugated (CCCTAA) 3

PNA probe (Panagene) diluted at 50 nM in 70% formamide, 10 mM Tris-HCl (pH7.2), and 1%

blocking reagent (Roche). Slides were denatured at 80ºC for 3 min at room temperature, and hybridization was performed at room temperature in a moist chamber in the dark for 2 hours.

Slides were washed twice for 15 min in 70% formamide and 10 mM Tris-HCl (pH 7.2) and three times for 5 min in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.05% Tween-20 at room temperature. Slides were washed in PBS and mounted in VECTASHIELD with DAPI (Vector laboratories).

Metaphase spreads were visualized on an epifluorescence Axioimager Z2 microscope and analyzed using the metasystem ISIS software.

2D gels

DNA (5 µg) extracted from HeLa cells treated with or without doxycycline as described above and transduced with Empty, TRF2, or Top-less expressing vectors was migrated on an 0.5% agarose in 1 TBE (15-cm gel at 130 V) until the xylene dye was 2 cm from the bottom of the gel. Bands were cut and placed horizontally for a second-dimension electrophoresis performed in 0.5 µg/ml ethidium bromide and 1 TBE (both in the gel and the running buffer).

Markers were run beside each sample band. Migration was performed at 50 V for 14 hours. After migration, DNA was transferred onto a membrane, telomeric DNA was revealed and data were analyzed as above (Trioxsalen experiments).

Overhang assay

The overhang assay was adapted from van Steensel et al., 1998(van Steensel et al., 1998).

Briefly, 10 µg of genomic DNA from HT1080 cells expressing shTERF2 and transduced with either the Empty vector or vectors expressing the TRF2 or Top-less proteins (see above) were digested with 125 and 175 units of HinfI and RsaI (Promega), respectively, overnight at 37°C.

After ethanol precipitation, the samples were divided in two; half was digested with 100 units of E. coli Exonuclease I (New England Biolabs) for 5 hours at 37°C. All samples were hybridized 32 P with 0.2 pmoles of a end-labeled single-stranded (CCCTAA) 3 probe overnight at 50°C.

Hybridized samples were loaded on a 10-cm-long, 0.9% 1 TBE agarose gel and migrated at 6

V/cm for 75 min at room temperature. The gel was then dried on 3 MM paper for 4 hours at 40°C

and exposed on a phosphorimager screen. Analysis was performed as described above on a Typhoon 9500. For denaturing conditions, an in-gel denaturing hybridization was performed on the dried gel, as described previously (Karlseder et al., 2002).

RESULTS

RAP1 is specifically required for telomere protection in senescent fibroblasts.

We passaged human primary lung fibroblasts (MRC-5) at 5% oxygen and we performed western blotting at different population doublings to determine the levels of RAP1 and TRF2 expression (Supplementary Fig. 1A). While, as expected (Fujita K. et al., 2010;Lou Z. et al., 2015) the TRF2 levels greatly decreased (around 80%) in senescent cells, the levels of RAP1 remained nearly constant, suggesting an important role played by RAP1 in these cells. Thus, we asked whether RAP1 is required for telomere protection in senescent cells. The expression of an shRNA against RAP1 in young and senescent cells efficiently downregulated its expression (Supplementary Fig. 1B). In order to monitor telomere protection, we performed

Telomere Dysfunction Induced Foci (TIF) assays by analyzing the colocalization of 53BP1, a DNA damage response (DDR) protein, with a telomeric PNA probe (Fig. 1A-B). The rate of TIFs augmented with increasing the number of cell divisions as previously reported [START_REF] Herbig | Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a)[END_REF]Kaul Z. et al., 2011;Suzuki M. et al., 2012;Fumagalli M. et al., 2014). In agreement with previous studies failing to detect telomere dysfunction upon RAP1 disruption (Martinez P. et al., 2010;Sfeir A. et al., 2010;Kabir S. et al., 2014) the downregulation of RAP1 in young and pre-senescent cells does not increase the rate of TIF. However, the TIF rate significantly increased in fully senescent RAP1-compromised cells, revealing that RAP1 takes over telomere protection against DNA damage checkpoint activation specifically in senescent cells (Fig. 1A-B). Telomere length analysis does not show telomere length alterations due to RAP1 loss,; thus, the increase of TIFs is unlikely to be the consequence of an excess of telomere shortening in RAP1-compromised cells(Supplementary Fig. 1.C-D).

RAP1 prevents telomere fusions in senescent fibroblasts.

Then we investigated whether RAP1 also protects telomeres of senescent cells from telomere fusions by using a PCR-based method that relies on subtelomeric DNA primers which are used to amplify fusions between different chromosome ends (Supplementary Fig. 2A) (Capper R. et al., 2007;Letsolo BM. et al., 2010). We performed PCR reactions with a set of three different subtelomeric primers that in total bind to the ends of approximately 22 chromosomes. Nearly no chromosome fusions were detected in young fibroblasts (fusion frequency of 1.5x10 -5 ; Fig. 2A-B and Supplementary Fig. 2B-C) with a modest increase in pre-senescent cells (frequency of 4.5x10 -5 ). These low rates of telomere fusion were not role of RAP1 in senescent cells. A similar observation was made by Martinez and colleagues in a double RAP1/TERT knockout mice (Martinez P. et al., 2016).

Reversal of senescence is impaired in RAP1 compromised senescent fibroblasts.

Finally, we asked whether RAP1 inhibition could impair the return to growth of senescent cells in case of checkpoint failure. Since it is possible to reverse replicative senescence through p53 inactivation [START_REF] Beauséjour | Reversal of human cellular senescence: roles of the p53 and p16 pathways[END_REF], senescent human primary fibroblasts were infected with either an shRNA against p21 CIP1 (shp21 CIP1 ) or shp21 CIP1 +shRAP1 (Fig. 4A). After approximately 10 days post infection, most of the shp21 CIP1 transduced cells restarted proliferation while losing their SA-β-gal staining (Fig. 4B). In contrast, the number of shp21 CIP1 +shRAP1 cells decreased without losing their SA-βgal staining (Fig. 4B). It is likely that the impaired restart of RAP1 compromised cells results from an increased rate of telomere aberrations. We conclude that senescent cells maintain a level of RAP1 expression sufficient to allow them to restart proliferation in case of checkpoint failure.

DISCUSSION

Here we demonstrate that the downregulation of RAP1 in human primary cells that have reached their replicative capacity give rise to telomeric fusions that are dependent on the classical NHEJ ligase IV repair pathway. Several studies in mouse and human cells have failed to detect a role for RAP1 in telomere protection (Martinez et al., 2010;Sfeir et al., 2010, Kabir et al., 2014). For instance, this was our case in young or pre-senescent cells where depletion of RAP1 did not have an adding effect on telomere fusions. The importance of the anti-fusion role of RAP1 was only evident when telomeres were shorter, in one case upon senescence and in another in HeLa cells treated with the telomerase inhibitor BIBR1532. Upon RAP1 depletion (Supplementary Fig. 3D-E and Fig. 3B), we observe neither telomere length shortening nor signs of telomere dysfunction apart from telomere fusions (Fig. 3D-F).

Altogether, our results unveil that during replicative senescence of human cells, the telomeres switch from a RAP1-independent to a RAP1-dependent mode of telomere protection. We propose that this switch is triggered by the appearance of short telomeres unable to fold into t-loops, which is likely to be the main anti-DNA damage repair mechanism mediated by TRF2 in young cells (Fig. 4C).

The PCR-base method used on this study to detect fusion events relies on the use of sub-telomeric primers. We used three subtelomeric primers in the same PCR reaction to increase the probability of fusion detection. The minimum predicted size of a fusion detected using the 16p probe (e.g. Fig. 2B) is around 7kb, however the majority of the amplicons were shorter than that. This would imply that at least one of the telomeres that fused has no telomeric repeats and even more some of the sub-telomeric DNA should be eroded as reported previously (Capper R. et al., 2007). Martinez and colleagues (2016) showed that RAP1 has a protective role on mouse telomeres but only in Terc-/-mice. In contrast to our cellular settings, telomere shortening was accelerated in double knockout mice (Terc-/-, Rap-/-) compared to Terc-/-. This accelerated telomere shortening upon RAP1 depletion gave rise to an increased number of telomere aberrations such as fusion events, signal free ends, multiple telomere signal and telomere sister chromatid exchanges. Together with our results, RAP1 appears to have the capability to protect against telomere fusions by different mechanisms: by inhibiting classical NHEJ (this study) and by preventing homologous recombination at chromosome ends resulting in fusogenic telomere-free ends (Rai R. et al., 2016;Martinez P. et al., 2016).

Interestingly, RAP1 has been shown to interact with the Sun1, a member of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex and to tether telomeres to the nuclear envelope specifically just after mitosis (Crabbe L et al., 2012). It is therefore possible that the depletion of RAP1 in arrested senescent cells, or in cells with short telomeres, released the telomeres from the nuclear periphery making them more mobile and prone to fusions.

A downregulation of RAP1 in senescent fibroblasts forced to divide due to checkpoint inhibition caused a massive cell death. One explanation could be that accumulated fused chromosomes in the double RAP1 and p21 CIP1 knockdown condition may trigger mitotic catastrophe, which has been described as a mechanism of cell death occurring during or after aberrant mitosis [START_REF] Vakifahmetoglu | Death through a tragedy: mitotic catastrophe[END_REF].

Our findings reveal that RAP1 acts as a mechanism of telomere protection specifically in replicative senescent cells. Whether this telomere capping role of RAP1 drives its conservation through evolution as a constitutive telomeric protein raises the question of the physiological rationale to prevent telomere fusions in senescent cell. engineered cell line with the doxycycline inducible knockout of RAP1 was a gift from Dr.

Songyang (Kim et al., 2017). Cells were grown in DMEM with 10% tetracycline-free serum.

To induce knockout of RAP1, we treated cells for 15 days with doxycycline (2 µg/mL, Sigma).

Telomere shortening was selectively induced by treatment with the telomerase inhibitor BIBR1532 (20 µM, Merck).

Lentivirus production and infection. Lentiviruses were produced by transient calcium phosphate transfection of 293T cells with the virus packaging plasmids, p8.91 and pVSVg, as well as with the lentiviral expression vector that contained the sequence of interest.

Titration was performed approximately 10 days after infection by means of puromycin (1 µg/ ml) selection of clones.

The following shRNA plasmids were purchased from Sigma and used for lentivirus production: pLKO-shScramble, pLKO-shTERF2IP, pLKO-shp21 CIP1 , pLKO-shLIG3, pLKO-shLIG4. Infection with various shRNAs was performed for a minimum of 4 days, and depending on experiment cells were kept in culture for up to 10-15 days after infection (10 days for telomere fusion assay in MRC-5, 15 days -for post-senescent cells). Efficiency of each shRNA was checked routinely by RT-qPCR or western blotting.

Antibodies. The following antibodies were used for western blotting: rabbit polyclonal anti-RAP1, 1:5000 (Bethyl Laboratories, Inc., A300-306A), rabbit polyclonal anti-TRF2, 1:5000 (Novus Biologicals, NB110-57130), mouse monoclonal anti-α-tubulin, 1:2000 (Merck, T9026), rabbit polyclonal anti-GAPDH, 1:1000 (Novus Biologicals 100-56875), mouse monoclonal anti-p21, 1 µg/ml (Abcam, ab16767), HRP goat anti-mouse IgG, 1:10 000 (Vector Laboratories, PI-2000) and HRP goat anti-rabbit IgG, 1:10 000 (Vector Laboratories, PI-1000).

For immunofluorescence we used the next antibodies: rabbit polyclonal anti-53BP1, 1:250 DNA extraction. Genomic DNA was extracted either by proteinase K, RNase A and phenol/ chloroform followed by ethanol precipitation [START_REF] Sambrook | Molecular Cloning: a Laboratory Manual[END_REF] or following instructions of DNA Blood and Tissue Kit (Qiagen).

(
Telomere fusion assay. We performed telomere fusion assay as described before (Capper R. et al., 2007;Letsolo BT. et al., 2010) with some modifications. Shortly, genomic DNA was digested with EcoRI (Promega), and accurate concentrations were measured using Qubit fluorometer (Thermo Fisher Scientific). For each sample at least 16 PCR reactions were performed using 50 ng of DNA per reaction, a mix of subtelomeric primers (21q1, 16p1, XpYpM) 0,2 µM each and the FailSafe™ PCR System (Lucigen) under following conditions:

26 cycles of 94 °C for 15 s, 58 °C for 30 s, and 68 °C for 10 min. PCR products were resolved on the 0,8 % agarose gel followed by southern blotting. Nylon membranes were hybridized with the corresponding radioactively labeled (αP32 dCTP) subtelomeric probes and a DNA ladder probe (SmartLadder MW-1700-10, Eurogentec), exposed and revealed on the Typhoon FLA 9500 Phoshphorimager (GE Healthcare Life Sciences). Molecular size of each of the bands was calculated by ImageQuant TL 8.1 software (GE Healthcare Life Sciences). Fusion frequencies were estimated as the number of fused telomeres per genome (per 6pg of human diploid genome for MRC-5 or per 10 pg of DNA for HeLa cells).

STELA. We performed STELA as it was described by Baird DM and colleagues (Baird DM. et al., 2003) with some modifications. Shortly, total genomic DNA was digested with EcoRI, quantified by Qubit fluorometer (Thermo Fisher Scientific). A ligase reaction was performed with 10 ng DNA and telorette linker sequence by T4 DNA ligase. 250 pg of ligated DNA was used for PCR with telomere-adjacent (XpYpE2) and teltail primers, and the FailSafe™ PCR System (Lucigen). We cycled the reactions under the following conditions: 26 cycles of 94 °C for 15 s, 58 °C for 30 s, and 68 °C for 10 min. PCR products were resolved on 0,8% agarose gels followed by southern blotting. Hybridized membranes were exposed and the signal detected on the Typhoon FLA 9500 (GE Healthcare Life Sciences). Analysis was performed using the software designed by Lai and colleagues (Lai TP. Et al., 2017).

Teloblot. To measure telomere length we performed southern blot. Total DNA was digested with HinfI/RsaI (Promega), and 5 µg per sample were migrated on 1% agarose gels. After transfer of DNA to the N+ Hybond membrane (GE Healthcare), each membrane was hybridized with the telomeric DNA probe (purified 650-bp telomeric fragment) obtained by random priming using the Klenow large fragment enzyme and radioactively labeled (αP32) dCTP nucleotides. The signal was later revealed and analyzed using the Typhoon FLA 9500 (GE Healthcare Life Sciences).

Telomere overhang assay. The overhang assay was performed as described by Benarroch-Popivker et al., 2016(Benarroch-Popivker et al., 2016). Briefly, 5 µg of genomic DNA from HeLa cells were digested with HinfI and RsaI (Promega), and incubated with 0.2 pmoles of the radioactively end-labeled single-stranded (CCCTAA)3 probe overnight at 50°C. Hybridized samples were loaded into 0,9 % agarose gel and run at 6 V/cm for 75 min. The gel was then dried on 3 MM paper for 4 hours at 40°C and exposed on a phosphorimager screen.

Telomere overhang signal was normalized to the total telomere signal, which was obtained by hybridization of the denatured gel with the same probe as the native gel. Analysis was performed using the Typhoon FLA 9500 (GE Healthcare Life Sciences).

Western blotting. Protein extracts were obtained by lysis in ice-cold RIPA buffer for 30 min followed by 30 min centrifugation at 4°C. 30 µg of proteins were separated on 4-20% acrylamide gradient SDS gels (BioRad), transferred on Amersham Protran 0.45 µm nitrocellulose membranes (GE Healthcare Life Sciences) for 90 min at 300 mA. Further the membranes were blocked in 5% skim milk in PBST buffer, and incubated thereafter with the primary and secondary antibodies. Membranes were developed using the Luminata Forte HRP substrate (Millipore) and exposed in the Fusion Solo apparatus (Vilbert Lourmat).

IF-FISH.

Imunnofluorecence-FISH in MRC-5 cells and further analysis of the images were performed as described in Mendez- [START_REF] Mendez-Bermudez | Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2[END_REF][START_REF] Mendez-Bermudez | Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2[END_REF]. For FISH the following telomeric PNA probe was used: Cy3-OO -CCCTAACCCTAACCCTAA. For measuring DDR we used anti-53BP1 antibody.

Metaphase chromosome analysis. To obtain chromosome spreads, HeLa cells, treated selectively with BIBR1532 and doxycycline as described above, were arrested in metaphase using 50 ng/ml colcemid (KaryoMAX, Invitrogen) for 2 hours at 37ºC. Afterwards, trypsinized cells were incubated with hypotonic solution (75 mM KCl) for 15 minutes at 37ºC, fixed in ice-cold methanol : glacial acetic acid (3:1), and spread on slides. FISH with the telomeric PNA probe was performed as described above. Stained metaphase chromosomes were visualized on the Zeiss Axiovert Z2 epi-fluorescent microscope and analyzed using the metasystem ISIS software.

Senescence-associated β-galactosidase (SA-β-gal) assay. We measured the percentage of SA-β-B-gal positive cells to evaluate the percentage of senescent cells in culture. In order to do so, we used the Senescence Detection Kit (Abcam) following manufacturer instructions.

Statistics

General discussion and future perspectives

In this manuscript, we presented the results obtained during the Thesis project on the RAP1 role in telomere protection.

Here we report two independent scenarios where RAP1 takes over telomere protection.

First, we mutated several lysines and arginines into alanines within the TRFH domain of TRF2. This mutant, called Top-less, had impaired topological capabilities, such as DNA wrapping and also affected t-loop formation (the number of t-loops was decreased in comparison to the control). Since t-loops are natural barriers against DNA repair and DDR, we further decided to explore how partial telomere deprotection, caused by Top-less, controls NHEJ. As expected, we observed severe end-to-end fusions upon TRF2 knockdown. This fusion phenotype can be rescued by overexpression of full-length TRF2 or Top-less. The contribution of the work performed during the Thesis project was to show that Top-less in combination with RAP1 inhibition did not rescue fusions. This finding suggested that topological properties of TRF2, mediated by TRFH domain, are important in NHEJ control, and when affected, can be backed up by RAP1.

Second, as the main work of the Thesis project, we explored how RAP1 protects telomeres in senescent cells. In relation to this, the current model of different telomere states has been proposed by Cesare and Karlseder [Cesare AJ. and Karlseder J., 2012]. In this model, three different telomere states are described as an outcome either of TRF2 dysfunction or subsequent events during replicative senescence. The closed state, which can be applied to young or dividing primary cells, is based on the discovery of t-loops that are found on the chromosome extremities and serve as barriers to DNA damage [Griffith JD. et al., 1999;Doksani Y. et al, 2013;Benarroch Popivker et al., 2016]. The intermediate state exists due to the progressive telomere shortening when t-loops cannot be formed. It is characterized by partial depletion of TRF2 and accumulation of DDR markers, such as increase in TIFs. The last state is uncapped telomeres with no detectable TRF2 and excessive DDR and chromosome fusions [Cesare JA. et al., 2012]. The intermediate state can be applied to the cells that enter into replicative senescence, and the last state is an extreme case when telomeres experience a crisis or become so critically short that it leads to fusion [Cesare JA. et al., 2013]. Importantly, telomeres in senescent cells do not fuse massively due to the presence of TRF2 [Karlseder J. et al., 2002]. In addition, a recent study shows that linear telomeres of mouse and HeLa of an average length of 20 kb, that do not form t-loops due to TRF2 downregulation, are sensitive to ATM activation, but yet can be protected from NHEJ [START_REF] Van Ly | Telomere Loop Dynamics in Chromosome End Protection[END_REF] in agreement with our precedent publication [Benarroch Popivker et al., 2016]. However, in spite of all the evidence presented above, yet there is no experimental proof of t-loop dynamics in senescent cells. It may be due to the fact that there are limitations in detection and resolution of t-loops even by means of the most modern technics such as super resolution microscopy. In addition, the role of human RAP1 in human replicative senescent cells has not been addressed so far. For all these reasons, in the second project, we focus on telomere protection of cells that experience progressive telomere shortening.

To overcome the limitation of ceased cell division in senescent cells, instead of metaphase spread we decided to take advantage of the telomere fusion assay in order to detect chromosome fusions. This assay relies on PCR with different subtelomeric probes. If the fusion occurs between two different chromosome ends, it will be amplified in PCR followed by Southern blotting of the PCR products. Among the advantages, this technique is powerful enough to detect fusions originating from 22 different chromosome ends all at once [Letsolo BT. et al., 2010]. It detects sister chromatid-type fusion when there has been a deletion of at least one of the telomere repeat arrays creating an imperfect inverted repeat, as well as fusion between heterologous chromosomes containing substantial arrays of headto-head telomere repeats [Capper R. et al., 2007]. The limitations of the assay are due to the fact that it cannot detect 1) sister chromatid-type fusions that create perfect inverted repeats;

2) fusions that have substantial deletion of DNA material enough to hamper the binding of one of the subtelomeric primers; 3) fusions involving chromosomes not covered by the subtelomeric primers that are used.

We used telomere fusion assay to detect chromosome fusions in human primary fibroblasts of different population doublings. We observed no or very few fusions in young cells, a moderate increase in fusion frequency in pre-senescent dividing cells and approximately 10-fold increase in senescence. Strikingly, senescent cells upon RAP1 knockdown demonstrated a further 2-fold increase in fusion frequency compared to the control. This was not the case either for young or pre-senescent cells, indicating that the switch between RAP1-independent and RAP1-dependent telomere protection occurs during replicative senescence. Furthermore, we have assayed telomere uncapping by the TIF assay in all these populations of cells. Similar to the results on fusion frequency, we observed a difference between control and RAP1 knockdown only in senescent cells. Whether this DDR activation is the cause or the consequence of the chromosome fusions that increased when RAP1 is dysfunctional, is clearly something that has to be investigated further. In mice, it was shown that RAP1 was dispensable for repression of DDR, in particular, ATM pathway; however, in the very same study, no chromosome fusions were detected when RAP1 was removed from telomeres [Sfeir A. et al., 2010]. One can speculate that RAP1 dysfunction can affect TRF2 and trigger thus DDR. In human, TRF2 expression decreases in senescent cells, whereas RAP1 level is less affected [Fujita K. et al., 2010;Swanson MJ. et al., 2016] (this Thesis). But this does not answer the question whether TRF2 or RAP1 binding to telomeres is decreased or vice versa. In senescent yeast, Rap1 re-localizes within the chromosome to modulate expression of its target genes [Platt JM. et al., 2013;[START_REF] Ye | Transcriptional outcome of telomere signalling[END_REF], as in mouse when telomeres get shorter in telomerase null cells [Martinez P. et al., 2016]. Human RAP1 binds to telomeres through interaction with TRF2, and they together form a stable complex [START_REF] Li | Identification of human Rap1: implications for telomere evolution[END_REF]. On the other hand, human RAP1 can bind telomeric repeats independently, but with low affinity [Arat N. and Griffith JD., 2012]. Notably, RAP1 may be necessary for more accurate and selective TRF2 recognition of telomeric DNA [START_REF] Janoušková | Human Rap1 modulates TRF2 attraction to telomeric DNA[END_REF]. In the future, we aim to investigate the binding of both TRF2 and RAP1 to telomeres in young and senescent cells by means of chromatin immunoprecipitation (ChIP) or immunofluorescence FISH with specific antibodies and telomere probes.

To dissect the anti-fusion mechanisms of human RAP1, we used HeLa cells with doxycycline-inducible RAP1 knockout alleles [Kim H. et al., 2017]. We treated these cells with the telomerase inhibitor BIBR1532 to trigger telomere shortening and observed again increase in the fusion frequency when RAP1 expression was diminished. Later, we found that chromosome fusions upon RAP1 knockout appear through c-NHEJ. Notably, we demonstrated that RAP1 removal neither triggers a decrease of the average telomere length or increases the number of critically short telomeres or promotes telomere 3'-overhang shortening and telomere loss. Therefore, fusions cannot be simply explained by telomere changes only.

It has been shown before that many fusion events involved chromosome ends with no detectable telomere sequences [Capper R. et al., 2007]. For instance, the fusions we detected using the PCR-based method in this work suggest that the fusions involve chromosome ends with no telomeres or even more with DNA loss extending to the subtelomeric region. In order to characterize the type of fusions and to determine the exact sequence loss at the time of the fusion, we are going to sequence them either by classical Sanger sequencing or using a novel approach to generate long read sequencing.

In search of mechanisms that drive fusion phenotype, we decided to use a genetic approach. We aim to decipher which domains of human RAP1 may be important for prevention of c-NHEJ. To do so, we designed several truncation mutants of human RAP1 (Figure 11), which lack separate domains or have a point mutation as the RAP1 mutant F336R (a mutation in the binding site of TRF2). We have done the first cell culture trial to check the expression of these mutants in HeLa (Figure 11). Therefore, in the future, we will perform the telomere fusion assay in HeLa expressing different RAP1 mutants.

Among the future work is the search for potential interactors of RAP1 that can contribute to the fusion phenotype besides TRF2. It was reported previously that human RAP1 can interact with several proteins. For instance, immunoprecipitation of endogenous RAP1 from HeLa cell nuclear extracts was able to specifically pull down TRF2, Ku86, Rad50, and RAP1[O'Connor MS. et al., 2004]. Ku86 and Rad50 are DNA repair proteins, which makes them appealing targets to test in regard to RAP1. Interestingly, the level of KU declines during replicative senescence [START_REF] Seluanov | Changes in the level and distribution of Ku proteins during cellular senescence[END_REF].

Additionally, RAP1 has been shown to interact with Sun1, which is a member of the LINC complex (Linker of Nucleoskeleton and Cytoskeleton) that bridges inner and outer membranes of the nuclear envelope [Crabbe L. et al., 2012]. Sun domain proteins have been involved in the tethering of telomeres to the nuclear envelope in yeast [Bupp JM. et al., 2007]. Remarkably, numerous studies have demonstrated that DSBs are targeted to the nuclear periphery for the repair [Oza P. and Peterson CL., 2010]. In this regard, yeast Mps3, a member of the Sun domain protein family, plays a central role in connecting peripheral localization, DSB repair and telomeres [START_REF] Schober | Yeast telomerase and the SUN domain protein Mps3 anchor telomeres and repress subtelomeric recombination[END_REF][START_REF] Oza | Mechanisms that regulate localization of a DNA double-strand break to the nuclear periphery[END_REF]Oza P. and Peterson CL., 2010]. Therefore, a possible scenario is that upon inhibition of human RAP1 in senescent cells telomeres become more mobile and prone to fusions.

1.

  Telomere basic: structure and replication Our understanding of telomere functions somehow started 80 years ago by the work of Herman Muller and Barbara McClintock. Muller was studying chromosome damage

Figure 1 .

 1 Figure 1. Different telomere structures. A. G-quadruplex. B. t-loop. C. i-motif. Illustration

Figure 2 .

 2 Figure 2. Structure of the telomerase holoenzyme. This structure includes a reverse transcriptase (TERT) and associated proteins, an RNA template (TERC), and a short piece of the telomere DNA [Illustration from Protein Data Bank, http://pdb101.rcsb.org/motm/227].

Figure 3 .

 3 Figure 3. Telomere-associated proteins among different species. Modified from [Giraud-Panis MJ. et al., 2013].

  . Fusions were also a c o m m o n c a u s e o f r e d u c e d c h r o m o s o m e n u m b e r a m o n g a n t s p e c i e s

Figure 5 .

 5 Figure 5. Multiple pathways to repair a DNA double-strand break.

  ` phe Giraud-Panis Correspondence eric.gilson@unice.fr (E.G.), giraud-panis@unice.fr (M.-J.G.-P.) In Brief Benarroch-Popivker et al. show that TRF2 wraps DNA around its TRFH domain, thereby controlling telomeric DNA topology, t-loop formation, and ATM inhibition. In TRF2 wrapping-deficient cells, protection of telomeres against fusion relies on the recruitment of RAP1. Highlights d TRF2 modifies DNA topology by wrapping 90 base pairs of DNA around its TRFH domain d A mutant deficient in DNA wrapping, Top-less, causes relaxation of telomeric DNA d Top-less telomeres are deprotected and harbor fewer t-loops but are not fused by NHEJ d RAP1 protects Top-less telomeres against fusions Benarroch-Popivker et al., 2016, Molecular Cell 61, 274-286 January 21, 2016 ª2016 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2015.12.009 INTRODUCTION

  Figure 1. TRFH Domain of TRF2 Condenses 90 bp of DNA

  Figure S4

Figure S2 .

 S2 Figure S2. The acetylation footprinting method: principle and validation. Related to Figure

Figure S3 .

 S3 Figure S3. Top-less: a mutant allowing separation between topology-related and unrelated

Figure

  Figure S5. TRF2 controls telomeric DNA topology. Related to Figure 4.

Figure S6 .

 S6 Figure S6. DDR activation in Top-less expressing cells. Related to Figure 5.

  2 and 3 times for 5 min with 0.1 M Tris-HCl pH 7.2, 0.15 M NaCl, 0.08% Tween-20, at room temperature and finally with PBS 1x. YOYO-1 (1:20000 in PBS1x) was dropped on samples and immediately washed with PBS 1x. Coverslips were then covered with PBS1x and directly used for imaging.dSTORM imaging and analysisThe stained coverslips were imaged the same day at room temperature in a closed chamber (Ludin Chamber, Life Imaging Services) mounted on an inverted motorized microscope (Nikon TI-E) equipped with a 100x 1.49 NA PL-APO objective and a Perfect Focus System (Nikon), allowing long acquisition in oblique illumination mode. Imaging was performed in an extracellular solution containing reducing agents and oxygen scavengers. For dSTORM, Alexa-647 was first converted into dark state using a 642 nm laser (Coherent) at 30-50 kw/cm2 intensity. Once the ensemble fluorescence was converted into the desired density of single molecules per frame, the laser power was reduced to 7-15 kw/cm2 and imaged continuously at 10 fps for 5,000 frames. The level of single molecules per frame was controlled by using a 405 nm laser (Omicron). The laser powers were adjusted to keep an optimal level of stochastically activated molecules during the acquisition. Single molecule fluorescence was collected by a TIRF-Quad filter set 405/488/561/640 (F66-04TN from AHF analysentechnik AG). The fluorescence was collected using a 512x512 EMCCD (Evolve, Photometrics). The acquisition and localization sequences were driven by MetaMorph 7.8.3 and Wavetracer 1.5 software (Molecular Devices) in streaming mode at 10 frames per second (100 ms exposure time) using the full chip of the camera. Single molecule localization and re-construction were performed offline using Wavetracer and GPU acceleration. The reconstructed images where analyzed by Image-J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Frequency of fusions in human primary fibroblasts. A. Fusion frequencies were measured by means of telomere fusion assay with 3 subtelomeric primers in the same reaction (21q1, XpYpM and 16p1). B. Representative membranes hybridized with the 16p probe are shown.

Figure 3 .

 3 Figure 3. Telomere fusions in HeLa upon inducible RAP1 knockout. A. Western blotting with anti-RAP1 and tubulin antibodies to evaluate the efficiency of knockout, which was induced by doxycycline (DOX; 1ug/ul final concentration) treatment for 15 days. Cells were treated selectively with the telomerase inhibitor BIBR1532 for 25 days. B. Southern blot (teloblot) hybridized with a telomeric probe to evaluate the efficiency of BIBR1532. C. Frequency of fusions in HeLa cells. D. Percentage of end-to-end fusions observed in HeLa knockout vs control upon BIBR1532 treatment. Number of chromosomes analyzed per condition: -DOX = 1366, +DOX = 1474. E. Percentage of other chromosome aberrations obtained from the conditions described in D. F. Examples of chromosome aberrations observed in D and E.

Figure 4 .

 4 Figure 4. Return to growth of post-senescent MRC-5 cells. A. Growth curves of MRC-5 fibroblasts. After reaching replicative senescence, cells were infected either with shp21CIP1 or shp21CIP1+shRAP1 and harvested 15 days post infection. B. SA-β-gal assay in senescent and post-senescent fibroblasts (day 0 and 15 of lentivirus infection). Approximately 300 cells were analyzed per condition. Percentage of SA-β-gal positive cells is indicated for each condition. C. Model of RAP1 telomere protection in young and senescent cells.

  s . F o r R T-q P C R w e u s e d t h e f o l l o w i n g p r i m e r s : T E R F 2 I P-F : CGGGGAACCACAGAATAAGA, TERF2IP-R: CTCAGGTGTGGGTGGATCAT, 36B4-F: AACTCTGCATTCTCGCTTCCT, 36B4-R: ACTCGTTTGTACCCGTTGATG, p21 CIP1 -F: TGGTAGGAGACAGGAGACCT, p21 CIP1 -R:AATACTCCCCACATAGCCCG, LIG3-F: GAT CAC GTG CCA CCT ACC TTG T, Lig3-R: GGC ATA GTC CAC ACA GAA CCG T, LIG4-F: CAC CTT GCG TTT TCC ACG AA-3, LIG4-R: CAG ATG CCT TCC CCC TAA GTT G. Primers used for telomere fusion assay: *21q1: 5ʹ-CTTGGTGTCGAGAGAGGTAG-3ʹ, *16p1: 5ʹ-TGGACTTCTCACTTCTAGGGCAG-3ʹ, *XpYpM: 5ʹ-ACCAG GTTTTCCAGTGTGTT-3ʹ. Primers for generation of subtelomeric DNA probes: XpYpO: 5ʹ-CCTGTAACGCTGT TAGGTAC-3ʹ, XpYpG: 5ʹ-AATTCCAGACACACTAGGACCCTGA-3ʹ, 21qseq1: 5ʹ-TGGTCTTATACACTGTGTTC -3ʹ, 21qsq1rev: 5ʹ-AGCTAGCTATCTACTCTAACAGAGC-3ʹ, 16p2: 5ʹ-TCACTGCTGTATCTCCCAGTG -3ʹ, 16pseq1rev: 5ʹ-GCTGGGTGAGCTTAGAGAGGAAAGC-3ʹ. Primers used for STELA: XpYpE2: TTGTCTCAGGGTCCTAGTG, telorette: TGCTCCGTGCAT CTGGCATCTAACCCT, teltail: TGCTCCGTGCATCTGGCATC.RNA extraction RT-qPCR. Total RNA was extracted following instructions of the RNeasy Mini Kit (Qiagen), and then 1 ug of RNA was reverse transcribed into cDNA using the High-Capacity RNA-to-cDNA kit (Thermo Scientific). Each qPCR reaction contained 10x diluted cDNA, 0.2 µM primers and SYBR green master mix (Roche, 4913914 001).

.

  Statistical analysis was performed by means of Prism 5 software (GraphPad). For comparison of two groups we used two-tailed Mann-Whitney U test, for multiple groups the Kruskal-Wallis test was used. P-values < 0.05 were considered significant (* p < 0.01, **p < 0.001, ***p < 0.0001).

  Figure1

Figure 11 .

 11 Figure 11. Human RAP1 mutants used in the study. A. A graphical representation of the RAP1 mutant design. * corresponds to the point mutation. B. A western blot showing expression of different mutants in HeLa cells.

Table S1 Oligonucleotides used in this study, Related to STAR Methods section: Real time qPCR. Table S2 siRNA used in this study, Related to STAR Methods section: Transient transfection

 S1

Acknowledgements Acknowledgements …………………………………………………………………... xii Table of contents ……………………………………………………………………...

(A) Distribution of deconvoluted volumes corresponding to the same set of TRF2/DNA complexes shown in Figure 1A. Histograms, expressed as percentage of events and corresponding to the raw data, were fitted with individual populations applying a Gaussian multipeak fitting. The solid line corresponds to the sum of the multi-fitting. Note that the volume corresponding to the mean value of the first peak is bigger than the calculate volume of the TRFH + 2 Myb domains calculated from the 3D X-ray crystallography data (pdb 3BUA and 1VFC respectively) using the CRYSOL software. Peak 1 is thus compatible with the volume of a dimer. (F) Distribution of deconvoluted volumes for TRFH/DNA complexes calculated from AFM data shown in Figure 1B. Histograms correspond to raw data and curves to the sum of a Gaussian multi-peak fitting. Note that the value corresponding to the main volume of the first peak is very close to the volume of the TRFH domain calculated from the 3D X-ray cristallography data (pdb nature of the species studied by migration in an acrylamide gel. One can observe the appearance of the slowly migrating four stranded Holliday junction (J). Since substrate (S) and product (P) were undistinguishable, quantification was done on the sum of the two species. Left panel: acrylamide electrophoretic analysis of aliquots at different time point. Right panel: quantitative analysis of the % of Holliday junction (% J) and % of the other species (% S+P) through time.

Error bars correspond to standard deviation between three experiments. Note the identical behavior for both proteins. siRNA with expression of either empty vector or TRF2 or Top-less or AΔB) was done by RT-qPCR and is respectively of 1, 0.13, 0.90, 0.79 and 0.97 fold of enrichment.

(F) Recruitment of 53BP1 on telomeres (TIFs) of HT1080 cells down-regulated for TERF2 by shRNA and expressing either TRF2, and the 7K, 2R, Top-less and AB mutants. Data show the mean ± SE and P values were calculated using the Mann-Whitney test (** P < 0.01, *** P <

Article 2 Human RAP1 prevents telomere fusion in senescent cells

Lototska L 1 , Songyang Z 2 , Royle NJ 3 , Jing Ye 4 , Mendez-Bermudez A 1,4 *, Gilson E 1,4,5

SUMMARY

RAP1 is an evolutionary conserved telomeric protein between yeast and mammals.

Although yeast Rap1 is a key telomere capping protein preventing NHEJ and consequently telomere fusions (Pardo B. and Marcand S., 2005), and mammalian RAP1 protects against NHEJ in vitro (Bae NS. and Baumann P., 2007;Sarthy J. et al., 2009), its role at mammalian telomeres in vivo is still controversial (Sfeir A. et al., 2010;Kabir S. et al., 2014;Martinez P. et al., 2016). An emerging view is that RAP1 behaves as a backup antifusion factor in mammalian cells when its interacting partner, TRF2, is dysfunctional (Benarroch-Popivker D. et al., 2016;Rai R. et al., 2016) or upon telomerase inhibition in mice (Martinez P. et al., 2016).

Here we demonstrate that RAP1 is required to protect telomeres specifically in replicative senescent human cells. Downregulation of RAP1 in these cells, but not in young or dividing pre-senescent cells, leads to telomere uncapping and fusions. The anti-fusion effect of RAP1 was further explored in a HeLa cell line when the RAP1 gene can be invalidated by doxycycline (Kim H. et al., 2017). The invalidation of RAP1 in these cells gives rise to telomere fusions only upon telomerase inhibition by BIBR1532 treatment. We further showed that the fusions triggered by RAP1 loss are dependent upon ligase IV, indicating that they are generated through classical NHEJ between telomere ends. Keywords: Telomeres, RAP1, NHEJ, chromosome fusions, replicative senescence increased upon RAP1 downregulation. In contrast, senescent cells were characterized by nearly 10 times higher fusion frequency as compared to young cells (frequency of 1.4x10 -4 ), a frequency further increased upon RAP1 downregulation (frequency of 2.6x10 -4 ).

RAP1 protects critically short telomeres from fusion.

To determine whether the RAP1-dependent telomere protection observed in senescent cells is due to the appearance of critically short telomeres, we used a HeLa cell line with a doxycycline-inducible knockout allele of RAP1 (Kim H. et al., 2017). We treated cells with doxycycline for 15 days with or without the telomerase inhibitor BIBR1532 (Fig. 3A). As expected, BIBR1532 caused a notable decrease in telomere length (Fig. 3B). Similar to MRC-5 senescent cells (Supplementary Fig. 1D-E), no further length shortening was observed when RAP1 was inhibited (Fig. 3B) even when critically short telomeres were assayed by single telomere length analysis (STELA) of the XpYp telomere (Baird DM. et al., 2003) (Supplementary Fig. 3A).

The PCR-based telomere fusion assay revealed that RAP1 downregulation triggers an increase in fusion frequency only in HeLa cells treated with BIBR1532 (Fig. 3C and Supplementary Fig. 3B). Next, we ask whether the fusions were the result of the classical or alternative NHEJ pathway by depleting DNA ligase IV (LIG4) or DNA ligase III (LIG3) respectively (Fig. 3C and Supplementary Fig. 3C-E) [START_REF] Oh | DNA ligase III and DNA ligase IV carry out genetically distinct forms of end joining in human somatic cells[END_REF]. Only cells downregulated for LIG4 were insensitive to RAP1 inhibition showing that, at least in part, the fusions triggered by RAP1 loss are dependent on the classical NHEJ pathway. Notably, in-gel telomere overhang assays in HeLa cells upon RAP1 invalidation did not show an obvious reduction in the 3' overhang length (Supplementary Fig. 3F-G) suggesting the telomere fusions triggered by RAP1 inhibition do not result from the appearance of blunted telomere ends.

Then, we analyzed the telomere abnormalities triggered by RAP1 inhibition in metaphase spreads of HeLa cells treated with BIBR1532 (Fig. 3D-F). Consistent with the fusion-PCR results, HeLa cells treated with BIBR1532 and doxycline exhibit an increase in the frequency of telomere fusions. Notably, RAP1 inhibition did not lead to telomere fragility (as recorded by multiple telomere signal or MTS) nor to telomere shortening.

Overall, these results indicate that RAP1 is required to protect critically short telomeres from classical NHEJ-mediated fusions, providing an explanation for the specific anti-fusion

Supplementary

Materials and methods

Cell lines and reagents. MRC-5 human primary lung fibroblasts were obtained from ATCC. MRC-5 cells were grown in DMEM supplemented with 10% fetal bovine serum penicillin (100 IU/ml), and streptomycin (100 µg/ml) at 37°C 5% CO2, 5% O2. HeLa CRISPR/Cas9 Supplementary Michelle Debatisse, 3 Eric Gilson, 1,2,9,10,11, * and Jing Ye 1,10, * SUMMARY Hard-to-replicate regions of chromosomes (e.g., pericentromeres, centromeres, and telomeres) impede replication fork progression, eventually leading, in the event of replication stress, to chromosome fragility, aging, and cancer. Our knowledge of the mechanisms controlling the stability of these regions is essentially limited to telomeres, where fragility is counteracted by the shelterin proteins. Here we show that the shelterin subunit TRF2 ensures progression of the replication fork through pericentromeric heterochromatin, but not centromeric chromatin. In a process involving its N-terminal basic domain, TRF2 binds to pericentromeric Satellite III sequences during S phase, allowing the recruitment of the Gquadruplex-resolving helicase RTEL1 to facilitate fork progression. We also show that TRF2 is required for the stability of other heterochromatic regions localized throughout the genome, paving the way for future research on heterochromatic replication and its relationship with aging and cancer. Note : All siRNAs were purchased from Dharmacon. Reference starting with "L" (On-Target Plus SMARTpool) is a mixture or 4 siRNAs against the gene of interest.

TOP2A-2 J-004239-06 CGAAAGGAAUGGUUAACUA TOP2A-3 J-004239-07 GAUGAACUCUGCAGGCUAA TOP2B-1 L-004240-00-0005 TOP2B-2 J-004240-07 GAAGUUGUCUGUUGAGAGA TOP2B-3 J-004240-08 CGAAAGACCUAAAUACACA SUV39H1-1 L-009604-00-0005 SUV39H1-2 J-009604-07 CUAAGAAGCGGGUCCGUAU SUV39H1-3 J-009604-08 GGUGAAAUGGCGUGGAUAU LIG4-1 J-004254-09 GCACAAAGAUGGAGAUGUA LIG4-2 J-004254-10 GGGAGUGUCUCAUGUAAUA G9a-1 L-006937-00-0005 G9a-2 J-006937-05 GGACCUUCAUCUGCGAGUA G9a-3 J-006937-06 GAACAUCGAUCGCAACAUC CONTROL D-001810-01 UGGUUUACAUGUCGACUAA