Dr Jean-Baptiste Charbonnier

Dr Pierre Verrelle D'

Dr Merci Au

Jozo Delic

Tu

Clément

Article -Dynamics of phospho-S27-S33-Ku70 at DNA DSBs : functional link to genome stability Bibliography List of Figures 1.Introduction

Keywords: NHEJ, Ku, DNA, Double strand break repair, genomic stability

DNA integrity is permanently challenged by a plethora of endogenous and exogenous threats. Double strand breaks (DSB) are the most toxic lesions when un-or misrepaired, since they can become a source of mutations and drive carcinogenesis.

Two main DNA repair pathways classical non-homologous end joining (cNHEJ) and homologous recombination (HR) have evolved to repair these lesions. Our group previously identified phospho-Ku70 overexpressed in a patient subset of treatment-resistant chronic lymphocytic leukemia (R-CLL). Multiple observations such as apoptosis resistance after genotoxic stress, error-prone repair junctions, genomic instability and a significant resection of telomere ends led to the assumption of a deregulated c-NHEJ in R-CLL. The Ku protein (Ku70/Ku80 heterodimer) is a core component of c-NHEJ with multiple other cellular functions. In this work we characterize the functions of phospho-Ku70 on the DSB site induced by irradiation or enzymatically. We show that phospho-Ku70 provides radioresistance and genomic stability, when compared to a non-phosphorylable Ku70 mutant. We further demonstrate that phosphorylation of Ku70 is important for DNA repair kinetics and for Ku70 release from the DNA after repair completion. In addition, we employ a site specific I-SCEI reporter assay to monitor efficiency of c-NHEJ and HR, and establish that phospho-Ku70 affects both c-NHEJ and HR repair pathways. Finally, we developed a humanized KI mouse model to study the role of phospho-Ku70 in vivo. Preliminary data on mouse embryonic fibroblasts indicate that non-phosphorylable Ku70 expressing cells have growth defects and increased apoptosis susceptibility.

In summary, we determine that the new form of phospho-Ku70 drives DNA DSB repair, radioresistance and genomic stability. An in-depth knowledge of the molecular processes of phospho-Ku70 in DNA DSB repair may be exploited in specific cancer treatment strategies in the future.

Résumé

L'intégrité de l'ADN est menacée en permanence par de nombreuses agressions endogènes et exogènes. Les cassures double brin (CDB) sont les lésions les plus toxiques lorsqu'elles sont non-ou mal-réparées car elles deviennent une source de mutations susceptible d'entraîner une carcinogenèse. Deux voies principales de réparation de l'ADN ont été développées pour réparer ces lésions : la jonction d'extrémités non-homologue classique (cNHEJ) et la recombinaison homologue (RH). Notre groupe a préalablement identifié phospho-Ku70 sur-exprimé dans une sous-population de patients atteint d'une leucémie lymphocytaire chronique (R-CLL) résistant à tout traitement. Des observations telles que la résistance à l'apoptose induite par le stress génotoxique, la réparation de jonctions sujette à erreur, l'instabilité génomique et une résection des extrémités des télomères ont conduit à l'hypothèse d'un cNHEJ dérégulé dans la R-CLL. La protéine Ku (hétérodimère Ku70 / Ku80) est un facteur central de c-NHEJ avec plusieurs autres fonctions cellulaires. Dans ce travail, nous avons caractérisé les fonctions de phospho-Ku70 sur le site de la CDB induites par irradiation ou activité enzymatique. Nous montrons que phospho-Ku70 assure une radio-résistance et une stabilité génomique par rapport à un mutant Ku70 non phosphorylable. Nous montrons en outre que la phosphorylation de Ku70 influence la cinétique de réparation de l'ADN et la libération de Ku70 de l'ADN après achèvement de la réparation.

Nous avons par ailleurs utilisé un test rapporteur I-SCEI pour évaluer l'efficacité de cNHEJ et HR, et nous avons pu montrer que le phospho-Ku70 affecte à la fois les voies de réparation c-NHEJ et HR. Enfin, nous avons développé un modèle de souris KI humanisé pour étudier le rôle de phospho-Ku70 in vivo. Les données préliminaires sur les fibroblastes embryonnaires de souris indiquent que les cellules exprimant le Ku70 non-phosphorylable présentent des défauts de croissance et une susceptibilité à l'apoptose.

En résumé, nous montrons que la nouvelle forme de phospho-Ku70 favorise la réparation d'ADN DSB, la radio-résistance et la stabilité génomique. La connaissance fine des processus moléculaires de phospho-Ku70 dans la réparation ADN DSB pourrait ouvrir la voie à des stratégies spécifiques de traitement du cancer à l'avenir.

Mots-clés: NHEJ, Ku, ADN, Réparation de cassures double brin, stabilité génomique

I Introduction

Chapter 1

General introduction

This manuscript deals with the regulation of DNA double strand break repair and cellular fate after genotoxic stress, such as irradiation. More particularly it considers the role of Ku70 and it's modified form phosphorylated at serine 27 and serine 33 within these processes. A proper DNA maintenance and DNA metabolism are central to the prevention of genomic instability and cancer development.

Mutations are at the origin of the biological variations found in cancer.

Even before the DNA structure was decoded, several observations linked an abnormal chromosome distribution to cancer. It was also recognized that cancer cells have more than the 46 chromosomes that are found in normal cells. Moreover it could be shown that exposure of D. melanogaster sperm to X-rays induced transmutation, that was visible as chromosomal aberrations and phenotypic variations in the fruitfly progeny. By the middle of the 20th century, it was thus suggested that exposure to DNA damaging agents can cause mutations, however a causality for cancer was not established, yet [1].

The link between mutations, DNA and cancer was mainly made after the breakdown of the DNA structure by Watson and Crick. The knowledge of the structural changes brought to DNA by exposure to chemical compounds, such as alkylating agents or polycyclic aromatic hydrocarbons, provided the first link of a causal relationship between DNA changes and carcinogenesis [1].

At this point DNA repair pathways in bacteria such as the enzymatic photoreactivation and later, the in eukaryotic cells conserved nucleotide excision repair and base excision repair, have been discovered. Through the identification of patients with xeroderma pigmentosum, that had an UV-sensitivity due to impaired DNA repair and a skin cancer predisposition, a causal link between DNA repair defects and cancer susceptibility was drawn. In this context, not only defects in the DNA repair machinery, but also in the genes that coordinate the DNA damage response, such as ATM, were found to cause radiosensitivity and malignant transformation [1,2]. Today the accumulation of mutations, and the caused genomic instability are considered as enabling characteristics in cancer development [START_REF] Hanahan | Hallmarks of cancer: The next generation[END_REF].

The Ku protein is a truly remarkable molecule that fills a long list of DNA maintenance functions and is considered as a tumour suppressor gene. We will therefore outline its general characteristics and history in the first place. Then the DNA repair mechanisms and the coordination of the DNA damage response will be determined, before tackling a particular case of DNA double strand break repair, necessary for immunoglobulin receptor rearrangement. We will acknowledge that Ku has functional and regulatory functions that go beyond DNA repair and will see that deregulated Ku is associated with human disease. The end of the introduction, will pay attention to the discovery of a novel form of Ku70 (phospho-S27,S33-Ku70), which was discovered in a subset of chronic lymphocytic leukaemia patients. These patients suffer from an aggressive form of the disease that needs immediate treatment and is linked to a bad survival prognosis.

Chapter 2

The Ku protein: History, protein domains and functional characteristics

The identification of the Ku protein-a historical review

The story of the Ku protein begins in the early 1980's, when two different Japanese research groups detected the presence of Ku-auto antibodies in the sera of patients with connective tissue diseases [START_REF] Tojo | Precipitating antibody to a soluble nuclear antigen "Ki" with specificity for systemic lupus erythematosus[END_REF], [START_REF] Mimori | Characterization of a High Molecular Weight Acidic Nuclear Protein Recognized by Autoantibodies in Sera from Patients with Polymyositis-Scleroderma Overlap.pdf[END_REF]. Due to this observation, they suggested the presence of auto antibodies against the Ku antigen as a diagnostic marker for polymyositis and scleroderma. Ku was shown to localize to the nucleus in samples of different mammal and rodent cells and Reeves et al. demonstrated Ku binding to deoxyribonucleic acid (DNA) by using murine monoclonal antibodies against the deoxyribonucleoprotein complex [START_REF] Reeves | Use of monoclonal antibodies for the characterization of novel DNA-binding proteins recognized by human autoimmune sera[END_REF]. In the following year knowledge of Ku gained substantial advancement when Francoeur et al. [START_REF] Francoeur | Identification of Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity[END_REF] demonstrated that the Ku protein, actually consists of a heterodimer formed by two closely related protein partners named Ku86 (further on named Ku80) and Ku66 (further on named Ku70), respectively. Ku70 and Ku80 were shown to be non-covalently

bound to each other, and to be highly conserved in different mammals as well as ubiquitously expressed in several organs. Further characterization on the DNA binding preferences of Ku by Mimori et al. [START_REF] Mimori | Mechanism of Interaction between Ku Protein and DNA*[END_REF] demonstrated that the heterodimer efficiently binds double stranded DNA (dsDNA) in a sequence non-specific manner and that the interaction increased dependent on the number of freely available DNA double strand ends. Taken together these data provide the starting point for thirty years of research to decode Ku's substantial role in DNA double strand break repair (DSB) pathways, V(D)J recombination, telomere maintenance etc.

Ku is now known to be present throughout eukaryotic evolution. Furthermore in archae and bacteria homologues of Ku have been identified, underlining its importance in the development in all domains of the phylogenetic tree of life [START_REF] Downs | A means to a DNA end: the many roles of Ku[END_REF].

The different protein domains of Ku70 and Ku80 and their functions

In the early 1990's molecular cloning techniques make it possible to decode Ku80's and Ku70's amino acid sequence [START_REF] Mimori | Isolation and characterization of cDNA encoding the 80-kDa subunit protein of the human autoantigen Ku (p70/p80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome[END_REF][START_REF] Reeves | Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen[END_REF] and reveal Ku70 to consist of 609 amino acids (aa) and Ku80 of 732 aa. Primary sequence analysis shows no extensive sequence homology between the subunits in higher eukaryotes. Nevertheless their relationship becomes evident when the primary sequences of yeast and lower eukaryotes is decoded, showing that around 22% of identical aa and 38% of similar aa are found in the C-terminal ends of both subunits [START_REF] William S Dynan | Interaction of Ku protein and DNAdependent protein kinase catalytic subunit with nucleic acids[END_REF]. Sequence alignment of Ku70 and Ku80 homologues from yeast to human reveal several sequence motifs, named primary homology regions (PHR). These regions are responsible for a conserved secondary and tertiary protein structure in both subunits and thus constitute the presumption that the Ku heterodimer evolved from a common ancestor gene [START_REF] Downs | A means to a DNA end: the many roles of Ku[END_REF][START_REF] William S Dynan | Interaction of Ku protein and DNAdependent protein kinase catalytic subunit with nucleic acids[END_REF][START_REF] Gell | Mapping of protein -protein interactions within the DNA-dependent protein kinase complex[END_REF]. Inspired by the sequence similarity between the C-termini, Dynan et al. suggested that the ancestral gene product may have formed a homodimer important for DNA binding, and that if so, Ku70 and Ku80 could form a quasisymmetrical protein-DNA complex. An assumption proven right by the published The N-terminal α/β -domains (also called von Willebrand-domain (vWA))

(Ku70, residues 34-250; Ku80, residues 6-238) make only small contributions to the dimer interface and DNA binding. They are potential sites of protein-protein interactions, such as repair or stress response factors that are recruited to DNA ends. Ku binds with an orientation preference to DNA meaning that Ku70 binds proximal and Ku80 distal to the DNA end. The N-terminus of Ku70 borders the DNA binding groove and is thought to impose a energetic barrier (more than a third of the 33 aa are negatively charged aspartate or glutamate residues) to the inward movement of the Ku heterodimer, helping Ku to be retained in proximity to the DNA end [START_REF] Walker | Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair[END_REF]. Precisely regulated sliding of Ku distal to the DNA break is a critical and well regulated process that activates DNA-PKcs and the DNA DSB-end-ligation process catalysed by the Lig4/XRCC4-complex during DNA repair [START_REF] Turchi | Cisplatin-DNA adducts inhibit translocation of the Ku subunits of DNA-PK[END_REF][START_REF] Yoo | Geometry of a complex formed by double strand break repair proteins at a single DNA end: recruitment of DNA-PKcs induces inward translocation of Ku protein[END_REF][START_REF] Kysela | Ku stimulation of DNA ligase IV-dependent ligation requires inward movement along the DNA molecule[END_REF][START_REF] Yaneva | Interaction of DNA-dependent protein kinase with DNA and with Ku-biochemical ans atomic force microscopy studied.pdf[END_REF].

The central βbarrel domains (core domains) of Ku70 and Ku80 form contacts across the dimer interface and are centred around a protein dyad that cradles the DNA break (see Fig. .

Even though Ku70 and Ku80 show structural similarities concerning their Nterminal and core domains, their C-terminal regions are structurally and functionally divergent [START_REF] Harris | The 3D Solution Structure of the C-terminal Region of Ku86 (Ku86CTR)[END_REF]. The most distal Ku70 C-terminal region (aa 561-609) has similarities with SAP (scaffold-attachment factor A (SAFA) and SAFB, apoptotic chromatin-condensation inducer in the nucleus (ACINUS) and PIAS)-domains, a helix-extended loop-helix motif, shown to bind on non-canonical DNA sequences and important for the organization of chromatin structure [START_REF] Walker | Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair[END_REF][START_REF] Aravind | SAP -A putative DNA-binding motif involved in chromosomal organization[END_REF][START_REF] Zhang | The Three-dimensional Structure of the C-terminal DNAbinding Domain of Human Ku70[END_REF]. This domain is linked to the heterodimeric main body by a flexible peptide chain (aa 536-560) [START_REF] Harris | The 3D Solution Structure of the C-terminal Region of Ku86 (Ku86CTR)[END_REF][START_REF] Zhang | The Three-dimensional Structure of the C-terminal DNAbinding Domain of Human Ku70[END_REF], and lies in proximity to the α/β -domain of Ku80 in absence of DNA. Upon DNA binding the SAP domain moves towards the continuous DNA

strand, distal to the break site, interacts with DNA and thereby stabilizes the Ku complex on the DNA (cf. Figure 2.2A) [START_REF] Rivera-Calzada | Structural model of full-length human Ku70-Ku80 heterodimer and its recognition of DNA and DNA-PKcs[END_REF].

The C-terminal region of the Ku80 subunit shows a globular structure and is an important site for protein-protein interaction [START_REF] Zhang | Solution structure of the C-terminal domain of Ku80 suggests important sites for protein-protein interactions[END_REF]. Upon DNA binding the Cterminus stays at the Ku core domain but is then displaced towards the arriving DNA-PKcs enzyme, a PI3-kinase (phosphatidyl-inositol-3-kinase), to favour interaction and trigger its kinase activity [START_REF] Gell | Mapping of protein -protein interactions within the DNA-dependent protein kinase complex[END_REF][START_REF] Han | Characterization of a Ku86 variant protein that results in altered DNA binding and diminished DNAdependent protein kinase activity[END_REF][START_REF] Muller | Regulation of DNA-dependent protein kinase activity in leukemic cells[END_REF][START_REF] B K Singleton | The C Terminus of Ku80 Activates the DNA-Dependent Protein Kinase Catalytic Subunit[END_REF], important for the coordination of DNA DSB repair [START_REF] Rivera-Calzada | Structural model of full-length human Ku70-Ku80 heterodimer and its recognition of DNA and DNA-PKcs[END_REF] (see Figure 2.2B). Interestingly the C-terminal Ku80 domain and the DNA-PKcs protein are not conserved throughout evolution. Even though found in vertebrates, they are not found in yeast or worms, thus arguing for a co-evolution of these two components [START_REF] Gell | Mapping of protein -protein interactions within the DNA-dependent protein kinase complex[END_REF][START_REF] B K Singleton | The C Terminus of Ku80 Activates the DNA-Dependent Protein Kinase Catalytic Subunit[END_REF]. In fact, this domain is part of a conserved motif shared by other DNA-repair proteins like NBS1 or ATRIP, that are required for recruitment of ATM and ATR, two other family members of the PI3-kinases [START_REF] Falck | Conserved modes of recruitment of ATM , ATR and DNA-PKcs to sites of DNA damage[END_REF].

Functional characterization of Ku as part of the XRCC gene family-links between DSB repair and V(D)J recombination

A milestone in the understanding of DNA repair pathways and essential players therein was set by genetic complementation analysis 1 in the 1990's. Different rodent cell lines with DNA repair deficiencies showing sensitivity to different genotoxic agents, ranging from alkylators, cross-linkers, radiomimetics, x-rays, γirradiation or others were identified [START_REF] Jones | Six complementation groups for ionizingradiation sensitivity in Chinese hamster cells[END_REF][START_REF] Thacker | The genetic basis of resistance to ionising radiation damage in cultured mammalian cells[END_REF][START_REF] Jeggo | Genetic analysis of ionising radiation sensitive mutants of cultured mammalian cell lines[END_REF] and led to the classification of 11 complementation groups (see Table 2.1) [START_REF] Malgorzata | Mammalian mutants defective in the response to ionizing radiation-induced DNA damage[END_REF][START_REF] Collins | Mutant rodent cell lines sensitive to ultraviolet light , ionizing radiation and cross-linking agents : a comprehensive survey of genetic and biochemical characteristics[END_REF]. The work performed by Penny Jeggo on the xrs-mutants, isolated from the rodent CHO (chinese hamster ovary) cell line, enable the uncovering of XRCC5 and its role in DNA double strand break repair and chromosome integrity [START_REF] Kemp | X-ray sensitive mutants of Chinese hamster ovary cells defective in double-strand break rejoining[END_REF][START_REF] Jeggo | X-Ray-sensitive mutants of Chinese hamster ovary cell line. Isolation and cross-sensitivity to other DNA damaging agents[END_REF][START_REF] Jeggo | X-ray sensitive mutants of Chinese hamster ovary cell line: radio-sensitivity of DNA synthesis[END_REF][START_REF] Jeggo | Genetic analysis of X-ray-sensitive mutants of the CHO cell line Jeggo[END_REF][START_REF] Kemp | Radiation-induced chromosome damage in Xray-sensitive mutants (xrs) of the Chinese hamster ovary cell line[END_REF][START_REF] B K Singleton | Molecular and biochemical characterization of xrs mutants defective in Ku80[END_REF]. Later Taccioli et al., and Smider et al., prove Ku80 to be the gene product of XRCC5 and establish the knowledge about a direct role for Ku in DNA repair and V(D)J-recombination 2 [START_REF] Smider | Restoration of X-ray Resistance and V ( D ) J Recombination in Mutant Cells by Ku cDNA[END_REF][START_REF] G E Taccioli | Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination[END_REF][START_REF] Errami | Ku86 defines the genetic defect and restores X-ray resistance and V(D)J recombination to complementation group 5 hamster cell mutants[END_REF]. Indeed, the link between V(D)J recombination and DSB repair becomes more and more evident, when the overlapping phenotypes in cell lines defective for the members of the complementation groups 4 (XRCC4; Ligase IV),5 (XRCC5; Ku80),6 (XRCC6; Ku70) and 7 (XRCC7; DNA-PKcs) were uncovered, underlining the action of all these genes in the same pathway during antigen receptor rearrangement and DSB repair [START_REF] Penny | Menage à trois: double strans break repair, V(D)J recombination and DNA-PK[END_REF]. 1 Complementation assays investigate whether two parental mutants with the same phenotype(for example IR-sensitivity) have a mutation in the same gene or whether two genes in the same pathway are affected. In the latter case hybrids (F1) of those parental mutants show a wild-type phenotype and the genes are said to be in the same complementation group, as they can complement when crossed; in contrast if the phenotype persists in the hybrids, one concludes that the same gene was affected in the parental mutants.

2 V(D)J recombination is a mechanism of random gene assembly to generate an unique antigen receptor during the maturation of B-and T-cells (see Chapter 4)

1.Introduction 11

The co-action of these proteins is further demonstrated by the physical interaction and activation of Ku with DNA-PKcs forming the heterotrimeric DNA-PK complex [START_REF] Tanya | The DNA-Dependent Protein Kinase: Requirment for DNA Ends and Association with Ku Antigen[END_REF][START_REF] Nicholas J Finnie | DNA-dependent protein kinase activity is absent in xrs-6 cells: Implications for site-specific recombination and DNA double-strand break repair[END_REF][START_REF] Nicolai | Complementation of the ionizing radiation sensitivity, DNA end binding, and (V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen[END_REF] and the interaction of DNA-PK with XRCC4/Lig4 in proximity to DSBs [START_REF] Grawunder | Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells[END_REF][START_REF] Nick Mcelhinny | Ku recruits the XRCC4-ligase IV complex to DNA ends[END_REF][START_REF] Chen | Interactions of the DNA ligase IV-XRCC4 complex with DNA ends and the DNA-dependent protein kinase[END_REF]. Furthermore, functional co-action of Ku80 and DNA-PKcs in V(D)J recombination and DSB repair are demonstrated, when XRCC5 mutants, lacking DNA-PKcs catalytic activity, are complemented with the XRCC5 gene and thus restore kinase activity and radiation resistance [13, 26-28, 45, 46]. Finally, Roth el al., draw parallels between the XRCC genes 4-7 and the DSB repair pathway reactions seen in Xenopus, termed non-homologous end joining (NHEJ) [START_REF] Roth | How to make ends meet The repair of double-stranded breaks in DNA and the recombination Ku protein , which binds DNA ends , and its associated protein kinase[END_REF][START_REF] Thode | A novel pathway of DNA end-to-end joining[END_REF][START_REF] Beyert | Nonhomologous DNA end joining of synthetic hairpin substrates in Xenopus laevis egg extracts[END_REF][START_REF] Thompson | Nomenclature of human genes involved in ionizing radiation sensitivity[END_REF]. We know now that all these factors (and others) are necessary for the DNA repair by NHEJ and that this pathway represents the most commonly used repair pathway in mammals. the essential needs of a cell/organism to develop, live and reproduce. Therefore it is of unconstrained importance that the genetic integrity is preserved. However, the DNA molecule is under constant threat by noxious ravages from endogenous and exogenous origin that can generate genetic alterations and mutations leading to cancer and cell death [START_REF] Lindahl | Instability and decay of the primary structure of DNA[END_REF][START_REF] Hoeijmakers | Genome maintenance for Preventing Cancer[END_REF]. It is thus of no surprise, that the cell has established protective mechanisms, like DNA repair and DNA surveillance pathways to maintain genomic integrity to preserve its kind.

In this chapter I will present the cellular toolbox to deal with different kinds of DNA lesions, before detailing the repair mechanisms that specifically assure repair of DNA DSB. As much in front, Ku has a particular role in DNA DSB repair, not only as an active member of the non-homologous end joining (NHEJ) machinery, but also as a coordinator in DNA repair pathway choice, and a mediator of and actor in the DNA damage response (DDR). and cell [START_REF] Keith W Caldecott | Single-strand break repair and genetic disease[END_REF][START_REF] Jackson | The DNA-damage response in human biology and disease[END_REF].

Single strand break repair (SSBR) and base excision repair (BER) are closely related processes. The lesions that activate these repair pathways usually do not disturb the DNA structure but are rather the entire loss of a nucleotide (SSBR) or the modification of the nucleobase by purine oxidations, cytosine to uracil deaminations or base methylations (BER) [START_REF] Lindahl | Instability and decay of the primary structure of DNA[END_REF][START_REF] Hoeijmakers | Genome maintenance for Preventing Cancer[END_REF][START_REF] Brown | Targeting DNA repair in cancer: Beyond PARP inhibitors[END_REF][START_REF] Krokan | Base Excision Repair[END_REF]. The BER removes the altered nucleobase using different DNA glycolases generating an abasic site (APsite) which is cleaved by an AP-endonuclease thus creating a SSB. The created SSB, further undergoes end processing, gap filling by the short or long patch repair and is finally ligated by the Lig3 or Lig1 enzymes. The SSBR, acts similar to BER, however the lesion is recognized through PARP-1, which subsequently recruits downstream repair factors [START_REF] Keith W Caldecott | Single-strand break repair and genetic disease[END_REF]. Single strand annealing (SSA) represents another form of mutagenic DSB repair. Like HR, SSA requires long-range end resection, but as opposed to HR does not require a donor sequence, does not entail strand invasion and is independent of RAD51. Instead, SSA uses the resected ends to find sequence homologies and mediates end joining between nucleotide repeats in the genome. In the repair product, one copy of the repeat and the intervening DNA sequence are deleted, thus resulting in loss of genetic information. SSA is considered to be a back-up of HR [START_REF] Ceccaldi | Repair Pathway Choices and Consequences at the Double-Strand Break[END_REF][START_REF] Mladenov | DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferationdependent regulation[END_REF].

Nucleotide excision repair (NER

Non-homologous end joining (NHEJ) Classical non-homologous end joining (c-NHEJ) is the predominant pathway for DNA double strand break repair in mammals. It acts throughout the cell cycle but is most active in G0/G1 and G2.

Compared to HR it has fast kinetics and repairs double strand breaks via blunt ligation, independent of sequence homology [START_REF] Ceccaldi | Repair Pathway Choices and Consequences at the Double-Strand Break[END_REF][START_REF] Mladenov | DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferationdependent regulation[END_REF][START_REF] Fell | The Ku heterodimer: Function in DNA repair and beyond[END_REF][START_REF] Adachi | DNA ligase IVdeficient cells are more resistant to ionizing radiation in the absence of Ku70: Implications for DNA double-strand break repair[END_REF][START_REF] Kakarougkas | DNA DSB repair pathway choice: An orchestrated handover mechanism[END_REF][START_REF] Mao | DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells[END_REF]].

An alternative NHEJ (a-NHEJ) pathway has been suggested to act in DSB repair, too. Yet, there is still a lot of debate whether this pathway represents a back-up mechanism or if it is a dedicated pathway. Like the c-NHEJ pathway the a-NHEJ acts throughout the cell cycle, but in contrast is dependent on initial DNA end resection like HR. This kind of repair is mutagenic and favours nucleotide insertions, deletions and chromosome translocations [START_REF] Ceccaldi | Repair Pathway Choices and Consequences at the Double-Strand Break[END_REF][START_REF] Deriano | Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage[END_REF][START_REF] Fell | The Ku heterodimer: Function in DNA repair and beyond[END_REF][START_REF] Sfeir | Microhomology-Mediated End Joining: A Back-up Survival Mechanism or Dedicated Pathway?[END_REF] (more details on this pathway in Chapter 3.1.4). It is mostly active when either c-NHEJ and HR fail to repair [START_REF] Ceccaldi | Repair Pathway Choices and Consequences at the Double-Strand Break[END_REF][START_REF] Ceccaldi | Homologous-recombinationdeficient tumours are dependent on Polθ-mediated repair[END_REF][START_REF] Wang | PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways[END_REF][START_REF] Mateos-Gomez | Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination[END_REF].

Ku is part of the c-NHEJ pathway, but is also an important regulator and inhibitor of a-NHEJ and HR (cf Chapter 3.2).

c-NHEJ step-by-step

The c-NHEJ pathway appears to be a straightforward mechanism as it "simply" rejoins broken DNA ends, without the necessity of a sister chromatid and homology 1.Introduction 18 search. Still c-NHEJ needs to follow certain steps to realign broken ends in the most accurate way, given the nature of the damage. Blunt ends for example, as generated after endonuclease cleavage can be rejoined directly, whereas IR induced damage causes "dirty" or modified ends that have to be processed prior to joining.

The general workflow of c-NHEJ consists of five steps: 1) Recognition of the break by Ku , 2) Synapsis (DNA bridging), 3) Processing of the ends, 4) Ligation of the processed ends and 5) Ku removal from the repaired DNA [START_REF] Deriano | Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage[END_REF][START_REF] Fell | The Ku heterodimer: Function in DNA repair and beyond[END_REF]. A schematic summary of all steps is provided in Figure 3.2).

1) Recognition of the break by Ku

The first step in c-NHEJ is the recognition of the DNA DSB by the Ku heterodimer. Ku is considered to be one of the most abundant proteins in the cell (approximately 400-500 x 10 3 molecules/cell) and is the very first protein recruited to a DSB [START_REF] Peter R Blier | Binding of Ku Protein to DNA[END_REF][START_REF] Paillard | Analysis of the mechanism of interaction of simian Ku protein with DNA[END_REF][START_REF] Pierre-Olivier Mari | Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4[END_REF]. The Ku heterodimer slides on DNA with its ring shaped structure, thereby making contacts to a few phosphates of the DNA backbone [START_REF] Walker | Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair[END_REF] and then serves as interaction point for the other NHEJ factors like DNA-PK cs [START_REF] Tanya | The DNA-Dependent Protein Kinase: Requirment for DNA Ends and Association with Ku Antigen[END_REF], XRCC4/Ligase4 [START_REF] Nick Mcelhinny | Ku recruits the XRCC4-ligase IV complex to DNA ends[END_REF], XLF/Cernunnos (further on called XLF (XRCC4-like factor) in this manuscript) [START_REF] Yano | Functional significance of the interaction with Ku in DNA double-strand break recognition of XLF[END_REF] and PAXX (paralog of XRCC4 and XLF) [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 endjoining in XLF-deficient mice[END_REF][START_REF] Satish | PAXX Is an Accessory c-NHEJ Factor that Associates with Ku70 and Has Overlapping Functions with XLF[END_REF].

Upon DNA-PK cs arrival Ku slides about 10bp distal to the break [START_REF] Yoo | Geometry of a complex formed by double strand break repair proteins at a single DNA end: recruitment of DNA-PKcs induces inward translocation of Ku protein[END_REF][START_REF] Yaneva | Interaction of DNA-dependent protein kinase with DNA and with Ku-biochemical ans atomic force microscopy studied.pdf[END_REF], making place for the kinase and triggering its catalytic activity [START_REF] B K Singleton | The C Terminus of Ku80 Activates the DNA-Dependent Protein Kinase Catalytic Subunit[END_REF] important for the orchestration of downstream effectors [START_REF] Spencer | The life and death of DNA-PK[END_REF][START_REF] Katherine S Pawelczak | Coordination of DNA-PK activation and nuclease processing of DNA termini in NHEJ[END_REF]. DNA-bound Ku becomes thus part of the heterotrimeric DNA-PK complex.

It has been a long issue of debate whether there are several Ku proteins binding to a DNA end like "beads on a string" [START_REF] Downs | A means to a DNA end: the many roles of Ku[END_REF][START_REF] Walker | Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair[END_REF][START_REF] De Vries | HeLa nuclear protein recognizing DNA termini and translocating on DNA forming a regular DNA-multimeric protein complex[END_REF] or if one single Ku heterodimer is sufficient per break site [START_REF] Roberts | Loading of the nonhomologous end joining factor, Ku, on protein-occluded DNA ends[END_REF][START_REF] Britton | A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA doublestrand break repair[END_REF]. Indeed, Ku does not form distinct DNA repair foci after irradiation, like for example γ-H2Ax or Rad51, suggesting that it does not accumulate in large numbers on DNA DSBs. In cellulo, using a particular extraction buffer, Britton et al. visualized Ku foci with a particular extraction buffer and found rarely more than two Ku heterodimers per DNA DSB [START_REF] Britton | A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA doublestrand break repair[END_REF].

2) Synapsis

Synapsis describes the tethering of the broken DNA strands and is important to Early in vitro and atomic force microscopy studies in the late 1990's have already shown that Ku can easily transfer between two strands of DNA, suggesting that DNA molecules are bridged by Ku during DNA repair [START_REF] Bliss | Ku selectively transfers between DNA molecules with homologous ends[END_REF][START_REF] Pang | Ku Proteins Join DNA Fragments as Shown by Atomic Force Microscopy[END_REF][START_REF] Ramsden | Ku protein stimulates DNA end joining by mammalian DNA ligases: A direct role for Ku in repair of DNA double-strand breaks[END_REF]. Later, functional studies affirmed that Ku80 loss in murine fibroblasts leads to higher DNA dynamics of the broken ends. This phenomenon is of particular importance as stresses like γ-irradiation can induce clusters of DNA DSBs, that if not properly aligned, can be the cause of chromosomal translocations in cellulo [START_REF] Soutoglu | Positional stability of single double-strand breaks in mammalian cells[END_REF]. In this context, Ku is necessary for the recruitment of XLF that assembles into long filaments with its family partner XRCC4, spanning and aligning DNA ends [START_REF] Andres | A human XRCC4-XLF complex bridges DNA[END_REF][START_REF] Ropars | Structural characterization of filaments formed by human Xrcc4-Cernunnos/XLF complex involved in nonhomologous DNA end-joining[END_REF]. By using super-resolution microscopy Reid et al. show that after Ku loading onto DNA, XLF/XRCC4/Lig4 filaments are recruited, mediating side-by-side pairing of two filaments until an end-to-end alignment is attained [START_REF] Reid | Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair[END_REF]. Interestingly, the mechanism presented by Reid et al. is independent of DNA-PK cs .

In contrast, electron microscopy and two photon fluorescence cross-relation spectroscopy, clearly have shown a role for DNA-PK cs for juxtaposition of DNA ends and downstream processes like processing and ligation [START_REF] Defazio | Synapsis of DNA ends by DNA-dependent protein kinase[END_REF][START_REF] Merkle | Analysis of DNA-Dependent Protein Kinase-Mediated DNA End Joining by Two-Photon Fluorescence Cross-Correlation Spectroscopy[END_REF]. These observations are in agreement with the two-stage model proposed by Graham et al. [START_REF] Graham | Two-Stage Synapsis of DNA Ends during Non-homologous End Joining[END_REF], which suggests that heterotrimeric DNA-PK on the DNA forms a first long range synaptic complex, which activates DNA-PK catalytic activity and recruits downstream effectors XLF/Lig4/XRCC4 to constitute a short range synaptic complex, which is then ready for ligation.

Discrepancy between the models of

3) Processing of the ends

Processing of the DNA ends renders them compatible for ligation by providing a 3'hydroxyl-and a 5'phosphate DNA-end. DNA breaks are not always directly connectable and must be processed prior to joining. For instance, IR damaged DNA ends often show 3'-phosphoglycolates, 5'-hydroxyl groups, abasic sites, thymine glycols or ring fragmentations [START_REF] Katherine S Pawelczak | Coordination of DNA-PK activation and nuclease processing of DNA termini in NHEJ[END_REF]. Several kinases/phosphatases (PNPK), nucleases (Artemis, WRN, APLF, APTX, Mre11) polymerases (DNA polymerase β, µ, λ, TdT), helicases (RECQ1) or phosphodiesterases (tyrosyl-DNA phosphodiesterase 1) are involved in end processing. They are recruited dependent on the unique chemistry of each break [START_REF] Brandi L Mahaney | Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining[END_REF][START_REF] Howard | Different DNA End Configurations Dictate Which NHEJ Components are Most Important for Joining Efficiency[END_REF].

The most prominent processing enzyme is Artemis, having important roles in V(D)J recombination and NHEJ repair, hence loss of Artemis is associated with increased radiosensitivity and a complete loss of peripheral T-and B-lymphocytes [START_REF] Rooney | Defective DNA repair and increased genomic instability in Artemis-deficient murine cells[END_REF][START_REF] Moshous | Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency[END_REF]. Artemis has a 5'-exonuclease and 5'-or 3'-endonuclease activity when recruited and is activated by phosphorylated DNA-PK cs [START_REF] Katherine S Pawelczak | Coordination of DNA-PK activation and nuclease processing of DNA termini in NHEJ[END_REF][START_REF] Brandi L Mahaney | Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining[END_REF][START_REF] Howard | Different DNA End Configurations Dictate Which NHEJ Components are Most Important for Joining Efficiency[END_REF][START_REF] Jiang | Differential phosphorylation of DNA-PKcs regulates the interplay between end-processing and end-ligation during nonhomologous end-joining[END_REF].

Ku has a role in recruiting several factors for the processing step like polymerase µ and λ [START_REF] Kiran N Mahajan | Association of DNA polymerase mu (pol mu) with Ku and ligase IV: role for pol mu in end-joining doublestrand break repair[END_REF][START_REF] Ma | A biochemically defined system for mammalian nonhomologous DNA end joining[END_REF], APLF [START_REF] Hammel | An Intrinsically Disordered APLF Links Ku, DNA-PKcs and XRCC4-DNA Ligase IV in an Extended Flexible Non-Homologous End Joining Complex[END_REF], or WRN [START_REF] Grundy | The Ku-binding motif is a conserved module for recruitment and stimulation of non-homologous end-joining proteins[END_REF][START_REF] Karmakar | Ku heterodimer binds to both ends of the Werner protein and functional interaction occurs at the Werner N-terminus[END_REF]. Interestingly, Ku itself has been identified to have a processing activity as well, excising 5'terminal abasic sites that would otherwise block the ligation step [START_REF] Steven A Roberts | Ku is a 5'-dRP/AP lyase that excises nucleotide damage near broken ends[END_REF][START_REF] Strande | Specificity of the dRP/AP lyase of Ku promotes nonhomologous end joining (NHEJ) fidelity at damaged ends[END_REF]. Reid et al. elucidate that the 5'phosphate group is important for juxtaposition of the ends and a recognition element for Lig4, underlining the importance for proper end processing prior to ligation. They further depict a model in which non-compatible end chemistry can favour the dissociation of the two broken ends, leading to a new round of processing and tethering until compatible ends are generated. They suggest that a certain time window is provided for this "trial and error" process before other repair pathways such as mutagenic alt-NHEJ are favoured to take the job [START_REF] Reid | Bridging of double-stranded breaks by the nonhomologous end-joining ligation complex is modulated by DNA end chemistry[END_REF].

1.Introduction 21

4) Ligation of the processed ends

Ligation of the broken ends is performed after DNA synapsis and as soon as the processing steps warrant compatible ends. Only one ligase, Lig4, executes ligation during c-NHEJ. Loss of Lig4 is associated with embryonic lethality in mice and hypomorphic mutations lead to radiosensitivity, immunodeficiency, microencephaly and developmental delay in humans [START_REF] Frank | DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway[END_REF][START_REF] Barnes | Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice[END_REF][START_REF] Riballo | Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient[END_REF][START_REF] Mark | DNA ligase IV mutations identified in patients exhibiting developmental delay and immunodeficiency[END_REF][START_REF] Buck | Severe combined immunodeficiency and microcephaly in siblings with hypomorphic mutations in DNA ligase IV[END_REF]. Lig4 interacts and is stimulated by the XRCC4 protein, which by itself has no enzymatic activity but still leads to radiosensitivity when lost [START_REF] Grawunder | Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells[END_REF][START_REF] Stamato | Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell[END_REF]. XRCC4 is a scaffolding protein and interacts with APLF, Ku70 [START_REF] Pierre-Olivier Mari | Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4[END_REF], XLF, PNK and DNA thus stabilizing the Lig4-XRCC4 complex (X4-L4) on the break site [START_REF] Brandi L Mahaney | Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining[END_REF]. Lig4 stabilization is further provided by DNA-PK dependent phosphorylation [START_REF] Gang | Phosphorylation and regulation of DNA ligase IV stability by DNA-dependent protein kinase[END_REF].

XRCC4 is part of a homologous superfamily and includes the structurally similar proteins XLF and the recently identified protein PAXX. XLF was identified in patients with severe immunodeficiency, radiosensitivity and deficiency in DNA end ligation [START_REF] Andres | A human XRCC4-XLF complex bridges DNA[END_REF][START_REF] Ropars | Structural characterization of filaments formed by human Xrcc4-Cernunnos/XLF complex involved in nonhomologous DNA end-joining[END_REF][START_REF] Ahnesorg | XLF interacts with the XRCC4-DNA Ligase IV complex to promote DNA nonhomologous end-joining[END_REF][START_REF] Buck | Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly[END_REF]. Besides its actions together with XRCC4 to align DNA ends, XLF was shown to activate XRCC4/Lig4 for the ligation of mismatched and non-cohesive ends up to 100-fold, probably by stabilizing the XRCC4/Lig4 complex [START_REF] Tsai | Cernunnos/XLF promotes the ligation of mismatched and noncohesive DNA ends[END_REF][START_REF] Roy | XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation[END_REF]. The other homologue of this family PAXX, has recently been discovered by 3 different laboratories and approaches [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] Craxton | XLS (c9orf142) is a new component of mammalian DNA doublestranded break repair[END_REF][START_REF] Xing | Interactome analysis identifies a new paralogue of XRCC4 in non-homologous end joining DNA repair pathway[END_REF]. Based on the following observations PAXX is sought to be a core protein in c-NHEJ: it co-purifies with Ku and DNA-PKcs, is recruited to DNA damage sites, loss leads to radiosensitivity and its Ku-dependent ligation activating characteristics [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] Craxton | XLS (c9orf142) is a new component of mammalian DNA doublestranded break repair[END_REF][START_REF] Xing | Interactome analysis identifies a new paralogue of XRCC4 in non-homologous end joining DNA repair pathway[END_REF]. Craxton et al. show that PAXX can stimulate the XLF-dependent ligation step by 7.6-fold on DNA substrates with non-cohesive ends, whereas no stimulatory effect was seen for blunt ends [START_REF] Craxton | XLS (c9orf142) is a new component of mammalian DNA doublestranded break repair[END_REF]. However, Tadi et al. challenges these observations by affirming PAXX only an accessory role in c-NHEJ, stating that PAXX's stimulatory function on DNA ligation is mostly masked by XLF and becomes only noticeable after omitting XLF function [START_REF] Satish | PAXX Is an Accessory c-NHEJ Factor that Associates with Ku70 and Has Overlapping Functions with XLF[END_REF].

The ligation step is conducted by Lig4 during c-NHEJ, however its activity is highly dependent on the interaction partners XRCC4 and XLF. The role of PAXX in ligation is still an issue of debate. for ubiquitination [START_REF] Lehman | DNA-dependent conformational changes in the Ku heterodimer[END_REF], another possibility could be other posttranslational modifications such as phosphorylation [START_REF] Jeffrey R Skaar | Mechanisms and function of substrate recruitment by F-box proteins[END_REF] that enhance ubiquitination of Ku. pairs. The importance of Ku in NHEJ is exemplified in Ku deficient cells that harbour erroneous end-joining using microhomologies or large deletions for repair that generate genomic alterations [START_REF] Bétermier | Is non-homologous end-joining really an inherently error-prone process?[END_REF][START_REF] Soutoglu | Positional stability of single double-strand breaks in mammalian cells[END_REF][START_REF] Michael J Difilippantonio | DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation[END_REF][START_REF] Li | Ku70: a candidate tumor suppressor gene for murine T cell lymphoma[END_REF][START_REF] Nussenzweig | Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: the effects of ionizing radiation on growth, survival, and development[END_REF].

These data show that c-NHEJ is not an error-prone process per se, but rather adapts to the nature of the broken DNA. Thereby c-NHEJ maximizes the annealing of complementary nucleotides, thus diminishing a maximum of genetic alterations [START_REF] Bétermier | Is non-homologous end-joining really an inherently error-prone process?[END_REF].

Homologous Recombination

Homologous Recombination is a cellular key pathway, which acts in meiosis when homologous chromosome sequences are exchanged to guarantee genetic variation in offspring. In mitosis homologous recombination assures proper DNA double strand break repair during replication or in S/G2 cell cycle phase.

In contrast to NHEJ the DNA ends are not "simply" rejoined, but are resected to license subsequent strand invasion of a homologous DNA sequence. The process of DSB-induced homologous recombination can be divided into three steps: presynapsis, synapsis and postsynapsis (cf Figure 3.3). 1.Introduction

Pre-synapsis

alternative/mutagenic/microhomology mediated NHEJ

A new "alternative" non-homologous end joining pathway is the most recently discovered double strand break repair pathway and is known under a variety of names: a-NHEJ, alt-NHEJ, backup-NHEJ or MMEJ (microhomology mediated end joining). A main characteristic of a-NHEJ is the alignment of microhomologies (MH) distal from the DNA break, associated with deletions directed by end resection. This pathway represents a mutagenic, error-prone pathway and is associated with chromosomal translocations and oncogenesis [START_REF] Catherine T Yan | IgH class switching and translocations use a robust non-classical end-joining pathway[END_REF][START_REF] Simsek | DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation[END_REF][START_REF] Simsek | Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4/ligase IV during chromosomal translocation formation[END_REF]. It is called alt-NHEJ according to its discovery in yeast and mammalian cells, where c-NHEJ was inhibited through deletion of its core factors, XRCC4 or Ku [START_REF] Kabotyanski | Double-strand break repair in Ku86-and[END_REF][START_REF] Simon | Saccharomyces cerevisiae Ku70 potentiates pathways DNA repair[END_REF][START_REF] Liang | Ku80-deficient cells exhibit excess degradation of extrachromosomal DNA[END_REF].

The general model for a-NHEJ involves 5 steps: (1) initial 5'-3' end resection of the DNA ends, (2) MH annealing, (3) removal of the resulting flaps,(4) gap filling and ( 5) ligation (see Figure 3.4).

a-NHEJ shares the initial end resection with HR [START_REF] Lan N Truong | Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells[END_REF][START_REF] Zhang | An essential role for CtIP in chromosomal translocation formation through an alternative end-joining pathway[END_REF][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]. Short-range resection, executed by MRN/CtIP, thus can reveal distal homology sites of two adjacent ssDNA polymers that share complementarity.

Mammalian cells can anneal as few as 1 nt homologies [START_REF] Simsek | Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4/ligase IV during chromosomal translocation formation[END_REF], but mismatches and low GC content in the MH can result in unstable pairing [START_REF] Sfeir | Microhomology-Mediated End Joining: A Back-up Survival Mechanism or Dedicated Pathway?[END_REF]. Tethering factors have thus been discussed and PARP1 (Poly [ADP-ribose] polymerase 1)

was shown to play a role in a-NHEJ synapsis [151] [152]. For example during class-switch recombination (CSR) PARP1 competes with Ku to bind DNA ends [START_REF] Wang | PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways[END_REF], therefore a-NHEJ is also often referred as PARP1-dependent NHEJ.

Following annealing 3' flaps removal distal from the DNA ends is important prior to DNA polymerase dependent fill-in synthesis. In yeast 3'flaps are removed by the endonuclease Rad1-Rad10 [START_REF] Ma | Yeast Mre11 and Rad1 Proteins Define a Ku-Independent Mechanism To Repair Double-Strand Breaks Lacking Overlapping End Sequences[END_REF]. In vertebrates Rad1-Rad10 corresponds to XPF/ERCC1, but ERCC1 -/-mice revealed only a minor dependence on this flap endonuclease [START_REF] Bennardo | Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair[END_REF]. Further analysis has to elucidate flap removal in vertebrates. melanogaster [START_REF] Ham Chan | Dual roles for DNA polymerase theta in alternative end-joining repair of double-strand breaks in Drosophila[END_REF][START_REF] Yu | Synthesis-dependent microhomologymediated end joining accounts for multiple types of repair junctions[END_REF], which later on was confirmed to be evolutionary conserved in mice [START_REF] Mateos-Gomez | Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination[END_REF] and in HR-deficient tumours [START_REF] Ceccaldi | Homologous-recombinationdeficient tumours are dependent on Polθ-mediated repair[END_REF]. The C-terminal domain of Polθ encodes a proofreading-deficient polymerase that copies DNA in a template dependent manner and unlike other polymerases promotes extension and lesion bypass synthesis of ssDNA, increasing the probability to form base pairing by MH.

Fill-in synthesis

Polθ's N-terminal helicase-ATPase domain is important for MH pairing events, and might be important to displace DNA bound RPA, considered as an inhibitor of alt-NHEJ and a promoter of HR [START_REF] Sfeir | Microhomology-Mediated End Joining: A Back-up Survival Mechanism or Dedicated Pathway?[END_REF][START_REF] Ham Chan | Dual roles for DNA polymerase theta in alternative end-joining repair of double-strand breaks in Drosophila[END_REF][START_REF] Wang | Microhomology-mediated end joining: new players join the team[END_REF]. The central domain of Polθ contains a Rad51 -interaction domain, which blocks Rad51-mediated recombination together with the N-terminal ATPase domain [START_REF] Ceccaldi | Homologous-recombinationdeficient tumours are dependent on Polθ-mediated repair[END_REF], preventing HR. Polθ thus has a central role in driving microhomology mediated alt-NHEJ by its erroneous 1.Introduction 28 polymerase functions and inhibition of HR. Polθ has tethering and annealing activities and is recruited to DNA breaks in a PARP1 dependent manner [START_REF] Mateos-Gomez | Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination[END_REF][START_REF] Kent | Mechanism of Microhomology-Mediated End-Joining Promoted by Human DNA Polymerase Theta[END_REF].

Ligation of the DNA ends is the last step of alt-NHEJ. Lig3 and to a lesser extent Lig1 drive alt-NHEJ mediated sealing of DNA ends in biochemical assays and in vivo [START_REF] Simsek | DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation[END_REF][START_REF] Audebert | Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining[END_REF][START_REF] Wang | DNA Ligase III as a Candidate Component of Backup Pathways of Nonhomologous End Joining DNA Ligase III as a Candidate Component of Backup Pathways of Nonhomologous End Joining[END_REF].

It is a long matter of debate whether alt-NHEJ represents a back-up mechanism or if it is a dedicated pathway. This is first, because it became evident in cells that were deficient for c-NHEJ core factors [START_REF] Kabotyanski | Double-strand break repair in Ku86-and[END_REF][START_REF] Simon | Saccharomyces cerevisiae Ku70 potentiates pathways DNA repair[END_REF][START_REF] Liang | Ku80-deficient cells exhibit excess degradation of extrachromosomal DNA[END_REF] and second because no alt-NHEJ specific key factors have been identified so far. In fact all a-NHEJ factors appear to have overlapping functions with other known repair pathways including HR (MRN/CtIP), interstrand cross-link repair (Polθ), nucleotide excision repair (Lig 1) and base excision repair (PARP1, Lig 3) [START_REF] Sfeir | Microhomology-Mediated End Joining: A Back-up Survival Mechanism or Dedicated Pathway?[END_REF][START_REF] Wang | Microhomology-mediated end joining: new players join the team[END_REF].

However, it seems more and more likely that alt-NHEJ represents a distinct pathway that can coexist with c-NHEJ and HR. Several lines of evidence are in favour of such a function, such as 1) studies in C-terminal RAG2 mutants that show active alt-NHEJ in c-NHEJ proficient cells [START_REF] Corneo | Rag mutations reveal robust alternative end joining[END_REF], 2) two papers released in 2011 [START_REF] Zhang | An essential role for CtIP in chromosomal translocation formation through an alternative end-joining pathway[END_REF][START_REF] Lee-Theilen | CtIP promotes microhomology-mediated alternative end joining during class-switch recombination[END_REF], that show a prominent role for CtIP to drive alt-NHEJ mediated chromosomal translocation and impaired class switch recombination (CSR) in c-NHEJ proficient murine systems, 3) Truong et al. that provide an alt-NHEJ -HR competition assay and find alt-NHEJ to coexist with HR independent of c-NHEJ [START_REF] Lan N Truong | Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells[END_REF],

at levels around 10-20% of HR in human cell models and 4) Shamanna et al. [START_REF] Raghavendra | WRN regulates pathway choice between classical and alternative non-homologous end joining[END_REF] who provide insight into the inhibitory effect of WRN on alt-NHEJ, by impairing recruitment of MRN and CtIP to the break. They also state that WRN mutations in patient fibroblast show multiple, variable chromosomal aberrations and an increase in cancer incidence, however no direct link to alt-NHEJ is drawn by the authors.

Most of the early studies in alt-NHEJ were performed in murine systems [START_REF] Simsek | DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation[END_REF][START_REF] Simsek | Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4/ligase IV during chromosomal translocation formation[END_REF][START_REF] Weinstock | Formation of NHEJderived reciprocal chromosomal translocations does not require Ku70[END_REF]. However, there might be some striking species dependent differences concerning chromosomal translocations by alt-NHEJ in murine vs. human model systems [START_REF] Ghezraoui | Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining[END_REF]. Thus, Ghezroaoui et al. surprisingly find that the translocation rate is significantly decreased when core c-NHEJ factor like XRCC4 or Lig4 are In summary, there exist two types of NHEJ: error-free and error-prone. The error prone a-NHEJ is now recognized as a unique pathway that drives chromosomal translocations in a murine background; however its impact in the human DNA repair processes still remains elusive and needs further clarification. There is evidence for a second type of c-NHEJ in humans that accounts for mutagenic end-joining that has slow kinetics, active in the G1-cell cycle phase.

Crosstalk of DNA DSB repair pathways

Given the ability of a cell to deploy different DNA double strand break repair pathways it is of big interest how pathway choice is regulated. Generally c-NHEJ and HR represent the two major repair pathways that restore DNA integrity after DNA DSBs. Only if those two pathways fail to repair, the more mutagenic pathways like alt-NHEJ or single-strand annealing fill in the task, accepting errors only to avert most severe consequences associated with a complete loss of repair [START_REF] Mladenov | DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferationdependent regulation[END_REF] (see Figure 3.5). Special interest has been attributed to the choice between HR and NHEJ. Tight regulation and cooperation of both pathways seem inalienable dependent regulation of end resection, the crossroad between NHEJ and HR [START_REF] Mladenov | DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferationdependent regulation[END_REF].

We will see that multiple factors fine tune end resection and that Ku has an important role within.

DNA end resection dictates repair pathway choice between HR and NHEJ during S/G2-phase

The repair-concepts of c-NHEJ and HR repair diverge already at an early time point. During the S/G2-phase HR is dependent on a resection step, whereas c-NHEJ is not. In fact, resection inhibits c-NHEJ and vice versa Ku/DNA-PKcs block resection [START_REF] Kakarougkas | DNA DSB repair pathway choice: An orchestrated handover mechanism[END_REF][START_REF] Kakarougkas | Co-operation of BRCA1 and POH1 relieves the barriers posed by 53BP1 and RAP80 to resection[END_REF][START_REF] Zhou | Quantitation of DNA double-strand break resection intermediates in human cells[END_REF][START_REF] Symington | Double-Strand Break End Resection and Repair Pathway Choice[END_REF].

1.Introduction

The end resection mechanism The core machinery and the resection mechanism are preserved throughout evolution, so that the majority of studies concerning end resection have pioneered in yeast as a model system [START_REF] Huertas | DNA resection in eukaryotes: deciding how to fix the break[END_REF].

Generation of long 3' overhangs is a precondition for HR and is obtained in twosteps: initial resection and long range resection [START_REF] Symington | Double-Strand Break End Resection and Repair Pathway Choice[END_REF] that act in a bidirectional manner. Initially resection is executed by the coordinated activities of MRN nucleases (MRE11-NBS1-Rad50) and CtIP (MRX and Sae2 in yeast) [START_REF] Huertas | DNA resection in eukaryotes: deciding how to fix the break[END_REF][START_REF] Hustedt | The control of DNA repair by the cell cycle[END_REF]. The intrinsic endonuclease activity of MRE11 nicks the DNA backbone approximately 300bp distal from the break, giving access to its exonuclease to proceed in 3'-5' direction (towards the break) [START_REF] Garcia | Bidirectional resection of DNA double-strand breaks by Mre11 and Exo1[END_REF]. Long range resection depends on the coordinated helicase and exonuclease activities of BLM and Exo1 that proceed in 5'-3' direction (away from the break) creating long stretches of ssDNA important for strand invasion [START_REF] Hustedt | The control of DNA repair by the cell cycle[END_REF][START_REF] Garcia | Bidirectional resection of DNA double-strand breaks by Mre11 and Exo1[END_REF][START_REF] Chanut | Coordinated nuclease activities counteract Ku at singleended DNA double-strand breaks[END_REF] (see Fig. 3.6).

A special role in resection control is attributed to CtIP, as it stimulates MRE11 activity and fine tunes whether end resection takes place or not [START_REF] Huertas | DNA resection in eukaryotes: deciding how to fix the break[END_REF][START_REF] Huertas | Human CtIP mediates cell cycle control of DNA end resection and double strand break repair[END_REF][START_REF] Sartori | Human CtIP promotes DNA end resection[END_REF].

Thereby CtIP integrates signals from the cell cycle and chromatin status by interactions with Cdks and BRCA1, that recruit CtIP to the DSB and activate it in S/G2 [START_REF] Huertas | Human CtIP mediates cell cycle control of DNA end resection and double strand break repair[END_REF][START_REF] Sartori | Human CtIP promotes DNA end resection[END_REF][START_REF] Maximina | CtIP-BRCA1 modulates the choice of DNA double-strand break repair pathway throughout the cell cycle[END_REF][START_REF] Chen | Cell Cycle-dependent Complex Formation of BRCA1.CtIP.MRN Is Important for DNA Doublestrand Break Repair[END_REF]. During G1 end resection is inhibited by 53BP1 [START_REF] Bothmer | 53BP1 regulates DNA resection and the choice between classical and alternative end joining during class switch recombination[END_REF].

Upstream regulation of end resection by 53BP1-RIF and BRCA1-CtIP

on the chromatin level So far, the most upstream mechanism for resection is regulated by 53BP1-RIF and BRCA1-CtIP. 53BP1 is an important block for resection and thus promotes c-NHEJ, whereas its counterpart BRCA1 promotes resection by CtIP and thus HR.

Upon DSB induction the DNA damage response (DDR) protein ATM, phosphorylates the histone H2AX over several megabases which recruits the mediator MDC1. MDC1 has been shown important for ATM and MRN recruitment to the DSB extending region. It also leads to the recruitment of the ubiquitin ligases RNF8 and RNF168, which are considered important for the recruitment of 53BP1 to the chromatin. 53BP1 oligomerizes via interaction with dimethylated histone of 53BP1 by ATM leads to its interaction with RIF1 in G1 promoting c-NHEJ and repressing BRCA1-CtIP mediated resection [START_REF] Daley | RIF1 in DNA Break Repair Pathway Choice[END_REF][START_REF] Chapman | RIF1 Is Essential for 53BP1-Dependent Nonhomologous End Joining and Suppression of DNA Double-Strand Break Resection[END_REF][START_REF] Di | Rif1 prevents resection of DNA breaks and promotes immunoglobulin class switching[END_REF][START_REF] Escribano-Díaz | A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice[END_REF][START_REF] Zimmermann | 53BP1 : pro choice in DNA repair[END_REF][START_REF] Zimmermann | 53BP1 Regulates DSB Repair Using Rif1 to Control 5' End Resection[END_REF]. During S/G2 phase interaction between RIF1 and 53BP1 is disrupted via cell cycle dependent phosphorylation of CtIP (T487) and BRCA1 [START_REF] Chapman | RIF1 Is Essential for 53BP1-Dependent Nonhomologous End Joining and Suppression of DNA Double-Strand Break Resection[END_REF][START_REF] Escribano-Díaz | A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice[END_REF], giving access for nucleases to resect. BRCA1/CtIP and 53BP1/RIF1 are mutually exclusive on the DSB, underlining their reciprocal actions in the decision concerning end resection [START_REF] Zimmermann | 53BP1 : pro choice in DNA repair[END_REF].

Regulation of resection by DNA-PKcs

As mentioned earlier DNA-PKcs orchestrates NHEJ by phosphorylation events, this also impacts on the DNA pathway choice [START_REF] Cui | Autophosphorylation of DNA-Dependent Protein Kinase Regulates DNA End Processing and May Also Alter Double-Strand Break Repair Pathway Choice Autophosphorylation of DNA-Dependent Protein Kinase Regulates DNA End Processing and May Also Alter Double-Strand[END_REF][START_REF] Neal | Inhibition of homologous recombination by DNA-dependent protein kinase requires kinase activity, is titratable, and is modulated by autophosphorylation[END_REF]. DNA-PKcs blocks resection by inhibiting the access of Exo1. This is due to the phosphorylation status of its ABCDE cluster (of DNA-PKcs), partly regulated by ATM [START_REF] Zhou | Quantitation of DNA double-strand break resection intermediates in human cells[END_REF][START_REF] Zhou | DNA-dependent protein kinase regulates DNA end resection in concert with Mre11-Rad50-Nbs1 (MRN) and Ataxia Telangiectasia-mutated (ATM)[END_REF]. Additionally, DNA-PKcs phosphorylates ATM on multiple sites hence reducing its catalytic activity to promote DNA end 1.Introduction 33 resection [START_REF] Zhou | Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM[END_REF]. DNA-PKcs and ATM therefore seem to regulate each other in order to fine tune the end resection decision.

Regulation of resection by Ku at the DSB Another level of regulation is provided by Ku and MRN (MRX in yeast). In fact Ku and MRN are the first DNA repair proteins that locate to the DNA break [START_REF] Hartlerode | Recruitment and activation of the ATM kinase in the absence of DNA-damage sensors[END_REF], activities of either one have to proceed in a regulated manner to assure proper DNA repair.

Studies in yeast show that Ku loss leads to more MRX recruitment, rapid resection and higher levels of HR [START_REF] Zhang | Role of Dnl4-Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination[END_REF][START_REF] Eun Lee | Saccharomyces and Ku70, Mre11/Rad50, and RPA Proteins Regulate Adaptation to G2 / M Arrest after DNA Damage[END_REF][START_REF] Yong Shim | Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks[END_REF]. On the other hand MRX deletion comes along with a 20-fold higher recruitment of Ku [START_REF] Yong Shim | Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks[END_REF], blocking the first resection step [START_REF] Wu | Recruitment and Dissociation of Nonhomologous End Joining Proteins at a DNA Double-Strand Break in Saccharomyces cerevisiae[END_REF]. Additionally Ku blocks the access for Exo1 to execute long range resection [START_REF] Yong Shim | Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks[END_REF][START_REF] Eleni | Ku prevents Exo1 and Sgs1-dependent resection of DNA ends in the absence of a functional MRX complex or Sae2[END_REF][START_REF] Sun | Human Ku70/80 protein blocks exonuclease 1-mediated DNA resection in the presence of human Mre11 or Mre11/Rad50 protein complex[END_REF]. These observations suggest a competition model between Ku and MRX, so that the choice of a repair pathway resides in which protein complex is faster assembled and activated on the DNA break.

However, in human models the competition model seems not to be adequate due to organism differences concerning pathway dependency (yeast are much more reliant on HR than NHEJ compared to mammals) and cellular protein concentration of Ku (humans have greatly enriched cellular concentrations of Ku, compared to yeast). Additionally, unlike in yeast Ku cannot be pushed away by MRN and its recruitment/release kinetics in S and non-S-phase are similar in human models [START_REF] Sun | Human Ku70/80 protein blocks exonuclease 1-mediated DNA resection in the presence of human Mre11 or Mre11/Rad50 protein complex[END_REF]. Ku therefore seems to be actively released from the DSB, instead of displaced, in human cells [START_REF] Sun | Human Ku70/80 protein blocks exonuclease 1-mediated DNA resection in the presence of human Mre11 or Mre11/Rad50 protein complex[END_REF][START_REF] Shao | Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination[END_REF]. The exact mechanism for Ku removal, (similar to the removal after completion of c-NHEJ) is not fully understood yet. Still, several posttranslational modifications have been suggested including phosphorylation and ubiquitination in a cell cycle dependent manner.

Lee et al. [START_REF] Lee | Phosphorylation of Ku dictates DNA double-strand break ( DSB ) repair pathway choice in S phase[END_REF] provide evidence that a phosphorylation cluster in the core domain of Ku70 leads to conformational change and release of Ku from the break site, compared to phospho-mutants that are retained on the break. They argue that phosphorylation decreases Ku's affnity for DNA and that partially generated ssDNA on the break (to which Ku has less affnity) might tip the balance towards Ku release.

1.Introduction 34

Ubiquitination already has been shown to play an important role in the release of Ku from DNA after c-NHEJ completion [START_REF] Feng | The E3 ligase RNF8 regulates KU80 removal and NHEJ repair[END_REF]. In agreement, Ismail et al. [START_REF] Hassan | The RNF138 E3 ligase displaces Ku to promote DNA end resection and regulate DNA repair pathway choice[END_REF] present a new ubiquitin ligase, RNF138, to be recruited to short stretches of ssDNA after initial MRE11 resection and to ubiquitinate Ku80 for its release.

Interestingly RNF138 is also implicated in CtIP activation and its recruitment, strengthening the idea of RNF138 as an important regulator of end resection and pathway choice [START_REF] Schmidt | Systematic E2 screening reveals a UBE2D-RNF138-CtIP axis promoting DNA repair[END_REF].

Lastly, Chanut et al. [START_REF] Chanut | Coordinated nuclease activities counteract Ku at singleended DNA double-strand breaks[END_REF] A resection dependent c-NHEJ was recently revealed Surprisingly a report from 2017 shows that DNA end resection is not exclusive to the S/G2-phase and that RPA coated ssDNA can be detected in G1. They show that these resected ends occurring on complex DSB are a substrate for a slow acting, slow fidelity c-NHEJ. The resection process however differs from the resection process described above, as it starts resection not distal but from the DSB end, being independent on MRE11 endonuclease activity. In this case, CtIP is activated by Plk3, allowing its interaction with BRCA1 to promote initiation of resection, a resection eventually completed by Artemis and Mre11 exonuclease activities [START_REF] Biehs | DNA double-strand break resection occurs during non-homologous end-joining in G1 but is distinct to resection during homologous recombination in G2[END_REF].

In summary, DNA end resection is thought to be one of the major determinants of pathway choice in the S/G2-phase. The process is regulated on several levels and balanced by the presence of factors of both HR and NHEJ which are fine tuned in accordance with the cell cycle, phosphorylation and ubiquitination events. Ku

Other determinants of pathway choice

Besides the cell cycle and DNA end resection genomic localisation of the DNA break (hetero-or euchromatin) and the complexity of the break have been suggested to guide a switch from NHEJ to HR [START_REF] Mladenov | DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferationdependent regulation[END_REF][START_REF] Kakarougkas | DNA DSB repair pathway choice: An orchestrated handover mechanism[END_REF][START_REF] Clouaire | DNA double strand break repair pathway choice: a chromatin based decision? Nucleus[END_REF].

Ku outcompetes HR dependent repair pathways and alt-NHEJ

We have seen that Ku plays a central role in regulating end resection and several studies showed that Ku acts in a dominant negative manner inhibiting HR-related and alt-NHEJ repair pathways [START_REF] Fattah | Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells[END_REF]. The Fanconia Anemia pathway repairs interstrand cross-links via HR and cell lines deficient in FA genes are sensitive to DNA damaging agents, whereas simultaneous deletion of Ku70 reversed the sensitivity by reactivation of HR [START_REF] Fell | The Ku heterodimer: Function in DNA repair and beyond[END_REF][START_REF] Pace | Ku70 corrupts DNA Repair in the Absence of the Fanconi Anemia Pathway[END_REF]. Similarly yeast with impaired MRE11 activity, are shown sensitive to replication stress, due to inhibition of break-induced replication repair (BIR), and are rescued by Ku70 mutants to restore BIR activity [START_REF] Fell | The Ku heterodimer: Function in DNA repair and beyond[END_REF][START_REF] Steven S Foster | Functional Interplay of the Mre11 Nuclease and Ku in the Response to Replication-Associated DNA Damage[END_REF].

Besides HR dependent pathways, Ku also blocks alt-NHEJ [START_REF] Fattah | Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells[END_REF]. Loss of Ku leads to an increase of alt-NHEJ [START_REF] Fattah | Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells[END_REF]. In accordance, Ku and DNA-PK inhibit PARP1

DNA binding and enzymatic activity impairing alt-NHEJ, respectively [START_REF] Wang | PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways[END_REF][START_REF] Cheng | Ku counteracts mobilization of PARP1 and MRN in chromatin damaged with DNA double-strand breaks[END_REF][START_REF] Ariumi | Suppression of the poly ( ADP-ribose ) polymerase activity by DNA-dependent protein kinase in vitro[END_REF][START_REF] Wael Y Mansour | The absence of Ku but not defects in classical non-homologous end-joining is required to trigger PARP1-dependent end-joining[END_REF].

DNA damage response (DDR)

The DNA damage response represents an orchestrated signalling cascade that senses DNA damage, promotes conditions for proper DNA repair and carries out Glu motifs surrounding the DNA damage, as well as the downstream targets, important for cell cycle regulation, or p53, which acts as a transcription factor for several survival and death effector proteins [START_REF] Zou | DNA Damage Sensing by the ATM and ATR kinases[END_REF][START_REF] Matsuoka | ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage[END_REF].

ATM and ATR thus can be seen as "master" regulators activating and orchestrating a large network of cellular processes to maintain genomic integrity [START_REF] Zou | DNA Damage Sensing by the ATM and ATR kinases[END_REF].

ATM is recruited to the DNA damage by the MRN-complex subunit NBS1, hence its kinase activity is activated [START_REF] Zou | DNA Damage Sensing by the ATM and ATR kinases[END_REF][START_REF] Shiloh | The ATM protein kinase: regulating the cellular response to genotoxic stress, and more[END_REF][START_REF] Lee | ATM Activation by DNA Double-Strand Breaks Through the Mre11-Rad50-Nbs1 Complex[END_REF][START_REF] Lee | Direct Activation of the ATM Protein Kinase by the Mre11/Rad50/Nbs1 Complex[END_REF] They furthermore show profound differences for these Ku70 mutants concerning apoptosis and cell cycle arrest [START_REF] Victoria | Ku Regulates Signaling to DNA Damage Response Pathways through the Ku70 von Willebrand A domain[END_REF][START_REF] Victoria L Fell | Ku70 phosphorylation mediates Aurora B inhibition and activation of the DNA damage response[END_REF]. All these studies underline the central role of Ku70/Ku80 in ATM and to a lesser extend ATR activation and the subsequent alterations in cell fate decisions.

1.Introduction

Cell cycle and its checkpoints

The cell cycle defines a series of cellular events with the aim of DNA replication and proper cell division to generate two daughter cells. Three periods are distinguished: interphase, mitosis and cytokinesis. Interphase can be further divided into gap 1 (G1)-phase, synthesis (S)-phase and gap 2 (G2)-phase. During G1 the cell activates its metabolism, increases transcription and protein production as well as its organelle content. During S-phase the whole DNA is doubled, so that each chromosome has replicated a sister chromatid. During G2-phase the cell size increases, proteins are produced and the cell prepares for mitosis and cytokinesis, when chromosomes and cytoplasm are divided. After cell cycle completion, two daughter cells emerge and resume a new round of cell cycle. Cells that have stopped dividing (i.e. neurons) are said to be in G0, senescent or quiescent. These cells can re-enter the cell cycle (G0 and quiescent cells) or remain silent until their death (senescence).

The passage through the different cell cycle phases is mediated by the oscillating activity of cyclins, small proteins that activate Cdks in a timely regulated pattern, and D2, CyclinE, Cdc25A and E2F [START_REF] Iliakis | DNA damage checkpoint control in cells exposed to ionizing radiation[END_REF].

A delayed entry into S-phase is assured by the G1/S-checkpoint and prevents the cell to initiate any DNA synthesis when DNA is damaged. This checkpoint acts via two branches, which are both initiated via the transducer kinases ATM, Chk2 and to a lesser extent ATR and Chk1. The acute response, consists in the phosphorylation of Cdc25 by Chk2, allowing Cdc25 ubiquitination and degradation; thus Cdk2 can not be dephosphorylated and stays locked in an inactive state.

The second branch consists in the transcriptional activation of the Cdk inhibitor p21, by p53, which is stabilized and protected from degradation after phosphorylation (S15,S20) by the transducer kinases [START_REF] Medema | Checkpoint control and cancer[END_REF][START_REF] Iliakis | DNA damage checkpoint control in cells exposed to ionizing radiation[END_REF][START_REF] Bartek | Pathways governing G1 / S transition and their response to DNA damage[END_REF][START_REF] Indra | The same, only different -DNA damage checkpoints and their reversal throughout the cell cycle[END_REF]. A schematic overview of this checkpoint is depicted in Figure 3.10 A.

During S-phase DNA synthesis for replication takes place and instead of halting the cell cycle at the transition stage from one phase to the next, the intra-Sphase checkpoint acts throughout the entire replication-phase. ATM dependent signalling is still engaged during this checkpoint, nevertheless a shift towards ATR signalling is observed with the generation of increasing amounts of ssDNA during DNA synthesis. Similar to the G1-phase checkpoint, inhibition of origin firing is reached by blocking Cdk2/CyclinA/E complex. This however, in contrast to the G1-checkpoint, is independent of p53, and p21. It is instead realized by Chk1/Chk2 dependent degradation of Cdc25A and Wee1 activation. Wee1 kinase is expressed during S-phase and inhibits Cdk2 through phosphorylation [START_REF] Iliakis | DNA damage checkpoint control in cells exposed to ionizing radiation[END_REF][START_REF] Indra | The same, only different -DNA damage checkpoints and their reversal throughout the cell cycle[END_REF] (see Figure 3.10 B).

The transition from G2-phase to mitosis is essentially due to the activity of the To resume the cell cycle after DNA repair, checkpoint signalling must be turned off, a mechanism called checkpoint recovery, best understood for the G2/Mtransition [START_REF] Medema | Checkpoint control and cancer[END_REF]. The activity of Plk1 is essential in this process, phosphorylating Chk2, Claspin and Wee, thus reversing their cell cycle blocking functions, as well as promoting nuclear translocation of Cdc25C [START_REF] René | Checkpoint recovery in cells: how a molecular understanding can help in the fight against cancer[END_REF]. Although, Plk1 is essential for cell cycle recovery it is not sufficient. Several phosphatases are important in checkpoint recovery (PP1, PP2A, PP4, PP5, PP6) by dephosphorylating important DDR-factors like ATM, ATR Chk2, Chk1, BRCA1 and γ-H2AX [START_REF] Freeman | Phosphatases in the cellular response to DNA damage[END_REF][START_REF] René | Checkpoint recovery in cells: how a molecular understanding can help in the fight against cancer[END_REF]. Among these, Wip1 has a central role as it recognizes specifically p(S/T)Q-motifs, phosphorylated by ATM and ATR, and inhibits p53 expression and activity [START_REF] René | Checkpoint recovery in cells: how a molecular understanding can help in the fight against cancer[END_REF][START_REF] Lu | The type 2C phosphatase Wip1: an oncogenic regulator of tumor suppressor and DNA damage response pathways[END_REF]. point activation in S and G2, compared to Ku80 proficiency after irradiation. This is mainly due to deregulated ATM and ATR signalling [START_REF] Zhou | Ku affects the ATM-dependent S phase checkpoint following ionizing radiation[END_REF][START_REF] Wang | Ku Affects the CHK1-dependent G 2 Checkpoint after Ionizing Radiation 1[END_REF]. Interestingly, Ku80 also impacts on the assembly of replication complexes, necessary to drive DNA synthesis during S-phase and Ku80 deficiency favours accumulation in G1phase (activated by increased p21 levels, and decreased Cdc25A and Cdk2 levels), independent of DNA damage [START_REF] Rampakakis | Ku is involved in cell growth, DNA replication and G1-S transition[END_REF]. Moreover, direct interaction between Ku70 and cyclin A1, A2 and E are observed leading to cell cycle dependent phosphorylation events by Cdk2 that drive proper cell cycle flow [START_REF] Ji | DNA damage response involves modulation of Ku70 and Rb functions by cyclin A1 in leukemia cells[END_REF][START_REF] Mukherjee | Phosphorylation of Ku70 subunit by cell cycle kinases modulates the replication related function of Ku heterodimer[END_REF]. Finally, a specific residue in the vWA domain of Ku70 (S155) can induce cell cycle arrest in G1/S and G2/M phase, driven by the interaction with Aurora B [START_REF] Victoria L Fell | Ku70 phosphorylation mediates Aurora B inhibition and activation of the DNA damage response[END_REF]. All of these studies show that Ku can impact on the cell cycle independently of its DNA repair function.

In summary, stalling of the cell cycle is an important means for a damaged cell to provide a "time window" for repair and is a tightly regulated process. Several studies confirm the role of Ku within proper cell cycle progression, but also show that it can, itself, trigger cell cycle checkpoint activation. However, when DNA damage exceeds the cells capacity to repair and hinders the cell cycle to resume, the cells fate is guided through senescence or cell death.

Senescence

Cellular senescence is a condition in which cells, despite being alive, are unable to proliferate. This phenotype has first been described by Hayflick and colleagues after they had observed replicative exhaustion in human primary fibroblasts in vitro [START_REF] Hayflick | The serial cultivation of human diploid cell strains[END_REF].

Senescence is considered as an irreversible stress response, associated with growth arrest of metabolically active cells that can not re-enter DNA replication. These cells thus remain in the G1 phase. Senescence therefore is different from quiescence

or terminal differentiation [START_REF] Fabrizio D'adda | Living on a break: cellular senescence as a DNA-damage response[END_REF][START_REF] Campisi | Cellular senescence: when bad things happen to good cells[END_REF].

Several morphological changes like, enlarged and flattend cells, with increased granularity, a vacuole rich cytoplasm, as well as a reorganisation of the subnuclear chromosomes and sometimes multinucleation come along with this cell state [START_REF] Gewirtz | Accelerated senescence: An emerging role in tumor cell response to chemotherapy and radiation[END_REF][START_REF] Rebecca | Cellular Senescence -its role in cancer and the response to ionizing radiation[END_REF][START_REF] Munoz-Espin | Cellular senescence: from physiology to pathology[END_REF]. Besides these structural markers, several biochemical and molecular biomarkers have been associated to senescence: β-Galactosidase expression, cell cycle inhibitor expression (p21, p53, p15, p27, p16, ARF, hyphophosyphorylated RB), absence of proliferation, constitutive DNA damage foci in heterochromatic regions (senescence-associated heterochromatic foci enriched in histone 3met-H3K9; SAHF), and the secretion of inflammatory cytokines and chemokines (senescence associated secretion phenotype; SASP) [START_REF] Munoz-Espin | Cellular senescence: from physiology to pathology[END_REF].

Triggers of senescence are manifold, and include shortening of the telomeres (replicative senescence, as described by Hayflick), exhaustive amount of DNA lesions (DNA damage induced senescence), overexpression of oncogenes like RAS (oncogene induced senescence) or chronic generation of ROS (stress induced senescence) [START_REF] Campisi | Cellular senescence: when bad things happen to good cells[END_REF][START_REF] Munoz-Espin | Cellular senescence: from physiology to pathology[END_REF].

Molecularly it is driven and maintained by two pathways, the p53 and the p16-RB pathway. Both interact and finally converge in the activation of cell cycle inhibitors and hypophosphorylation of RB (see Figure 3.11). Stimuli that activate the ATM/ATR (DDR) primarily induce senescence through the p53-p21 pathway, but can also engage the p16-RB pathway dependent on the cell type, species or origin of stress [START_REF] Campisi | Cellular senescence: when bad things happen to good cells[END_REF][START_REF] Munoz-Espin | Cellular senescence: from physiology to pathology[END_REF]. In humans both, have to be inhibited to prevent the onset of senescence [START_REF] Smogorzewska | Different telomere damage signaling pathways in human and mouse cells[END_REF][START_REF] Shay | A role for both RB and p53 in the regulation of human cellular senescence[END_REF].

The biological purpose of senescence is the elimination of damaged cells, which is particularly important in cancer. It is therefore traditionally considered as a tumour suppressor mechanism on a cellular level. Limiting the accumulation of damage, by growth arrest on one hand and the secretion of inflammatory signals Ku in senescence Studies in mouse derived knock out cells for Ku70 and Ku80

have revealed a role for Ku in replicative exhaustion, as these cells demonstrate increased premature senescence [START_REF] Gu | Growth retardation and leaky SCID phenotype of Ku70-deficient mice[END_REF][START_REF] Nussenzweig | Requirement for Ku80 in growth and immunoglobulin V(D)J recombination[END_REF]. Moreover Ku80 deficient mice show signs of premature ageing and senescence, a phenotype that could be reversed by the simultaneous deletion of p53, with the price of an increased B-cell lymphoma incidence in those mice, underlining the close association between senescence and carcinogenic processes [START_REF] Lim | Analysis of ku80-Mutant Mice and Cells with Deficient Levels of p53[END_REF][START_REF] Vogel | Deletion of Ku86 causes early onset of senescence in mice[END_REF]. Lastly, the expression of a human phosphomimetic mutant of S155 Ku70 increased the expression of p21 and the β-Galactosidase activity in MEFs and a human cell line, further emphasizing the direct role of

Ku in the onset of senescence [START_REF] Victoria L Fell | Ku70 phosphorylation mediates Aurora B inhibition and activation of the DNA damage response[END_REF]. Interestingly, all studies providing evidence for a role of Ku in senescence were performed without exogenous DNA damage,

showing that the Ku associated senescence phenotype is independent of its role in DNA DSB repair.

In summary, senescence is a mechanism that is induced upon several cellular stresses, thereunder excessive DNA lesions. As a tumour suppressor mechanism its purpose is the elimination of damaged cells leading to tissue regeneration. Several lines of evidence provide proof for the role of Ku in the onset of senescence, this function however seems to be independent of its repair function.

Cell death pathways

Besides senescence, programmed cell death pathways represent a means to get rid of potentially harmful cells during the DDR. Different cell death programs, such as apoptosis, necrosis, necroptosis, ferroptosis, mitotic catastrophe etc., are distinguished, dependent on their morphological and molecular differences in execution.

Special attention should be given to apoptosis in this section, as it represents the principal cell death mechanism triggered upon DNA damage. We will see that Ku has a prominent role within this process independent of its DNA repair function.

Additionally mitotic catastrophe is a significant cause of cell death in tumour cell lines after treatment with irradiation [START_REF] Gudkov | The role of p53 in determining sensitivity to radiotherapy[END_REF][START_REF] Roninson | If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells[END_REF] and shall be presented at the end of this section.

Apoptosis

Apoptosis was first described in the nematode C.elegans where programmed cell death at particular time points was observed during development. It is a mechanism that occurs under physiologic conditions and is a homoeostatic mechanism This platform serves inactive procaspases 8 or 10 to oligomerize and initiate selfcleavage [START_REF] Wynand P Roos | DNA damage and the balance between survival and death in cancer biology[END_REF][START_REF] Michael | Ionizing radiation as a response-enhancing agent for CD95-mediated apoptosis[END_REF][START_REF] Scaffidi | Two CD95 ( APO-1 / Fas ) signaling pathways[END_REF]. Subsequently caspase 8 and 10, activate the effector caspases 3 and 7, which cleave and inactivate essential cellular proteins for cell cycle, DNA repair, cell structure or signalling [START_REF] Matt | The DNA damage-induced cell death response: a roadmap to kill cancer cells[END_REF].

After irradiation induced DNA damage, it is the intrinsic apoptosis that is mostly activated [START_REF] Matt | The DNA damage-induced cell death response: a roadmap to kill cancer cells[END_REF][START_REF] Maier | Cellular pathways in response to ionizing radiation and their targetability for tumor radiosensitization[END_REF].

It

Mitotic catastrophe

Mitotic catastrophe represents a main event to drive cell death in cancer cell lines exposed to irradiation [START_REF] Roninson | If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells[END_REF][START_REF] Vakifahmetoglu | Death through a tragedy: mitotic catastrophe[END_REF]. It is characterized by a prolonged block of the G2/M-checkpoint, loss of clonogenicity and a delayed death [START_REF] Roninson | If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells[END_REF][START_REF] Vakifahmetoglu | Death through a tragedy: mitotic catastrophe[END_REF]. In contrast to apoptosis, mitotic catastrophe, is a mitosis linked death pathway [START_REF] Vakifahmetoglu | Death through a tragedy: mitotic catastrophe[END_REF] and is thus associated with the appearance of temporarily viable aneuploid, multi-and micronucleated giant cells [START_REF] Vitale | Mitotic catastrophe: a mechanism for avoiding genomic instability[END_REF]. However mitotic catastrophe does not represent a unique cell death pathway. Today it is generally accepted that mitotic catastrophe is a "pre-stage" of cell death, mainly executed by apoptosis [START_REF] Vakifahmetoglu | Death through a tragedy: mitotic catastrophe[END_REF][START_REF] Vitale | Mitotic catastrophe: a mechanism for avoiding genomic instability[END_REF][START_REF] Castedo | Cell death by mitotic catastrophe: a molecular definition[END_REF].

As described in chapter 3.10, multiple cell cycle checkpoints guarantee a time window for repair, after DNA damage, and it is only during weakened checkpoint activity, that damage acquired in interphase can be expanded to mitosis. For example, checkpoint adaption describes the entry of mitosis in the presence of persistent DNA damage, when a sustained checkpoint-induced G2/M-arrest can no longer be maintained. Furthermore, these cells can either succumb to mitotic catastrophe in metaphase, or proceed to the next interphase by mitotic slippage, when similar to adaption, the mitotic checkpoints are weakened or an aberrant round of mitosis is conducted without proper segregation of sister chromatids and cytokinesis [START_REF] Vakifahmetoglu | Death through a tragedy: mitotic catastrophe[END_REF][START_REF] Castedo | Cell death by mitotic catastrophe: a molecular definition[END_REF]. Cells that exit mitosis are either tetraploid, due to failed cytokinesis or are highly aneuploid [START_REF] Vitale | Mitotic catastrophe: a mechanism for avoiding genomic instability[END_REF]. Most of these cells are non-viable and undergo mitotic catastrophe during the next cycle, however each round can drive the production of new potentially tumourigenic cells with high genetic instability.

1.Introduction 51

Mitotic catastrophe is considered as an onco-suppressive mechanism, that culminates in the clearance of potentially harmful cells [START_REF] Vitale | Mitotic catastrophe: a mechanism for avoiding genomic instability[END_REF].

It is thought that inhibition of mitotic catastrophe increases the survival of aneuploid and genetically unstable cells, thereby contributing to carcinogenesis and the karyotypic heterogeneity of cancer cells [START_REF] Vitale | Mitotic catastrophe: a mechanism for avoiding genomic instability[END_REF].

Autophagy

To provide a full picture of the DNA damage response, autophagy and its role at the intersection of cell death and survival, as well as its genome protection function shall be presented.

The term autophagy translates from the Greek auto meaning "oneself" and phagy meaning "to eat" and describes the catabolic degradation and recycling of cellular components, to meet with the anabolic and bioenergetic needs of the cell [START_REF] Aristides | DNA Damage Response and Autophagy: A Meaningful Partnership[END_REF][START_REF] Hewitt | Repair, Reuse, Recycle: The Expanding Role of Autophagy in Genome Maintenance[END_REF]. There exist three different types of autophagy, from which macroautophagy is the best studied. This type of autophagy sequesters cytoplasmic cargo into double membrane vesicles called autophagosomes, which ultimately fuse to lysosomes [START_REF] Hewitt | Repair, Reuse, Recycle: The Expanding Role of Autophagy in Genome Maintenance[END_REF].

Autophagy is a ubiquitous mechanism that is highly conserved in eukaryotic cells with the aim to preserve homoeostasis It is induced upon a variety of stress conditions, such as nutrient deprivation, growth factor withdrawal, energetic stress, oxidative stress or DNA damage. Deregulation of autophagy is involved in a variety of diseases including cancer [START_REF] Rodriguez-Rocha | DNA damage and autophagy[END_REF].

The autophagic machinery is a carefully orchestrated process where around 30 members of the Atg-gene family and a plethora of associated proteins take part [START_REF] Hewitt | Repair, Reuse, Recycle: The Expanding Role of Autophagy in Genome Maintenance[END_REF]. The autophagic process can be divided into three steps. First, during initiation, an initial double membrane called the phagophore is build up by association of ULK1 with Atg13, FIP200 and Atg101. This step is mainly regulated by an inhibitory phosphorylation of ULK1 by mTORc1, which is the main inhibitor of autophagy. mTORc1 on the other hand is inhibited through phosphorylation by JNK [START_REF] Aristides | DNA Damage Response and Autophagy: A Meaningful Partnership[END_REF][START_REF] Hewitt | Repair, Reuse, Recycle: The Expanding Role of Autophagy in Genome Maintenance[END_REF]. When cellular homoeostasis is balanced, Beclin-1 is sequestered by the anti-apoptotic protein Bcl-2 and it is only after JNK dependent phosphorlyation of Bcl-2, that Beclin-1 is released [START_REF] Rodriguez-Rocha | DNA damage and autophagy[END_REF].

The role of autophagy as an independent cell death pathway is highly controversial, and it is rather seen as a survival mechanism, to provide cellular save-energy building blocks during unfavourable environmental conditions [START_REF] Gozuacik | Autophagy as a cell death and tumor suppressor mechanism[END_REF]. This said, the constitutive activation of autophagy after an overwhelming amount of DNA damage, can however shift the balance to an uncontrolled autophagy, that may be a prerequisite for the activation of cell death pathways [START_REF] Wynand P Roos | DNA damage and the balance between survival and death in cancer biology[END_REF]. In this context the DNA bound FOXO3 protein triggers transcriptional activation of LC3 and BNIP, both important for autophagy, as well as the sustained activation of the ATM/ATR-Chk1/Chk2-p53-axis, that ultimately activates p53-dependent cell death by necroptosis and apoptosis [START_REF] Wynand P Roos | DNA damage and the balance between survival and death in cancer biology[END_REF].

Interestingly, besides its role in homoeostasis, cell survival and cell death, au- Despite being a cytoplasmic process placed at the edge of cell survival and cell death, autophagy now gets more and more into focus as a part of DNA repair regulation, and genomic stability as part of the DDR.

Coordination and cell fate decisions upon the DDR

During this chapter we have seen that a damaged cell has several cell fate alternatives after damage induction, such as irradiation. Still we do not know which parameters are involved in this choice. The decision whether a cell attempts DNA repair, initiates apoptosis or stays in an arrested state are highly dependent on the cell type, cell cycle stage, the environmental conditions, as well as the amount of DNA damage. Apart from lymphocytes that immediately trigger apoptosis after irradiation, and fibroblast that preferentially undergo senescence, most cell types follow a determined hierarchy of decision points. Gudkov et al., proposes that these cells show a cell cycle arrest to allow an attempt of repair by the DNA repair machinery and given successful repair, the cell can restart proliferation. In case of incomplete or erroneous repair, due to repair factor deficiencies or a devastating amount of damage, cells trigger apoptosis or senescence [START_REF] Gudkov | The role of p53 in determining sensitivity to radiotherapy[END_REF]. Importantly, failures in the DDR can reactivate proliferation even in cells where DNA damage persists and thus drive genetic instability [1]. The consequence is on one hand a mutator phenotype, which further drives instability or on the other hand triggers a mitotic catastrophe and cell death [START_REF] Gudkov | The role of p53 in determining sensitivity to radiotherapy[END_REF]. We have also seen in this chapter that the Ku protein is a main regulator of DNA DSB repair and NHEJ, but that it also shows repair-independent activities during the DDR. The decision points described above are summarized in Figure 3.13, additionally Ku is delineated at those points where it has an attributed function. (see Figure 4.1A) . Beyond that, further variety is given by the action of endo-and exonuclease activity, as well as by a nucleotide transferase that adds nucleotides in a random manner between the segments [START_REF] Murphy | Janeway's Immunobiology[END_REF]. We will see in this chapter, that the cNHEJ factors have crucial functions in V(D)J recombination and that deficiencies can have severe impact on the developing T-and B-lymphocytes.

1.Introduction 56

V(D)J recombination relies on the cNHEJ machinery

In chapter 2.3 we have already mentioned that V(D)J recombination and the NHEJ show similar dependencies on the XRCC-genes. A lack of these genes results in DNA DSB repair deficiency accompanied by weakened V(D)J recombination. Two major discoveries constituted the understanding that these pathways are closely interwoven [START_REF] Villartay | Congenital defects in V(D)J recombination[END_REF]: first, the observation that mice with a severe combined immunodeficiency (scid) and a defect in circulating B-and T-lymphocytes showed a general radiosensitivity [START_REF] Bosma | A severe combined immunodeficiency mutation in the mouse[END_REF][START_REF] Fulop | The scid mutation in mice causes a general defect in DNA repair[END_REF], as well as, second the demonstration that CHO XRCC DNA repair mutants showed a severe defect in coding and signal joint formation after RAG 1/2 expression in vitro [START_REF] Taccioli | Impairment of V ( D ) J Recombination in Double-Strand Break Repair Mutants[END_REF].

V(D)J rearrangements do not occur randomly though rather reorganize in a highly Artemis that catalyses the opening of the hairpin coding joint. This usually results in a 3'overhang, and Artemis further increases junctional diversity with its exo-and endonuclease activity. Coding end processing is further assured by the TdT (terminal-deoxinucleatidyl-transferase), recruited by Ku80 [START_REF] Bogue | V(D)J recombination in Ku86-deficient mice: Distinct effects on coding, signal, and hybrid joint formation[END_REF][START_REF] M M Purugganan | Ku80 is required for addition of N nucleotides to V(D)J recombination junctions by terminal deoxynucleotidyl transferase[END_REF], that catalyses the addition of nucleotides randomly. Finally, the XRCC4/Lig4 complex ligates the coding joint, that codes for the antigen receptor and the excised signal joint, which will be lost in later cell divisions [START_REF] Villartay | Congenital defects in V(D)J recombination[END_REF][START_REF] Schatz | V(D)J Recombination: Mechanisms of Initiation[END_REF]. KO models [START_REF] Nussenzweig | Requirement for Ku80 in growth and immunoglobulin V(D)J recombination[END_REF][START_REF] Bosma | A severe combined immunodeficiency mutation in the mouse[END_REF][START_REF] Gao | A Critical Role for DNA End-Joining Proteins in Both Lymphogenesis and Neurogenesis[END_REF][START_REF] Frank | Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV[END_REF][START_REF] Zhu | Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates[END_REF][START_REF] Ouyang | Ku70 is required for DNA repair but not for T cell antigen receptor gene recombination In vivo[END_REF][START_REF] Gu | Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination[END_REF]. Analysis of the coding and signal joints further reveal large deletions in XRCC4 CHO mutant cells of up to 20bp for coding joints and only 10% of correct signal joints [START_REF] Taccioli | Impairment of V ( D ) J Recombination in Double-Strand Break Repair Mutants[END_REF]. Incorrect rearrangements were further observed in mice embryonic stem (ES) cells of XRCC4 deficient [START_REF] Gao | A Critical Role for DNA End-Joining Proteins in Both Lymphogenesis and Neurogenesis[END_REF] and MEF's of Lig4 deficient embryos [START_REF] Frank | Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV[END_REF]. Similarly a reduction in coding and signal joint formation [START_REF] Smider | Restoration of X-ray Resistance and V ( D ) J Recombination in Mutant Cells by Ku cDNA[END_REF][START_REF] Errami | Ku86 defines the genetic defect and restores X-ray resistance and V(D)J recombination to complementation group 5 hamster cell mutants[END_REF][START_REF] Nussenzweig | Requirement for Ku80 in growth and immunoglobulin V(D)J recombination[END_REF][START_REF] Taccioli | Impairment of V ( D ) J Recombination in Double-Strand Break Repair Mutants[END_REF] with an accumulation of hairpin ending coding joints 1.Introduction and blunt-full length signal joints [START_REF] Zhu | Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates[END_REF] is observed in Ku80 deficient CHO cells and mice. Ku70 deficiency shows comparable characteristics with a reduction of coding joint formation of up to 100-fold compared to Ku70 wt cells and 2-3-fold compared to DNA-PKcs mutated scid mice [START_REF] Ouyang | Ku70 is required for DNA repair but not for T cell antigen receptor gene recombination In vivo[END_REF][START_REF] Gu | Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination[END_REF]. Interestingly, DNA-PKcs deficient mice and CHO mutant cells demonstrate aberrant coding joint formation, but unlike XRCC4, Lig4, Ku80 and Ku70, have only few impairment in signal joint formation [START_REF] Bosma | A severe combined immunodeficiency mutation in the mouse[END_REF][START_REF] Michael | The defect in murine severe combined immune deficiency: Joining of signal sequences but not coding segments in V(D)J recombination[END_REF][START_REF] Malynn | The scid defect affects the final step of the immunoglobulin VDJ recombinase mechanism[END_REF][START_REF] Blunt | Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation[END_REF][START_REF] Blunt | Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse[END_REF]. This phenotype is shared with immuno-deficiencies due to mutations in the Artemis gene [START_REF] Moshous | Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency[END_REF][START_REF] Nicolas | A human severe combined immunodeficiency (SCID) condition with increased sensitivity to ionizing radiations and impaired V(D)J rearrangements defines a new DNA recombination/repair deficiency[END_REF] and is explained by the structure of blunt signal ends that can be directly ligated, without prior processing by DNA-PKcs mediated Artemis activity [START_REF] Touvrey | Distinct effects of DNA-PKcs and Artemis inactivation on signal joint formation in vivo[END_REF][START_REF] Lewis | DNA elements are asymmetrically joined during the site-specific recombination of kappa immunoglobulin genes[END_REF]. Even though, XLF mutations in humans are associated with profound immunodeficiencies and phenotypes similar to the Ligase 4 syndrome, the targeted mutation of this gene in mice has only a partial effect on lymphocyte numbers and V(D)J recombination [START_REF] Li | Lymphocyte-Specific Compensation for XLF/Cernunnos End-Joining Functions in V(D)J Recombination[END_REF][START_REF] Vera | Cernunnos Deficiency Reduces Thymocyte Life Span and Alters the T Cell Repertoire in Mice and Humans[END_REF]. It is thus suggested that XLF function is not necessary for any activation of catalytic processes, but that it might rather stabilize the PCC complex, a function shared with ATM and the RAG complex [START_REF] Li | Lymphocyte-Specific Compensation for XLF/Cernunnos End-Joining Functions in V(D)J Recombination[END_REF][START_REF] Vera | Cernunnos Deficiency Reduces Thymocyte Life Span and Alters the T Cell Repertoire in Mice and Humans[END_REF]. A hypothesis, that was confirmed by a strong lymphopenia and defective coding joint formation in double deficient KO mice for XLF/ATM [START_REF] Zha | ATM damage response and XLF repair factor are functionally redundant in joining DNA breaks[END_REF] and mice expressing a C-terminal lacking RAG2 (RAG2 c/c ) in a XLF deficient background [START_REF] Lescale | RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair[END_REF]. The role for the newest member of the cNHEJ family, PAXX, is redundant to XLF, so that single deficiency of PAXX shows no effects in signal and coding joint formation [START_REF] Satish | PAXX Is an Accessory c-NHEJ Factor that Associates with Ku70 and Has Overlapping Functions with XLF[END_REF][START_REF] Kumar | PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line[END_REF] and normal lymphocyte development [START_REF] Liu | PAXX promotes KU accumulation at DNA breaks and is essential for endjoining in XLF-deficient mice[END_REF]. However double deficiency of XLF and PAXX triggers a complete block of coding joint formation, similar to the defects seen in XRCC4 or Ku80 and have highly imprecise single joints [START_REF] Liu | PAXX promotes KU accumulation at DNA breaks and is essential for endjoining in XLF-deficient mice[END_REF][START_REF] Kumar | PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line[END_REF][START_REF] Lescale | Specific Roles of XRCC4 Paralogs PAXX and XLF during V ( D ) J Recombination[END_REF]. Unlike XLF -/-cells, PAXX -/- cells are not dependent on ATM and DNA-PKcs kinase or RAG activities [START_REF] Liu | PAXX promotes KU accumulation at DNA breaks and is essential for endjoining in XLF-deficient mice[END_REF][START_REF] Lescale | Specific Roles of XRCC4 Paralogs PAXX and XLF during V ( D ) J Recombination[END_REF],

suggesting that PAXX has no role in PCC stabilization. However the association of Ku with PAXX is essential for V(D)J recombination in an XLF -/-background [START_REF] Liu | PAXX promotes KU accumulation at DNA breaks and is essential for endjoining in XLF-deficient mice[END_REF][START_REF] Lescale | Specific Roles of XRCC4 Paralogs PAXX and XLF during V ( D ) J Recombination[END_REF]. Liu et al. [START_REF] Liu | PAXX promotes KU accumulation at DNA breaks and is essential for endjoining in XLF-deficient mice[END_REF] speculate that PAXX modulates the stability of the DNA-PKcs/Ku-DNA complex, promoting end-synapsis and ligation in an XLF deficient background. This is further supported by the observation that PAXX, stimulates the XRCC4/Lig4 catalytic activity in a Ku-dependent way, when XLF function is as a lyase, deubiquitination and helicase activities. Figure 5.1 summarizes all (so far) known functions for Ku, and we will focus a bit more detailed how Ku acts in these cellular processes.

Enzymatic functions of Ku

Ku possesses a lyase activity associated with NHEJ. Thereby Ku is involved in the excision of abasic sites at the DNA double strand 5' end, to provide proper ends for ligation [START_REF] Steven A Roberts | Ku is a 5'-dRP/AP lyase that excises nucleotide damage near broken ends[END_REF][START_REF] Strande | Specificity of the dRP/AP lyase of Ku promotes nonhomologous end joining (NHEJ) fidelity at damaged ends[END_REF]. The second known enzymatic activity of Ku (Ku70) is its deubiquitination activity on sequestered Bax. Thus, upon apoptotic stimuli Ku70 releases de-ubiquitinated Bax, which can translocate to mitochondria and drive apoptosis [START_REF] Avigail D Amsel | Regulation of the proapoptotic factor Bax by Ku70-dependent deubiquitylation[END_REF][START_REF] Rathaus | DeubiKuitylation: A novel DUB enzymatic activity for the DNA repair protein[END_REF]. Moreover Ku performs DNA helicase activity to unwind DNA duplexes in 3' to 5' direction, important for its role in DNA replication [START_REF] Tuteja | Human DNA Helicase-Ii -a Novel DNA Unwinding Enzyme Identified as the Ku Autoantigen[END_REF][START_REF] Alexander E Ochem | Functional Properties of the Separate Subunits of Human DNA Helicase II / Ku Autoantigen[END_REF][START_REF] Matheos | Ku antigen, an origin-specific binding protein that associates with replication proteins, is required for mammalian DNA replication[END_REF].

Ku in transcription

Several lines of evidence point to a role of Ku in transcription, due to proteinprotein interactions. For example the C-terminus of Ku80 binds to RNA polymerase II elongation sites, through transcription elongation proteins, and thus regulates global transcription levels in a human cell line [START_REF] Mo | Subnuclear Localization of Ku Protein : Functional Association with RNA Polymerase II Elongation Sites[END_REF]. Further the role in transcription regulation is independent of Ku80's function in DNA repair, as a Ku80 mutant lacking C-terminal residues 687-728 and transcription activity had 1.Introduction 63 no effect on DNA end joining in vitro [START_REF] Woodard | Distinct Roles for Ku Protein in Transcriptional Reinitiation and DNA Repair *[END_REF]. Moreover, associated with DNA-PKcs, Ku80 represses transcriptional activity of RNA Polymerase I, which is further increased when DNA ends are present [START_REF] Kuhn | DNAdependent protein kinase: a potent inhibitor of transcription by RNA polymerase I[END_REF] and might represent a mechanism to repress transcription during DNA repair [START_REF] Downs | A means to a DNA end: the many roles of Ku[END_REF].

Ku in the mobility of genetic elements

Mobile genetic elements are transposons, retrotransposons and retroviruses. All of them exist as linear dsDNA. For instance, retroviruses integrate their genome into a host, after reverse-transcribing their RNA into cDNA with the help of an integrase. By a mechanism similar to V(D)J, a nucleophilic attack of the 3'OH group towards the host genome induces a SSB, to create an insertion site for the cDNA [START_REF] Downs | A means to a DNA end: the many roles of Ku[END_REF]. Ku can potentiate retrotransposon Ty1 integration into the genome of Saccharomyces cerivisae [START_REF] Downs | Involvement of DNA end-binding protein Ku in Ty element retrotransposition[END_REF], and DNA-PKcs and Ku80 are important factors for retrotransposition of retroviruses in human cells [START_REF] Li | Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection[END_REF][START_REF] Daniel | A role for DNA-PK in retroviral DNA integration[END_REF].

Ku in DNA replication

DNA replication doubles the genome, generating two identical daughter strains important for cell proliferation. It is initiated at specific locations, called the origins of replication, where the pre-replication complex is formed.

The heterodimer Ku binds to the origins of replication in a cell cycle dependent manner [START_REF] Ruiz | OBA/Ku86: DNA binding specificity and involvement in mammalian DNA replication[END_REF], peaking in late G1/S-phase and dissociates after origin firing [START_REF] Novac | In vivo association of Ku with mammalian origins of DNA replication[END_REF].

It interacts with several origin recognition complex proteins, such as ORC2, ORC3, ORC4 and ORC6 and permits proper loading of the subunits to form the ORCcomplex, a prerequisite for the assembly of the pre-replication complex [START_REF] Sibani | -Hadjopoulos. Ku80 binds to human replication origins prior to the assembly of the ORC complex[END_REF] on Ku70 is released and the heterodimer can de novo participate in the formation of the pre-replication complex [START_REF] Mukherjee | Phosphorylation of Ku70 subunit by cell cycle kinases modulates the replication related function of Ku heterodimer[END_REF].

Ku on telomeres

Telomeres are specific DNA-protein complexes localized at the end of chromosomes. Telomeres consist of TTAGGG repeats that range around 10 kb in human cells, to 100 kb in mice. Their end consists of a 3' TTAGGG overhang, that is several hundreds of base pairs long and that can fold back to invade the preceding double stranded region, generating a telomere-loop (t-loop). This structure is recognized and stabilized by the shelterin complex in humans, which consists of 6 protein subunits, notably TRF1 (telomere-repeat binding factor 1), TRF2

(telomere-repeat binding factor 2), TIN2 (TRF1-interacting nuclear factor 2), TPP1, POT1 (protection of telomere 1) and RAP1 (repressor-activator protein 1) [START_REF] Lazzerini | Stop pulling my strings -what telomeres taught us about the DNA damage response[END_REF]. Telomeres are important structures to stabilize chromosome ends that would otherwise be recognized as DNA strand breaks, consequently deficiencies have detrimental consequences. Especially deficiency of TRF2 destabilizes the t-loop and culminates in Ku dependent c-NHEJ mediated chromosomal end-to end fusions and genetic instability [START_REF] Lazzerini | Stop pulling my strings -what telomeres taught us about the DNA damage response[END_REF][START_REF] Giulia | Ku70 stimulates fusion of dysfunctional telomeres yet protects chromosome ends from homologous recombination[END_REF]. Paradoxically, Ku70/80 are found to be telomere associated factors [START_REF] Hsu | Ku is associated with the telomere in mammals[END_REF] and Ku inhibition triggers rapid telomere attrition by intrachromosomal recombination with the formation of t-circles and genomic instability [START_REF] Wang | Ku86 represses lethal telomere deletion events in human somatic cells[END_REF][START_REF] Sandra | Ku's essential role in keeping telomeres intact[END_REF][START_REF] Myung | Regulation of telomere length and suppression of genomic instability in human somatic cells by Ku86[END_REF][START_REF] Jaco | Role of human Ku86 in telomere length maintenance and telomere capping[END_REF]. Thus, on one hand, Ku protects telomeres from rapid telomere shortening, but on the other hand can trigger fusion of uncapped telomere ends through c-NHEJ.

Ku on telomeres, similar as on DNA DSB, has a dominant-negative effect on other repair pathways, thus restricting recombination and alt-NHEJ [START_REF] Lazzerini | Stop pulling my strings -what telomeres taught us about the DNA damage response[END_REF][START_REF] Giulia | Ku70 stimulates fusion of dysfunctional telomeres yet protects chromosome ends from homologous recombination[END_REF][START_REF] Sfeir | Removal of Shelterin Reveals the Telomere End-Protection Problem[END_REF].

Moreover a recent paper gives insight onto how Ku's DNA repair function can be suppressed at functional telomeres. Ku70 helix 5 (α 5) interacts with TRF2, which thus inhibits heterotetramerization of two Ku heterodimers on DNA ends.

The authors propose an elegant model, where TRF2 forms a physical barrier for 1.Introduction

65

DNA end tethering, thus inhibiting c-NHEJ on functional telomeres [START_REF] Ribes-Zamora | TRF2 Interaction with Ku Heterotetramerization Interface Gives Insight into c-NHEJ Prevention at Human Telomeres[END_REF].

The role of Ku on telomeres represents a double edged sword, as Ku mediated c-NHEJ is the main event for end-to-end fusions promoting genomic instability.

However loss of Ku favours telomere shortening and uncapping of telomeres. It thus appears that, in the first place, Ku is an important player in telomere maintenance and protection and that telomere specific factors, mask its c-NHEJ function.

Chapter 6

Ku associated with disease

Ku associated with autoimmune disease

In the beginning of this manuscript we have mentioned that Ku has been identified as the target of autoantibodies in connective tissue diseases (CTD), such as systemic lupus erythematosus, systemic sclerosis, polymyositis and Syorgen's syndrome [START_REF] Mimori | Characterization of a High Molecular Weight Acidic Nuclear Protein Recognized by Autoantibodies in Sera from Patients with Polymyositis-Scleroderma Overlap.pdf[END_REF][START_REF] Reeves | Use of monoclonal antibodies for the characterization of novel DNA-binding proteins recognized by human autoimmune sera[END_REF][START_REF] Mimori | Characterization of the DNAbinding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders[END_REF][START_REF] Mimori | Clinical Significance of Anti-Ku Autoantibodies -A Serologic Marker of Overlap Syndrome[END_REF]. Additionally Ku is found to be a thyroid autoantigen in Graves disease, an autoimmune disorder that causes hyperthyroidism [START_REF] Chan | Cloning and characterisation of a cDNA that encodes a 70 kDA novel human thyroid autoantigen[END_REF].

It might seem not evident that a nuclear protein could become an antigen for autoantibody development, as it is "hidden" in the nucleus. In fact, different research groups presented data of Ku being associated to the extracellular membrane [START_REF] Bellur S Prabhakar | Cell Surface Expression of the 70-kD Component of Ku , a DNA-binding Nuclear Autoantigen[END_REF][START_REF] Monferran | The membrane form of the DNA repair protein Ku interacts at the cell surface with metalloproteinase 9[END_REF][START_REF] Muller | The Double Life of the Ku Protein[END_REF].

Finally, the expression of Ku antibodies, in combination with other markers can be used in CTD diagnostics [START_REF] Mahler | Clinical and serological associations of autoantibodies to the Ku70/Ku80 heterodimer determined by a novel chemiluminescent immunoassay[END_REF][START_REF] Belizna | Anti-Ku antibodies: Clinical, genetic and diagnostic insights[END_REF].

Ku associated with cancer

Genome instability and mutation is commonly known as a hallmark of cancer, giving cancer cells a selective advantage to outgrow and dominate the local tissue environment [START_REF] Hanahan | Hallmarks of cancer: The next generation[END_REF]. As stated earlier, cellular repair pathways maintain genomic 1.Introduction 68 stability. Therefore deficiencies, especially in DSB repair pathways, predestine genomic translocations or gene amplifications, jeopardizing the cellular homoeostasis [START_REF] Fell | The Ku heterodimer: Function in DNA repair and beyond[END_REF][START_REF] Khanna | DNA double-strand breaks: signaling, repair and the cancer connection[END_REF].

Studies in mice clearly show Ku70 and Ku80 to prevent the appearance of chromosomal aberrations, chromosome fusions and aneuploidy [START_REF] Michael J Difilippantonio | DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation[END_REF][START_REF] Li | Deletion of Ku70, Ku80, or both causes early aging without substantially increased cancer[END_REF][START_REF] Zarir | The nonhomologous DNA end joining pathway is important for chromosome stability in primary fibroblasts[END_REF][START_REF] Fabrizio | Effects of DNA nonhomologous end-joining factors on telomere length and chromosomal stability in mammalian cells[END_REF] and thus place them as central caretakers in genome maintenance.

Despite the observed instability, deletion of Ku only leads to a slight increase in cancer incidence, mainly due to B-and T-cell lymphomas in mice [START_REF] Li | Ku70: a candidate tumor suppressor gene for murine T cell lymphoma[END_REF][START_REF] Nussenzweig | Requirement for Ku80 in growth and immunoglobulin V(D)J recombination[END_REF][START_REF] Li | Deletion of Ku70, Ku80, or both causes early aging without substantially increased cancer[END_REF].

Interestingly however, loss of Ku and another caretaker protein dramatically increases tumours development in mice. For instance, the combined deficiency of Ku80 and the tumour suppressor p53 accelerates the lymphoma onset and rate, underlining their cooperation in the limitation of oncogenic transformation in murine B-cells [START_REF] Michael J Difilippantonio | DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation[END_REF][START_REF] Lim | Analysis of ku80-Mutant Mice and Cells with Deficient Levels of p53[END_REF]. Similar to p53/Ku80 loss, haploinsufficiency of Ku80 in a murine PARP1 -/-background favours hepatocellular cancer formation due to accumulating aneuploidy and chromosomal aberrations [START_REF] Tong | Synergistic Role of Ku80 and Poly ( ADP-ribose ) Polymerase in Suppressing Chromosomal Aberrations and Liver Cancer Formation Synergistic Role of Ku80 and Poly ( ADP-ribose ) Polymerase in Suppressing[END_REF].

In contrast to mice spontaneous mutation or a complete loss of either of the Ku subunits has not been reported for humans up to date, arguing that it constitutes a lethal condition in humans. However, several studies associate Ku expression to the progression level of different tumour samples (see Table 6.1).

Several therapeutic approaches targeting Ku have been proposed. For example, Ku that translocates to the cell surface in human multiple myeloma cell samples, can be blocked with antibodies, sensitizing these cells towards apoptosis after irradiation and doxorubicin treatment [START_REF] Tai | Translocation of Ku86/Ku70 to the multiple myeloma cell membrane: functional implications[END_REF]. Additionally, targeting Ku70 with siRNA enhances irradiation and Topoisomerase II inhibitor treatment in cancer cell lines [START_REF] Ayene | Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and radiation[END_REF]. And finally, histone-deacetlyase inhibitors regulate Ku70-Bax interaction and mediate cell death in a neuroblastoma cell line [START_REF] Subramanian | Ku70 acetylation mediates neuroblastoma cell death induced by histone deacetylase inhibitors[END_REF].

1.Introduction 69

Epidemiology

Chronic lymphocytic leukemia is the most common form of leukaemia occurring in the western world [START_REF] Wendtner | Chronic Lymphocytic Leukemia Guideline[END_REF]( 22-30% of leukaemia being diagnoses as CLL [START_REF] Redaelli | The clinical and epidemiological burden of chronic lymphocytic leukaemia[END_REF])

and the incidence-rate is between 1-5.5 per 100.000 people worldwide [START_REF] Redaelli | The clinical and epidemiological burden of chronic lymphocytic leukaemia[END_REF]. CLL is a disease that affects mostly the elderly with a median age at diagnosis of approximately 64-70 years [START_REF] Redaelli | The clinical and epidemiological burden of chronic lymphocytic leukaemia[END_REF]. A gender and race specific shift is observed, with males being more often affected than women, and whites more often affected than blacks or Asians [START_REF] Redaelli | The clinical and epidemiological burden of chronic lymphocytic leukaemia[END_REF][START_REF] Nabhan | The impact of race, ethnicity, age and sex on clinical outcome in chronic lymphocytic leukemia: a comprehensive Surveillance, Epidemiology, and End Results analysis in the modern era[END_REF][START_REF] Kipps | Chronic lymphocytic leukaemia[END_REF]. Based on statistics of the National Cancer Intelligence Network 5-year survival rates are approximately 70% for males and 75% for females [354].

Diagnosis and prognostic factors

The diagnosis of CLL includes the evaluation of a B-cell blood count (> 5x10 9 Bcells/L called lymphocytosis), blood smear (small, mature-appearing lymphocytes with a narrow border of cytoplasm and dense nucleus without discernible nucleoli) and the assessment of the immunophenotype (CD5 high , CD19 high , CD23 high , CD20 low , CD79b low ) of the circulating B-cells [START_REF] Kipps | Chronic lymphocytic leukaemia[END_REF]355].

The clinical disease course can be highly variable in CLL patients, ranging from an almost indolent form with patients being free of symptoms and fully active for decades, to patients that rapidly develop high-risk disease with the requirement for treatment shortly after diagnosis and a rapid therapy-or disease related mortality [START_REF] Kipps | Chronic lymphocytic leukaemia[END_REF]. Several prognostic markers have been associated with the disease course and help to identify those patients that need therapy relatively shortly after diagnosis • Serum markers: CD23 and thymidine kinase may predict survival or progression-free survival [355]. β 2 -microglobulin expression (> 3.5 mg/L blood) is associated with poor prognosis [START_REF] Kipps | Chronic lymphocytic leukaemia[END_REF].

• Bone marrow infiltration: The tumour burden is associated with the type of bone marrow infiltration (diffuse vs. non-diffuse). A diffuse pattern is often associated with advanced disease [START_REF] Kipps | Chronic lymphocytic leukaemia[END_REF]. Moreover bone marrow aspirates can help evaluate cytopenias of red blood cells or thrombocytes [355].

Staging

Two clinical staging systems are used to distinguish low and high risk disease patients into 3 prognostic groups. The Rai staging system is used in the US, 1.Introduction 74 Five zones are examined on the presence of papable, enlarged lymph nodes of > 1 cm (cervical (head and neck), axillary, inguinal nodes(including superficial femorals), spleen, liver ) whereas the Binet classification is often used in Europe. Both tests rely on a physical examination of the patient and a standard laboratory blood test. High risk patients are identified through the assessment of advanced cytopenias in both staging systems. The different clinical features that identify CLL risk groups, as well as their life expectancy according to the Rai and Binet staging system are depicted in Table 7.1 [START_REF] Kipps | Chronic lymphocytic leukaemia[END_REF].

Treatment

The initiation of treatment is highly dependent on the disease status. Generally asymptomatic patients which have been assessed in the low or intermediate risk category based on the Rai and Binet staging are generally not drug treated. As soon as evidence for disease progression is given by a lymphocyte doubling time 1.Introduction 75 less than one year or progressive palpable lymphadenopathy and/ or splenomegaly, treatment should be considered.

Treatment options include chemotherapy, a combination of immuno-and chemotherapy and small molecules that target B-cell receptor or BCL-2 signalling pathways, which promote growth and survival of malignant B-CLL cells [START_REF] Kipps | Chronic lymphocytic leukaemia[END_REF].

Chemotherapy The traditional treatment relies on the use of chemotherapeutics that target the DNA, such as the purine analogs (fludarabine, pentostatin or cladribine), as well as DNA alyklators including chlorambucil, cyclophosphamide or bendamustine. Common side effects of these therapies are immunodepression and an increased risk of infections.

Chemoimmunotherapy Several clinical trials highlight the benefit of a combination therapy using chemotherapeutics in combination with the anti-CD20 monoclonal antibodies rituximab, obinutuzumab or ofatumumab. These trials show higher response rates and progression free survival (PFS) with a combinational therapy than a stand-alone chemotherapy. Subsequently the US FDA (united states federal drug administration) has approved the chemotherapeutic combination therapy with those three antibodies as a first-line treatment for CLL patients.

Ofatumumab is additionally approved as a single agent for relapsed or refractory disease. Other clinical trials evaluating the combination of bendamustine and rituximab or obinutuzumab are currently under way.

Small molecules

Inhibitors of BCR signalling

The BCR signalling pathway is a prominent target site, as it is considered to play a crucial role in CLL pathogenesis [START_REF] Kipps | Chronic lymphocytic leukaemia[END_REF]. Ongoing studies now examine the combined therapy with or without anti-CD20 antibodies and with or without ibrutinib to enhance clinical outcomes. Adverse effects of venetoclax are gastrointestinal disturbances, neutropenia, and the tumour lysis syndrome.

Other prognostic markers like apoptosis resistance to irradiation, NHEJ activity and telomere maintanance come into focus and define a novel subpopulations in CLL -data from the lab

Research by Jozo Delic concerning the impact of the ubiquitin-proteasome system on apoptosis after irradiation in healthy lymphocytes [START_REF] Delic | Ubiquitin pathway involvement in human lymphocyte gamma-irradiation-induced apoptosis[END_REF] and later lymphocytes dervied from CLL patients [START_REF] Masdehors | Increased sensitivity of CLL-derived lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin[END_REF][START_REF] Delic | The proteasome inhibitor lactacystin induces apoptosis and sensitizes chemo-and radioresistant human chronic lymphocytic leukaemia lymphocytes to TNF-alpha-initiated apoptosis[END_REF] led to the discovery of two specific subsets of CLL B-cells. These subpopulations are either radioresistant to cell death Until today, 308 CLL patient samples over a period of 25 years have been evaluated, and show around 18% to be radioresistant (R-CLL) and 82% to be radiosensitive (S-CLL). Men are proportionally higher represented (20%) in the R-CLL group than women (around 16.8%) [START_REF] Delic | DNA Damage Response / Signaling and Genome ( In ) Stability as the New Reliable Biological Parameters Defining Clinical Feature of CLL[END_REF].

Importantly patients in the R-CLL group have an aggressive course of disease, making front-line treatment inevitable and do nevertheless rapidly succumb to death (oral communication, Jozo Delic). In accordance, the median survival for R-CLL patients is significantly shorter than for S-CLL patients (10.593±1.611 and 15.946±1.014, p¡0.0001 Log-rank, Wilcoxon and Tarone-ware-tests), underlining the prognostic relevance of this marker (see Figure 7.2).

As mentioned earlier chromosomal aberrations in CLL can lead to the loss of the the mutation or loss of ATM and p53 can be attested. Examination of the p53mutation status in 12 R-CLL patients and 10 S-CLL, show 0/10 S-CLL and only 4/12 R-CLL patients to be p53-mutated [START_REF] Deriano | Apoptose et stabilité de l'ADN dans la Leucémie Lymphoïde Chronique-B : Implication du système de réparation des cassures double brin de l'ADN par recombinaiso n non homologue[END_REF]. Evaluation of 10 R-CLL patients with p53-wt show no loss of heterozygosity for the ATM gene [START_REF] Blaise | Chromosomal DNA and p53 stability, ubiquitin system and apoptosis in B-CLL lymphocytes[END_REF]. In summary, the radioresistance observed in the R-CLL subset can't be explained, solely by the presence of mutations in these tumour suppressor genes. Subsequently, the molecular background of this deregulation is found in the overactivation of the NHEJ pathway (see Chapter 3.1.1), with a 2-to 4-fold higher DNA-PKcs activity and a 2-to 3-fold higher DNA end binding activity of Ku in R-CLL samples than S-CLL samples [START_REF] Deriano | Human chronic lymphocytic leukemia B cells can escape DNA damage -induced apoptosis through the nonhomologous end-joining DNA repair pathway[END_REF]. Moreover the NHEJ pathway used in the R-CLL cells has highly mutagenic characteristics with associated extended deletions when tested in an in vitro assay [START_REF] Deriano | Mutagenicity of non-homologous end joining DNA repair in a resistant subset of human chronic lymphocytic leukaemia B cells[END_REF].

Likewise a significant resection of the 3' telomere ends and an increased telomere dysfunction with telomere loss and telomere duplication are associated with the R-CLL phenotype compared to the S-CLL phenotype. Overresected telomeres constitute a danger for genomic stability, as soon as they have lost the ability to fold back into a t-loop, protecting them from erroneous DNA repair activities. In R-CLL samples increased numbers of telomere-induced foci containing γ-H2AX and 53BP1, the association of activated DNA-PKcs and Ku70 on telomere repeats as well as increased dysfunctional telomeres are observed. These findings strongly suggest an uncapping of chromosome ends and loss of protection in R-CLL, activating DNA-repair and a damage response at telomeres in R-CLL lymphocytes.

The presence of 53BP1, which is normally excluded from functional telomeres, and the colocalization of DNA-PKcs and Ku70, strongly point to an activation of NHEJ, that was previously shown to be deregulated in R-CLL cells [START_REF] Brugat | Telomere dysfunction-induced foci arise with the onset of telomeric deletions and complex chromosomal aberrations in resistant chronic lymphocytic leukemia cells[END_REF]. Interestingly, Ku has taken a particular role in all of these studies, as it is found to bind DNA end structures with higher affinity in R-CLL extracts compared to S-CLL extracts. Given the key role of Ku in DNA maintenance and metabolism, a causal relationship between those observations is supposed.

1.Introduction

1.Introduction

Identification of phospho-S27-S33 Ku70

The studies conducted on the R-CLL and S-CLL subsets reveal several differences in the NHEJ process due to an overactivation of DNA-PKcs and an increased Ku binding. However no differences in the expression level of DNA-PKcs, Ku70 or Ku80 between R-CLL and S-CLL patients are found [START_REF] Deriano | Human chronic lymphocytic leukemia B cells can escape DNA damage -induced apoptosis through the nonhomologous end-joining DNA repair pathway[END_REF]. Therefore PI3-kinases with wortmannin or DNA-PKcs and ATM specific inhibitors, show them to be redundant in the phosphorylation of p-Ku70 [START_REF] Bouley | A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines[END_REF].

The biochemical characterization of p-Ku70 is further completed by functional assays on DNA repair and cell cycle kinetics. For this a special vector system that represses endogenous Ku70 by shRNA expression, with a simultaneous expression of an exogenous Ku70 gene either in its wild type-transfected (S27,S33), phosphomutant (A27,A33) or phospho-mimetic form (E27,S33) is expressed in a p53wt

Posttranslational modifications (PTM) regulate cellular processes

The posttranslational modification (PTM) of proteins is an important mechanism to control cellular processes without the need of de novo protein synthesis.

These modifications are thus an elegant means for the cell to rapidly and reversibly change protein characteristics, adapting to the current cellular state. For instance, during the cell cycle (as described in Chapter 3.9), the oscillating activity of CDk's, phosphorylating their target proteins, drives a cell through the different phases. Similarly DNA repair is greatly regulated by these modifications.

Different post-translational protein modifications are distinguished, ranging from small chemical groups, like a phospho-group, a acetyl-group or a methyl-group to small proteins, like ubiquitin, SUMO or NEDD8. All these modifications have a preferred protein target sites, for example phosphorylation occurs on hydroxy (OH)-containing aa like tyrosine, threonine or serine, whereas acetylation is found on lysine residues and methlyation targets lysine or arginine residues. All these modifications are accomplished by the actions of two kinds of enzymes, working oppositely. For example, kinases phosphorylate proteins, phosphatases dephosphorylate them. Similarly, acetyl-or methly-trasferases add acetyl-or methylgroups to proteins and are antagonized by deacetyl-or demethylases [START_REF] Oberle | Regulation of the DNA Damage Response to DSBs by Post-Translational Modifications[END_REF].

Furthermore ubiquitination is a ubiquitous process to regulate protein functions and degradation. Ubiquitin is a 76-aa containing protein, that can be attached individually (monoubiquitinqtion) or multiply forming ubiquitin chains (multi-or polyubiquitinqtion) on proteins. Addition of ubiquitin is conducted by a conserved family of ubiquitin ligases, and deubiquitinating enzymes (DUB) remove these protein modification. Similarly, SUMOylation and NEDDylation are conducted [START_REF] Oberle | Regulation of the DNA Damage Response to DSBs by Post-Translational Modifications[END_REF].

All of these different PTMs have been mentioned throughout this manuscript and have far-reaching importance in the control of DNA repair and metabolism.

1.Introduction 86

Ku70 phosphorylation sites reviewed

Several publications demonstrate Ku associated post-translational-modifications.

For instance, ubiquitination processes extract the heterodimer from DNA ends and acetylation controls Bax release for apoptosis induction. Overall, 117 PTMs have currently been described for Ku70 [START_REF] Phosphosite | PhosphoSite-Ku70[END_REF]. Phosphorylation is an important regulator of DNA repair and the orchestration of a DDR, particularly induced by the PI3-kinases ATM/ATR and DNA-PKcs.

Early studies have already shown that the Ku heterodimer is phosphorylated on serine residues in vivo [START_REF] Yaneva | A 10S particle released from deoxyribonucleasesensitive regions of HeLa cell nuclei contains the 86-kilodalton-70kilodalton protein complex[END_REF] and that phosphorylation events are catalysed by DNA-PKcs [START_REF] Boubnov | scid cells are deficient in Ku and replication protein A phosphorylation by the DNA-dependent protein kinase . These include : scid Cells Are Deficient in Ku and Replication Protein A Phosphorylation by the DNA-Dependent Protein Kinase[END_REF]. Today, 35 phosphorylation sites are identified for Ku70, from which 6 (S6, S27, S33, S51, S155, Y530) have been studied more closely [START_REF] Phosphosite | PhosphoSite-Ku70[END_REF].

The hitherto identified phosphorylation sites of Ku70 are shown in Figure 7.8.

Additionally potential phosphorylation sites in the Ku core region are described (see below), but are not listed by PhosphoSite.

1.Introduction 87

Phosphorylation on S6 and S51, are the first sites that have been described in the literature but are not necessary for DNA repair, DNA end binding or V(D)J recombination, even though they are target sites for DNA-PKcs dependent phosphorylation in vitro [START_REF] Jin | Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions[END_REF][START_REF] Doug W Chan | DNA-Dependent Protein Kinase Phosphorylation Sites in Ku 70 / 80 Heterodimer[END_REF]. These residues have later been found to be critical regulators in DNA-PKcs and Bax dependent cytotoxic cell death in neurons [START_REF] Liu | The DNA-PK Catalytic Subunit Regulates Bax-mediated Excitotoxic Cell Death by Ku70 Phosphorylation[END_REF].

More recently the site specific mutation of the S155 residue to a phosphomutant (A155) or phosphomimetic (D155) shows severe cellular consequences. Whereas the cells expressing the phosphomutant have a survival advantage after irradiation, the phosphomimetic expressing cells are highly radiosensitive. Moreover the phosphomimetic is severely impaired in growth, has a pronounced cell cycle arrest, a senescence phenotype and a highly active DNA damage response even without damage induction. Interestingly, although this study reveals a severely impaired DDR, in these phosphorylation-mutants, no defects in DNA repair efficiency and kinetics are established by comet assay or a plasmid repair luciferase assay [START_REF] Victoria | Ku Regulates Signaling to DNA Damage Response Pathways through the Ku70 von Willebrand A domain[END_REF][START_REF] Victoria L Fell | Ku70 phosphorylation mediates Aurora B inhibition and activation of the DNA damage response[END_REF].

Another residue that has no impact on DNA repair, but has a regulatory function on apoptosis is Y530, that is phosphorylated by Src-kinases. Phosphorylation blocks subsequent acetylation of Ku70, thus preventing Bax release and apoptosis [START_REF] Morii | Src acts as an effector for Ku70-dependent suppression of apoptosis through phosphorylation of Ku70 at Tyr-530[END_REF].

Using a knowledge based approach, Lee et al. [START_REF] Lee | Phosphorylation of Ku dictates DNA double-strand break ( DSB ) repair pathway choice in S phase[END_REF] target the pillar and bridge region, that form the ring structure of Ku70 for the introduction of site specific mutations at sites T307, T306, T305, T316 and T314 (termed 5A mutant). This mutant has an increased DNA end binding associated with the inhibition of DNA end resection, and an decreased commitment to HR, compared to its phosphomimetic counterpart 5D and the wt protein. However the sites, mutated in this study have not been confirmed to be veritable or inducible phosphorylation sites in vitro or in vivo.

Finally, our group has discovered the phosphorylation sites at serine 27 and 33 and demonstrates for the first time an impact on the DNA repair kinetics by these sites, which are found to be phosphorylated in vivo in a dose dependent manner by irradiation [START_REF] Bouley | A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines[END_REF].

Aim of this project

During the last decade the laboratory has intensively studied the cellular characteristics of lymphocytes derived from CLL patients. The extensive research on their apoptosis resistance guided the way towards the discovery that CLL derived lymphocytes can be divided into two subsets, either resistant (R-CLL) or sensitive (S-CLL) towards IR induced apoptosis. Interestingly, several observations made in the laboratory suggest an imbalance of the NHEJ pathway in the R-CLL compared to the S-CLL derived lymphocytes. First, the R-CLL subsets are shown to upregulate their NHEJ pathway to the cost of repair quality, accumulating repair junctions with extended deletions compared to S-CLL cell samples or healthy lymphocytes. Secondly, a profound dysfunction in telomere maintenance is determined in the R-CLL subset compared to S-CLL. In this context a significant resection of the 3' telomeric overhang and the accumulation of NHEJ factors, like 53BP1 or Ku70, are manifested on the chromosome ends of R-CLL cell samples, arguing for an activation of the NHEJ pathway. Lastly, the identification of two phosphorylation sites (S27, S33) of Ku70, that are found overexpressed in R-CLL compared to S-CLL derived lymphocytes, is the first identified molecular differentiator between R-CLL and S-CLL subsets. Furthermore using a cellular in vitro system longer persisting DNA damage and a persisting G2-arrest in phosphomutant (A27,33) were observed, compared to the wild-type transfected (S27,33) cells.

In this study we want to further characterize the cellular role of phospho-Ku and more particularly its role in DNA DSB repair. The central questions underlying this study are thus:

• Is the phosphorylation of Ku70 specific to IR or is it a general response to genotoxic stress?

• How does the phosphorylation of Ku influence on the cellular phenotype?

• What impact has the phosphorlyation of Ku on the DNA DSB repair?

2.Aim of this project 92

More precisely, at the molecular level, our questions are as follow:

-Is phospho-Ku70 found on the DNA after stress induction? -Does phospho-Ku70 have an impact on DNA repair by NHEJ and HR? -Does the phosphorylation of Ku impact on Ku release from the DNA after damage induction? These questions are approached by a cellular in vitro model in which expression of phospho-Ku70 is inhibited or mimicked.

To better understand the role of phospho-Ku70 in lymphocyte development, V(D)J recombination and its potential role in cancer development it is however imperative to study its function in vivo. Therefore, a humanized knock-in mouse model is created and some primary studies on extracted mouse embryonic fibroblast (MEFs) are conducted.

Given the central role of DNA repair in cancer development, the study of factors like phospho-Ku70 can potentially identify new targeting strategies for a more personalized medical approach.

Part III

Materials and Methods

Chapter 1

Materials and Methods

Cell lines and culture conditions 1.Cell lines

The cancer cell lines U2OS (osteosarcoma) and ZR75.1 (mammary duct) have been purchased at the ATCC platform. The U2OS HREJ cell line contains two reporter systems to measure NHEJ and HR efficiency (U2OS HREJ) and were a kind gift by Bernard Lopez (CNRS UMR 8200, Institut de Cancérologie Gustave-Roussy, Villejuif). The human fibroblast cell line GC92 contains a reporter system to measure NHEJ efficiency and was a kind gift from Pascale Bertrand (LREV Team, CEA Fontenay-aux Roses) and Bernard Lopez [START_REF] Rass | Role of Mre11 in chromosomal nonhomologous end joining in mammalian cells[END_REF]. The non cancerous mammary epithelium derived HME (hTert immortalized) cell line was a kind gift from RA Weinberg [START_REF] Elenbaas | Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells[END_REF].

Cell culture

All cell lines used were passed twice a week at around 1 -1.5*10 6 cells in 75cm 2 flasks. Cells were counted at each passage to assure stable growth conditions. Every 6-8 weeks the cell lines were changed to prevent cell line derivation. Culture conditions and composition of media are summarized in Table 1.1.

All cell lines were kept in culture media (10566016 or 10565018 Thermo Scientific, GIBCO) supplemented with fetal bovine serum (12657029, Thermo Scientific, GIBCO) and Antibiotic-Antimycotic (100x) (15240062,Thermo Scientific, GIBCO). The HME cell line was additionally supplemented with insulin (I9278, Sigma), EGF (E9644, Sigma) and hydrocortisone (H0888, Sigma).

Splitting cells

To passage adherently growing cells, medium was aspired and cells were washed two times with PBS -/-(14190250, Thermo Scientific, GIBCO), before adding 2mL

Tryple Express (12604013, Thermo Scientific, GIBCO) to loosen cell-cell interaction. Cells were incubated at 37 • C for several minutes, until they began to detach from the bottom of the flask. Trypsinized cell suspension was taken up in 5mL medium in a centrifugation tube and centrifuged (1200rpm, 5min) (5804R, Eppendorf).

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97

Supernatant was aspired and 10 mL fresh medium were added.

Cell counting was performed using approx. 10 µL of cell suspension diluted 1:1 with trypan blue, which were added to a counting chamber and counted automatically by a cell counter machine (TC20, Bio-Rad). Cells were split in a late exponentially growing state.

Thawing cells

Cell lines were stored in a mixture of 10 % DMSO, 50% fetal bovine serum and 40% culture medium in -196 • C nitrogen stocks.

Cells were quickly thawed in a 37 • C water bath and added to 5mL culture medium before centrifugation at 1200rpm for 5min (5804R, Eppendorf). Cells were seeded into 75cm 2 flasks containing 10mL medium.

Stable cell line generation using pEBV vectors

To study the phosphorylation of Ku70 a vector system established and kindly provided by Denis Biard (CEA, Institute of Emerging Diseases and Innovative Therapies (iMETI), Fontenay-aux-Roses, France) was used to generate stable cell lines that either express: S27,S33-Ku70 (phospho-wt), A27,A33-Ku70 (phosphomutant) or E27,E33-Ku70 (phospho-mimetic). Endogenous Ku70 is repressed using shRNA encoded on the same pEBV-vector. Using this system we generated cell lines that do not express endogenous but rather exogenous Ku70. The vectors replicate in a semi-conservative manner during cellular S-Phase, dependent on the presence of host replication-initiation factors, like Orc2, Orc3, Orc4 and Mcm [START_REF] Biard | Diplôme National d'HABILITATION A DIRIGER DES RECHERCHES de l[END_REF]. The replication is limited to one doubling per cell cycle. During chromosome segregation the vector anchors to a chromosome and therefore assures "one by one" segregation to daughter cells.

Transfection

Plasmid purification after bacterial culture was performed using the QIAGEN Plasmid Midi Kit (12143,QIAGEN), following manufacturer's instructions.

Cell transfection for all mutants was performed the same day, using the same transfection (114-15, Polyplus) and antibiotic selection conditions. In general, cells were seeded in a 12-well plate, to attain approx. 70% confluence the following day. The expressing cells were freshly transfected before each live cell acquisition. All cell lines were kept under constant antibiotic selection pressure. Table 1.2 summarizes the transfection conditions for each cell line.

Irradiation

Irradiation was performed using the γ-irradiator IBL-637 (Cs137). Dose rate used was 4.96 Gy/min for all experiments. Analysis was performed using R (Script, Allan Sauvat).

Cell cycle and hyperploidy

Cell cycle analysis was performed at several time points after irradiation. Cells were seeded the day before irradiation into 6-well plates at the following densities: 0.6*10 6 cells (NT, 6h, 11h), 0.3*10 6 (15h,24h) and 0.2*10 6 (48h,72h). Cells were irradiated and collected after the indicated time points. Cell pellets were resuspended in 1 mL cold 5% FCS /PBS -/-and 3mL of ice-cold 75% ethanol where added dropwise during vortexing the cell suspension. Collected samples were stored at -20 • C. The day before analysis cell samples were washed 2x with PBS -/-and centrifuged at 1200rpm, 5 min. Cell pellets were than taken up in 400µL of PBS -/-containing 25 µg/mL propidium iodide (P 4170, Sigma Aldrich) and 50 µg/mL RNAse A (Boehringer). Cell suspension was kept in the dark at 4 • C, ON. Data were acquired on a cytometer (FACScalibur, BD Biosciences).

Analysis of the fcs-files was performed with the FlowJo software, version 7.6.5 and VX.

Cell cycle synchronization

Cells were synchronized with 300µM mimosine (Sigma Aldrich, M0253),ON. Mimosine containing medium was substituted with normal cell medium 1h before irradiation.

γ-H2AX-foci detection via immunoflourescence

Cells were grown on 8-well cover slides (PEZGS0816, Millicell EZ Slides) for 2 days until 70-80% confluence was reached. Slides were irradiated at 4Gy and left in the 3. Materials and Methods

102

incubator for the indicated time points (0.5h, 4h or 24h). Cells were then washed 2x with PBS -/-and fixed with paraformaldehyde (PFA) 4% during 15min. After a washing step (2x) with PBS -/-, 0,1% Triton X-100 (X100, Sigma Aldrich) was added for 10min. to permeabilize the cells. Saturation of the cells was done with 10% goat's serum in PBS -/-at RT. After 1h, saturation solution was substituted with H2AX (phospho-S139) antibody (05-636, Merck Millipore) diluted 1:500 in 10% goat's serum in PBS -/-. After 1h of incubation cells were washed with Triton X-100 0,025% in PBS -/-(3x). An Alexa Flour488-coupled secondary antibody (A-11094, Thermo Scientific) was added during 1h at RT, slides were kept in the dark. After 3 washes with Triton X-100 0,025% in PBS -/-, Hoechst 33342 (1µg/mL) was added to the slides and incubated at 37 • C for 20min. Slides were washed with 0,025% in PBS -/-(3x) and mounted with Flouromount-G (0100-01, Southern Biotech). Data acquisition was performed using a microscope (Zeiss, Imager Z2) and a Plan Apochromat 63x (numerical aperture 1.4) oil objective.

Data analysis was performed using CellProfiler (Script Vincent Paget).

Chromosomal aberrations

Cells were seeded at day (D)-5 at a concentration of 0.5*10 6 cells. At D-3 cells were irradiated at 2Gy. To block cells in mitosis colcemid (0.03 µg/mL) was added for 2h at D-0. Cells were then washed with PBS -/-(2x). Cells were then trypsinized and 1.5*10 6 cells centrifuged (1200rpm, 5min). Cells were taken up in 300µL PBS -/-, before adding 1mL of KCl (0.06 M, 37

Chromosomal translocations

Preparation of metapahases was performed as described above (Section 1.10).

HME cells were irradiated at 4Gy and metaphases prepared after 24h.

Slides were pre-treated with RNAse A (120µL per slide in 2xSSC buffer (AM9763, Thermo Scientific)) during 45min, 37 • C to eliminate RNA that could interfere with the hybridization. To remove remaining cytoplasm a pretreatment with pepsine (P6887, Sigma Aldrich) was performed. Slides were incubated for 30sec-1min, 37 • C in 0.01M HCl, pH 2; 2µg/mL pepsin. Slides were washed with PBS -/-(2x), then with PBS +/+ for 5min. To fix pepsin treated metaphases, slides were incubated in 1% formaldehyde/ PBS +/+ for 10min. at RT. Slides were washed with PBS -/-, 5min. Dehydration was performed in 3 steps, at 70%, 90% and 100% ethanol at RT for 3min. and left to dry for 2-3min.

mFISH probe denaturation and hybridization were performed according to the manufacturer's protocol (24XCyte, D-0125-120-DI, MetaSystems). Metaphases were mounted with Flouromount-G aqueous (F4680, Sigmal Aldrich). Data were acquired using the Zeiss Imager Z2 microscope. Data analysis was performed with ISIS software (Metasystem).

Whole cell protein extraction

For whole cell protein extraction (WCE) cell pellets stocked at -80

Protein extraction for co-immunoprecipitation (Co-IP)

The protein extraction for Co-IP is divided in 3 parts, each after which cell lysates can be stored at -30

Bradford assay for protein quantification

Protein cell extracts were quantified using the Pierce Coomassie (Bradford) protein assay kit (23200, Thermo Scientific). 10µL of standards and samples were added to a 96-well plate and 150µL of assay reagent added. After 5 min. on a vertical shaker, samples and standards were analyzed as duplicates by measuring the light absorbance at 660nm with a plate reader (Magellan, Tecan). Data were exported and analyzed using Microsoft Excel 2010.

Protein extraction for chromatin binding assay

Cells were seeded at 0,5*10 6 cells in 10cm

Western Blot

The aim of a Western Blot is to transfer and immobilize the separated protein fractions gained by the SDS-PAGE to a membrane by adding an electric current.

For these experiments we used a wet blotting device of Biorad.

Two sponges, 2 Whatman Paper were incubated for 2 min with ice-cold freshly prepared transfer buffer. At the same time a 0.45µm PVDF-membrane (Immobilion-P,IPVH00010, EMD Millipore) was activated in ethanol and rinsed with water, before incubation in transfer buffer for another 2 min.

To assemble the blot the gel was covered by a PVDF membrane, placed in between two Whatman Papers and sponges and inserted into the blotter. The transfer was performed at 300mA, in a tranfer chamber kept at 4 • C 3. Materials and Methods 107

Protein Immunodetection

After western blotting the membrane was removed from the blotter and blocked ON at 4 • C or for 1h, RT in TBST/5 %BSA.

Primary antibodies were prepared using the dilutions depicted in Table 1.3.

After incubation primary antibody is rinsed for 5 min with H 2 O (3x) and incubated for 1h with the secondary antibody.

After the rinsed 1x for second incubation membranes were rinsed 10 min. with TBST and 2x with H 2 O.

The detection was carried out using the ECL Prime Protein Detection Reagent (RPN2232, GE Healthcare) in accordance to the manufacturer's protocol. Western Blots were revealed on chemiluminescence films (Amersham Hyperfilms ECL,28906837, GE Healthcare) in a dark chamber.

Antibodies

All antibodies used for Western Blotting are listed in Table 1.3.

Shotgun proteomics

Data were acquired in collaboration with the Proteomics Center Rotterdam (Dr.

J.Demmers). A Label-free quantification method was chosen, meaning that samples had not to be labelled before analysis. Data analysis was performed with Perseus.

Materials and Methods

109

NHEJ-HR efficiency

The NHEJ-HR efficiency was performed using transfected (Ku70Ser, Ku70Ala, Ku70Glu) U2OS HREJ and GC92 cells. These cells contain a single copy of a HR and NHEJ reporter to measure the efficacy of DNA break repair after I-SCE1 endonuclease expression, respectively. Cells were washed with PBS +/+ (2x), trypsinized and counted with a Malassez chamber. 0.25*10 6 were seeded into 6-well plates and transfected using JetPEI either with a plasmid coding for the endonuclease I-SCEI or a control. After 72h, cells were detached with sterile-filtered EDTA-PBS -/-(20mM). Cell pellets were centrifuged (1600rpm,5min) (5415R, eppendorf), before fixing with PBS -/-/PFA 2%. Cells were then washed with PBS -/-, before blocking with PBS -/-/BSA 2% during 5min. After centrifugation (1600rpm, 5min) (5415R, eppendorf) cells were taken up in PBS -/-/BSA 0.5% containing either 0.5µg CD4-Alexa Flour 647 (557681,BD Pharmigen), 0.5µg

H2Kd-PE (553566,BD Pharmigen) or no antibody. Cells were incubated during 45min at RT, protected from the light. After centrifugation (1600rpm,5min) (5415R, eppendorf), cells were washed and taken up in 300µL PBS -/-for FACS analysis. Data were acquired using the cytometer (FACScalibur, BD Biosciences).

Analysis was performed using FlowJo 7.6.5. NHEJ and HR efficiency was obtained by subtracting the signal of the control plasmid expressing samples from the I-SCEI expressing samples. H2Kd was used as a control for reporter expression in the GC92 cells. Control and I-SCEI expressing plasmids were prepared using QIAGEN Maxi Plus Kit (12963, QIAGEN).

Live cell imaging

Live cell imaging experiments were performed using U2OS cells expressing GFP-Ku70-mutants (Ku70Ser, Ku70Ala) transfected freshly before each experiment.

To stain the nucleus, 1µg/mL Hoescht33342 (14533, Sigma Aldrich) was added 30min before laser irradiation. Background GFP-fluorescence intensity was measured before irradiation for each cell before irradiation with a 405nm diode laser 3. Materials and Methods 110 beam. Cells were followed up to 240 min after irradiation, in intervals of 10min.

Data were acquired using the video-confocal inverted microscope Nikon A1 and a Plan Apochromat 60x (numerical aperture 1.3) oil objective. Data analysis was conducted using the NIS Elements software (Nikon).

Software

Phenotypic characterization of the phosphomutants 1.2.1 Cell growth is impaired in Ku70 phospho-mutant cell lines after irradiation

Multiple studies have shown that Ku deficiency leads to a growth defect [START_REF] Li | Ku70: a candidate tumor suppressor gene for murine T cell lymphoma[END_REF][START_REF] Rampakakis | Ku is involved in cell growth, DNA replication and G1-S transition[END_REF][START_REF] Li | Ku86 is essential in human somatic cells[END_REF]. We evaluated the onset of a growth deficiency over several days in nontreated and irradiated (2 and 4 Gy) cells. In non-treated conditions no significant effect on growth is observed between the different Ku70-mutants (Figure 1.3 A).

However after irradiation, by the seventh day of culture, cell numbers of Ku70Ala and Ku70Glu are 37.5% (2Gy), 60% (4Gy) and 25% (2Gy) , 50% (4Gy) less compared to Ku70Ser cells, respectively (Figure 1.3 B,C).

Clonogenic survival is impaired in Ku70 phosphomutant cell lines

Ku70 deficiency was further shown to induce a profound radiosensitivity [START_REF] Li | Ku70: a candidate tumor suppressor gene for murine T cell lymphoma[END_REF][START_REF] Gu | Growth retardation and leaky SCID phenotype of Ku70-deficient mice[END_REF].

To determine whether the Ku70 mutations interfere with radiosensitivity, clonogenicity of the cell lines was assessed. Unexpectedly the plating efficiency between the Ku70 mutant already differed significantly between Ku70Ser and Ku70Ala or Ku70Glu, respectively. Whereas about 36% of the plated cells formed colonies in the Ku70Ser group, only 24% or 28% of Ku70Ala and Ku70Glu cells did so (see 1% and 0.03% of Ku70Ser transfected cells give clonogenic progeny, compared to 13%, 0.5% and 0.015% of Ku70Ala and 8%, 0.3%, 0.004% of Ku70 Glu cells, respectively. These data suggest that Ku70Ala and Ku70Glu have an initial defect in colony formation and that this deficiency is further enhanced by irradiation.

Cell death by apoptosis is enhanced in Ku70 phosphomutant cell lines

Based on the observations of a slowed cell growth and enhanced radiosensitivity in the mutated Ku70Ala and Ku70Glu cells compared to Ku70Ser after irradiation, we further evaluated the sensitivity to cell death in these cell lines. Total cell death and apoptosis after irradiation and treatment with the radiomimetic were evaluated at day 3 and day 4 with a double staining using DiOC

Hyperploidy and subsequent mortality in non-phosphorylable

Ku70Ala expressing cells upon irradiation

The cell cycle was analysed over 72h after γ-irradiation (2Gy and 4Gy). The same cell cycle profile is observed for all cell lines. Indeed, similar block in S-(6h) and G2-phase (2Gy; 15h and 4Gy; 15-24h) were observed; the absence of a G1/S-block is probably due to due to an inherent p53-deficiency due to hMDM2 overexpression in U2OS cells (see Figure 1.7 A-D).

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We observed a certain amount of hyperploid cells, that accumulate beyond the G2-peak at late time points (48-72h), in all cell lines after 4Gy irradiation. We recognized that the cell line expressing Ku70Ala had approximately 40-60% increased amounts of super-G2 (n>4) compared to Ku70Ser. Ku70Glu expressing cells had around 10-30% higher super-G2 content compared to Ku70Ser (see Fig- Secondly, the evaluation of the phenotypic responses of the established cell model towards irradiated conditions clearly shows that Ku70Ser expressing cells have relative radioresistance (growth curve, clonogenic assay, cell death) when compared to the non-phosphorylated Ku70Ala and in a lesser extent Ku70Glu. Thirdly, a marked increase in hyperploid cells, that are genetically unstable and succumb mitotic catastrophe, manifests after 4Gy irradiation and several days in the Ku70Ala cells compared to the phospho-Ku70 expressing Ku70Ser cells. Fourthly, the phenotypes described above are all enhanced after irradiation and thus let strongly suggest that DNA DSB repair is impaired in Ku70Ala and Ku70Glu mutants compared to the wild-type expressing Ku70Ser cells. Figure 1.9 summarizes the results in this section.

The phenotypic characterization moreover gives the opportunity to draw some conclusions on the functional nature of the phosphorylation site, by comparing the cellular responses between Ku70Ala and Ku70Glu expressing cells. Phosphorylation is a posttranslational modification, which can either result in a conformational change of the protein due to the introduction of a negative charge, or can provide the capacity to interact with cellular factors, that specifically recognize the phosphorylated region [START_REF] Oberle | Regulation of the DNA Damage Response to DSBs by Post-Translational Modifications[END_REF]. By mimicking the phospho-site (Ku70Glu) we introduce a negative charge and thus mimic the phosphorylated protein conformation, however we can not mimic the phosphate group (PO 3- 4 -group) itself. By introducing a non-charged alanine as amino acid (Ku70Ala) we mimic the non-phosphorylated 4.Results 124 state of the protein in its initial confirmation. Given these considerations we can suppose that effects on phospho-Ku70 that are due to its charge and protein confirmation, would have opposing effects between Ku70Ala and Ku70Glu cells, however if the phosphorylated region is important we would rather have similar effects for Ku70Ala and Ku70Glu. The provided data let preferentially suggest, that it is the phosphorylated region rather than the protein confirmation that has an impact on phospho-Ku70 functionality, as no opposing effects are observed between the Ku70Ala and Ku70Glu-mutants. Ku70Glu expressing cells have rather a milder phenotype with less pronounced characteristics than Ku70Ala, when compared to Ku70Ser.

Due to these considerations the further analysis, regarding the repair of DNA DSB mainly concentrates on the Ku70Ser and Ku70Ala expressing cells. To characterize the function of phospho-Ku70 on the DSB site, we first analyzed their repair kinetics measuring the γ-H2AX foci by immunoflourescence after irradiation (4Gy) (see Figure 1.10 A). Figure 1.10 B, shows quantification of the obtained foci. In non-treated conditions the amount of γ-H2AX foci is similar in the Ku70Ser and Ku70Ala expressing cell lines (median= 5 foci/cell). After 0.5h the amount of γ-H2AX foci reaches its maximum, both cell lines show a median of 24 foci/ cell. At 4h after irradiation the amount of γ-H2AX foci decreases in both cell lines, however Ku70Ala cells show a slightly higher but significant (p< 0.001) amount of remaining γH2AX foci (median= 13; 25%-percentile= 8; 75%-percentile= 22), compared to the Ku70Ser expressing cells (median= 11; 25%per-centile= 7; 75% percentile= [START_REF] Falzon | EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single-to double-strand transitions in DNA[END_REF]. At 24h, γ-H2AX foci further decrease in both cell lines (median= 9). Similarly, western blot analysis of γ-H2AX shows a stronger signal at 4 and 6h after irradiation for Ku70Ala than Ku70Ser expressing cells (Figure 1.10 C). The strong signal for γ-H2AX in Ku70Ser and Ku70Ala after 24h (Western Blot) might be due to irradiation associated apoptosis.

Altogether these data suggest that phosphorylation of Ku70 accelerates repair capacity between 4 to 6h after irradiation compared to non-phosphorylated Ku70Ala.

phospho-Ku70 localizes to DNA damage and in the NHEJ-repair complex

To better understand the differences seen in γ-H2AX foci resolution and the increased genomic instability after irradiation in Ku70Ala expressing cells, we wanted to check whether phospho-Ku70 localizes to damaged DNA. Two complementary approaches, a chromatin binding assay and evaluation by immunoflourescence were conducted. Indeed, both approaches show a co-localization of phospho-Ku70 to damaged DNA. This happens in a DNA damage-dependent manner when cells were treated with the DSB inducer phleomycine (see Figure 1. 11 A,B). Additionally, we could show that after damage induction by microirradiation (405nm), phospho-Ku70 co-localizes with the DNA DSB repair marker γ-H2AX at 30min. DNA DSB repair, we assessed its co-binding with some of the principle NHEJ factors. Figure 1.12 A demonstrates the interaction of phospho-Ku70 with Ku80, Ligase 4 and PAXX. However Ku70Ala and Ku70Glu interact with Ku80, Ligase 4 and PAXX, too (see Figure 1.12 B). Furthermore a proteomics approach in the Ku70Ser transfected HME cell line, revealed no significant differences between the binding of Ku70 or phospho-Ku70 to the NHEJ core factors XRCC5 (Ku80), PRKDC (DNA-PKcs), Lig4 (Ligase 4) or XRCC4 (see Figure 1.12 C). Altogether these data indicate that phospho-Ku70 is not necessary for the formation and recruitment of NHEJ factors to the break site, but that it is a part of the NHEJcomplex, interacting with key players. It is thus possible that phospho-Ku70 impacts on the DSB repair efficiency and quality.

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Dissociation kinetics of Ku70Ala from the DNA DSB is decelerated compared to Ku70Ser

The Ku protein is the very first protein to be recruited to a DSB. It is a major DNA DSB sensor as well as a recruitment factor for core NHEJ proteins to assure proper repair. After the ligation of the break the Ku protein dissociates from the DNA. Proper recruitment and dissociation of the Ku heterodimer is a necessary requirement to provide faithful DNA repair and homoeostasis We measured the recruitment and release kinetics of Ku70Ser and Ku70Ala on DSB sites by using a live cell imaging approach with induced DSBs by microirradiation (UV 405nm).

The same conditions as in Figure 1.11 C, that were already shown to induce DSBs were applied. To follow Ku70 kinetics we transfected GFP-coupled Ku70Ser and Ku70Ala into U2OS cells. After microirradiation, cells were followed up to 240min. and pictures were taken every 10min. (see Figure 1.13 A). As shown in Figure 1.13 B, recruitment kinetics are similar in Ku70Ser and Ku70Ala cells, reaching their maximum between 30 and 50min. However the fold induction of Ku70 on the DSB reaches approximately 3.1-fold for Ku70Ala, whereas the maximal recruitment is 2.6-fold for Ku70Ser. Dissociation of Ku70Ser from the DNA is much faster and reaches 50% at 150min. Basal levels for the Ku70Ser expressing cells are reached between 190 and 200min. In contrast, Ku70Ala cells reach 50% only after 220min, and do not reach basal levels within the 240min. These data indicate that phosphorylated-Ku70 is released from the DNA strand break more rapidly than Ku70Ala. Based on the results we obtained on the γ-H2AX kinetics we suppose that Ku70Ala remains on the DSB because of persisting non-repaired DSB and that Ku70Ser is released after the fulfilment of DSB repair by NHEJ.

One of the mechanisms that were described to be required for Ku release is neddylation, a prerequisite for subsequent ubiquitination of the Ku heterodimer [START_REF] Brown | Neddylation Promotes Ubiquitylation and Release of Ku from DNA-Damage Sites[END_REF].

Intrigued by the observation that phosphorylation of Ku70 favours its release, we asked whether inhibition of neddylation with MLN 4924 similarly blocks Ku70Ala and Ku70Ser from their release and if DNA DSB repair is equally impaired after Based on these results we suggest that the differences in Ku70 release depicted in Figure 1.13 B are due to persisting DSB in Ku70Ala cells compared to Ku70Ser. We argue that phosphorylation on S27 and S33 Ku70 is necessary for the proper release of Ku70Ser by neddylation dependent ubiquitination from the DNA, as well as DSB repair.

Phosphorylation of Ku70 regulates NHEJ and HR repair activity

The Ku protein is an important regulator of DNA DSB repair. It is an inalienable factor for NHEJ, and has a dominant negative effect towards other DNA repair pathways, such as HR [START_REF] Shao | Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination[END_REF]. We tested whether phospho-Ku70 promotes quantitatively measurable differences in NHEJ and HR efficacy, compared to nonphosphorylated-Ku70Ala cells. We used two cell systems, GC92 [START_REF] Rass | Role of Mre11 in chromosomal nonhomologous end joining in mammalian cells[END_REF] and U2OS HREJ, which have both an integrated reporter construct, to measure the repair after expression of the endonuclease I-SCEI. The activity of NHEJ is measured via the expression of CD4, that is induced upon I-SCEI transfection and after excision of the internal fragment containing the H2Kd and CD8 genes (see Figure 1.14 A).

H2Kd expression was monitored before transfection to be about 97-99% in all cell lines (see Appendix B, Figure 5 A) as a control. Equal expression of I-SCEI was verified by western blot (see Appendix B, Figure 5 B). As a negative control an 4.Results empty transfection plasmid was used (see Appendix B, Figure 5 A).

The percentage of cells expressing CD4 in Ku70Ser and Ku70Ala cells after I-SCEI induction, depicted in Figure 1.14 A, shows an elevated expression in the Ku70Ala cells compared to Ku70Ser. This suggests that NHEJ activity is increased in Ku70Ala cells compared to Ku70Ser after enzymatic DSB induction.

Moreover we tested whether G1-dependent NHEJ in GC92 Ku70Ala cells, had a delayed DNA DSB repair activity compared to GC92 Ku70Ser cells, similar to

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what we have previously observed for the non-synchronized U2OS cells after irradiation. For this we synchronized the transfected GC92 cells in the G1 cell cycle phase with mimosine over night and irradiated them 1h after mimosine removal (see Appendix B, Figure6). As depicted in Figure 1.14 B, the Ku70Ala expressing cells showed a slowed down foci resolution after 2 and 4h, similar to the observations seen in non-synchronized U2OS cells (see Figure 1.10 B). These data thus indicate that even when NHEJ efficiency after enzymatic DSB induction is higher in the Ku70Ala cells, this repair process is aberrant after induction of complex DSBs after irradiation, resulting in persisting DSB when compared to Ku70Ser cells. Ku70Ala cells thus have a DSB repair defect that is specific to NHEJ activity after irradiation. HR activity was measured through the expression of GFP, after a recombination event. As for the GC92 cells, equal expression of I-SCEI was evaluated via western blot (Appendix B, Figure 4 C) and an empty transfection plasmid was used as negative control (Appendix B, Figure 4 A). To exclude any cell cycle dependent events, cycle distribution was measured before and after transfection. The cell cycle was similar in all cell lines (Appendix B, Figure 4 B). The percentage of cells expressing GFP is slightly, but significantly increased in the Ku70Ser expressing cells compared to the Ku70Ala expressing cells (see Figure 1.14 C). This indicates that Ku70Ser expressing cells have an elevated activity of HR, compared to Ku70Ala cells. This observation is not due to a higher proportion of these cells in S/G2. To conclude, these data indicate that the phosphorylation of Ku70 can influence the activity of the two main DNA DSB repair pathways, NHEJ and HR.

Aberrant DSB repair in Ku70Ala cells is at the origin of chromosomal aberrations and chromosomal translocations

The observations made on a delayed γ-H2AX foci resolution, an increased amount of hyperploidy, and the persistence of the Ku70Ala mutant on the DSB after We analysed genomic instability in two different cell lines, U2OS (osteosarcoma) and HME (mammary gland) expressing Ku70Ser and Ku70Ala. Chromosomal aberrations in the U2OS cell lines were measured by counting dicentrics, acentrics and ring structures visible before and after irradiation (2Gy,48h) using DAPI, centromer and telomere staining (see Figure 1.15 A, B) which allowed us to deduce the amount of chromosome breaks. As depicted in Figure 1.15 C and D, similar amounts of chromosomal aberrations/breaks are found under non-treated conditions in Ku70Ser and Ku70Ala expressing cells. After irradiation, the Ku70Ala expressing cells show a greater amount of chromosomal aberrations/breaks than phosphorylated Ku70Ser expressing cells, 160 to 118 DSB, respectively. These observations further underline the increased genomic instability that is severely enhanced in non-phosphorylable Ku70Ala expressing cells and together with the observation of a slowed down repair process (γ-H2AX) argue for a repair defect in the Ku70Ala expressing cells.

To consolidate this result we tested the amount of chromosome translocations in the non-cancerous HME cell line, immortalized by the expression of hTert (Telomerase reverse transcriptase). In contrast to the U2OS cells (∼ 79 chromosomes), this cell line has a quasi-normal diploid karyotype with 46 chromosomes. This makes it possible to conduct a multi-FISH approach, where each chromosome is "painted" with a unique colour. After transfection with the Ku70mutants Ku70Ser and Ku70Ala (Figure 1.16 A), the chromosome numbers (46, Ku70Ser; 45-47 Ku70Ala) and karytope aberrations were similar in both transfected cell lines, except that a proportion of Ku70Ala cells showed an additional translocation der(13)+(5;13;13) (40%) (Figure 1.16 C, NT condition). After irradiation (4Gy, 24h) both cell lines showed an increase in chromosome translocations beyond those identified during non-treated conditions (Figure 1.16 C, IR condition). Ku70Ser cells had a mean of 2.2 translocations/metaphase (45-57 4.Results 134 chromosomes/metaphase), compared to the Ku70Ala cells with 3 translocations/metaphase (45-51 chromosomes /metaphase) (Figure 1.16 B). However, both cell lines also showed metaphases without additional chromosomal translocations after irradiation, this was the case for 55% (11 metaphases; total of 20 metaphases) in Ku70Ser cells and 23.8% (5 metaphases; total of 21 metaphases). Besides chromosomal translocations we also found aberrations, such as deletions, chromosome fragments, acentric fragments and double minutes, all signs of genomic stability in both cell lines.

In summary these results underline, in two different cell systems, that Ku70Ala cells accumulate a higher proportion of chromosome aberrations and translocations in a majority of irradiated cells compared to Ku70Ser cells. We suggest that this is due to a delayed repair process at the DSB. [START_REF] Reeves | Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen[END_REF], der(18)t(11;18), +20, der(22)t(10;22) [START_REF] Yaneva | Interaction of DNA-dependent protein kinase with DNA and with Ku-biochemical ans atomic force microscopy studied.pdf[END_REF] ; NT Ku70Ala: 46,XX, ins(1), ins(8), -10, del [START_REF] Reeves | Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen[END_REF], der(18)t(11;18), +20, der(22)t(10;22), der(13)t(5;13;13) [START_REF] Downs | A means to a DNA end: the many roles of Ku[END_REF]; after irradiation only those translocations that are distinct compared to the nontreated condition have been counted. Other markers of genomic instability: dmin, double minute; del, deletion; ace, acentric fragment; C-Frag, chromosomal fragment; mar, marker Chapter 2

4.Results

Generation of a humanized knock-in (KI) mouse model to study p-Ku70's role in vivo

To study the role of Ku70 in the pathophysiology of chronic lymphocytic leukaemia and cancer development more particularly, an in vivo model seemed best suited.

We therefore collaborated with the Mouse Clinical Institute (Strasbourg, France) for the development of a humanized KI mouse expressing Ku70Ser and Ku70Ala.

For the development of such mice the sequence homology, concerning the human residues 27 and 33 were studied in mice. The overall protein sequence homology is about 90% with 83% of aa being identical. Compared to human, mice have a 2 aa shifted sequence of Ku70 (see Figure 2.1 A). The mutation strategy was to humanize the area around aa 21-31 in mice (corresponsing to aa 23-33 in human) and to mutate G25,S31 in mice to either S25,S31 (Ku70Ser) or A25,A31 (Ku70Ala).

The principal strategy of the KI mouse model generation is depicted in Figure 2.1 B. Briefly, the mouse embryonic stem (MES) cells were obtained from a Bl6/N mouse (wt mouse) background. A gene targeting vector coding for the humanized Ku70Ala and Ku70Ser alleles was added to the MES for homologous recombination. Recombination efficiency of approx. 4%, tested by neomycine resistance 4.Results 140 and PCR screening, were obtained. The positive selected MES, were than transferred into blastocysts of a BALB/cN background to obtain chimeric mice. The male chimeras were backcrossed to female wt-mice (expressing the Cre-protein to autoexcise the neomycine resistance) to obtain a first heterozygous generation (F1), expressing a murine wt-Ku70 allele and a humanized KI-Ku70Ala or KI-Ku70Ser allele, respectively. To obtain homozygous mice, expressing humanized KI-Ku70Ala and KI-Ku70Ser, heterozygous F1 mice were crossed, leading to the generation of three possible scenarios for F2 progeny: homozygous wt-Ku70 (Ku70 -/-), heterozygous wt-Ku70/KI-Ku70Ala (KI-Ku70Ala KI/-) or KI-Ku70Ser (KI-Ku70Ser KI/-), homozygous KI-Ku70Ala (KI-Ku70Ala KI/KI ) or KI-Ku70Ser (KI-Ku70Ser KI/KI ) expressing mice.

The ratios for born viable homozygous KI-Ku70Ala KI/KI and KI-Ku70Ser KI/KI mice were equal, without any gender specific differences. Of 48 newborns after 6 crossings, 20 were female (42%) and 28 male (58%) for the KI-Ku70Ser mice. For KI-Ku70Ala mice, out of 40 newborns after 6 crossings, 21 were female (52%) and 19 male (48%).

Previous reports of Ku70 KO mice, showed that these mice have an important growth delay leading to 40-60% smaller littermates, than controls [START_REF] Li | Ku70: a candidate tumor suppressor gene for murine T cell lymphoma[END_REF]. We thus evaluated growth of newborns over 3 weeks, but did not observe any differences in body ratios between KI-Ku70Ser KI/KI and KI-Ku70Ala KI/KI mice (see Figure

A,B).

To further determine and verify the cellular aspects that we have seen in the human cancer cell line U2OS, we generated mouse embryonic fibroblasts (MEFs) of homozygous KI-Ku70Ser KI/KI and KI-Ku70Ala KI/KI embryos. We determined the growth of these cells and their susceptibility to cell death after irradiation.

Preliminary results on the cell doubling monitored over 7 days, shows a growth defect in KI-Ku70Ala KI/KI cells compared to KI-Ku70Ser KI/KI cells with and without genotoxic stress (irradiation 2Gy). This could argue for a intrinsic genomic instability of these cells, compared to KI-Ku70Ser. In agreement with these 4.Results in mice. The residues underlined in pink are the additionally mutated residues in mice to obtain a humanized protein sequence, surrounding the phosphorylation sites. The sequences in blue (Ku70Ser) and red (Ku70Ala), correspond to the final aa sequence of the Ku70 protein in each of the KI models, respectively. (B) Generation of a KI model. With the help of a targeting vector, containing the nucleotide sequence corresponding to the substituted aa, mouse embryonic stem cells were transfected. Selection with a antibiotic resistance and subsequent PCR analysis revealed successfully recombined ES cells. These could be transferred into an empty donor blastocyst and carried out by a surrogate mother mouse. The born chimeras are composed of cells, from the recombined ES cells and from the donor blastocyst. To select mice that have integrated the KI in their reproductive organs, they were backcrossed with the wt-mouse to give heterozygous progeny (F1). Crossing of the F1 generation can ultimately produce homozygous mice that express KI-Ku70Ser or KI-Ku70Ala on both gene alleles.

findings, is the evaluation of cell death by DIOC/IP, in non-treated and in 2Gyirradiated cells, cell death is increased in KI-Ku70Ala compared to KI-Ku70Ser cells after non-treated conditions and genotoxic stress with irradiation (2Gy, 4Gy) or phleomycine (100µM, 1h). This study was initiated on the basis of a differential apoptosis resistance revealed between R-CLL and S-CLL patient subsets. Intensive research in this field uncovered that resistance to apoptosis may be due to upregulation of an erroneous type of NHEJ, characterized by an increase in the amount of junctional deletions [START_REF] Deriano | Mutagenicity of non-homologous end joining DNA repair in a resistant subset of human chronic lymphocytic leukaemia B cells[END_REF] and chromosomal aberrations [START_REF] Blaise | High levels of chromosome aberrations correlate with impaired in vitro radiation-induced apoptosis and DNA repair in human B-chronic lymphocytic leukaemia cells[END_REF] in R-CLL cells compared to S-CLL. Furthermore a telomeric deregulation characterized by shortened chromosome ends and the presence of Ku70 and 53BP1 at telomeres of R-CLL patients further underlined the potential effects of a deregulated NHEJ in these patient derived samples [START_REF] Brugat | Telomere dysfunction-induced foci arise with the onset of telomeric deletions and complex chromosomal aberrations in resistant chronic lymphocytic leukemia cells[END_REF]. Finally, R-CLL cells were shown to overexpress a PTM on Ku70 (phospho-S27-,S33-Ku70) and a first study evaluated the impact of this phosphorylation on DNA repair [START_REF] Bouley | A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines[END_REF]. This current study aimed to further characterize the impact of phospho-Ku70 on the dynamics on the DSB site. We first established that phospho-Ku70 is an actor in radioresistance and characterized our cellular model on phenotypic aspects. Then we observed that phospho-Ku70 is necessary for proper DNA release after DNA repair, that it impacts on both NHEJ and HR repair pathway efficiency and that it maintains genomic stability compared to non-phosphorylable Ku70Ala expressing cells. Moreover this study initiated the generation of a KI mouse model with the aim to study the role of phospho-Ku70 in vivo.

4.Results

in vitro study

Phosphorylation of Ku70 confers radioresistance

In this study we established an in vitro system using human cell lines that enabled us to evaluate the biological impact of Ku70 phosphorylation. To overcome Ku-deficiency associated cell death in a human cell system [START_REF] Li | Ku86 is essential in human somatic cells[END_REF], we had to use a system in which we can downregulate endogenous Ku70, while at the same time expressing exogenous Ku70. The exogenously expressed Ku70 mutants contained the desired mutations for wt (Ku70Ser), non-phosphorylable (Ku70Ala) and phosphomimetic (Ku70Glu).

Even before the Ku protein was identified to be a NHEJ core factor, the impact of radiation on Ku70 and Ku80 defective CHO cells was demonstrated [START_REF] Malgorzata | Mammalian mutants defective in the response to ionizing radiation-induced DNA damage[END_REF][START_REF] Collins | Mutant rodent cell lines sensitive to ultraviolet light , ionizing radiation and cross-linking agents : a comprehensive survey of genetic and biochemical characteristics[END_REF][START_REF] Jeggo | Studies on mammalian mutants defective in rejoining doublestrand breaks in DNA[END_REF].

Since that time multiple studies have evaluated the radioresistance that is conferred by both subunits in different models ranging from mouse [START_REF] Ouyang | Ku70 is required for DNA repair but not for T cell antigen receptor gene recombination In vivo[END_REF][START_REF] Gu | Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination[END_REF] to human [START_REF] Omori | Suppression of a DNA doublestrand break repair gene, Ku70, increases radio-and chemosensitivity in a human lung carcinoma cell line[END_REF][START_REF] Kazi | Mutations to Ku reveal differences in human somatic cell lines[END_REF] due to Ku's role in DNA DSB repair. In this present study we establish that the mutation of the serine residues on position 27 and 33 cause a growth defect (Figure 1.3), apoptosis induction (Figure 1.5) and a radioresistance phenotype (Figure 1.4). We suggest that all of these observations are linked through a DNA repair defect present in the mutated Ku70 expressing cell lines, as they are noticed after double strand break induction with γ-irradiation or phleomycine.

Several studies have linked Ku70 to apoptosis-regulation, independent to its DNA repair function. We think that this is not the case for the phospho-Ku70 protein and its mutants. Ku70 dependent apoptosis induction is established through its interaction with the pro-apoptotic protein Bax, regulated by PTM [START_REF] Liu | The DNA-PK Catalytic Subunit Regulates Bax-mediated Excitotoxic Cell Death by Ku70 Phosphorylation[END_REF][START_REF] Subramanian | Ku70 acetylation mediates neuroblastoma cell death induced by histone deacetylase inhibitors[END_REF][START_REF] Subramanian | HDAC6 Deacetylates Ku70 and Regulates Ku70-Bax Binding in Neuroblastoma[END_REF][START_REF] Gama | Hdm2 is a ubiquitin ligase of Ku70-Akt promotes cell survival by inhibiting Hdm2-dependent Ku70 destabilization[END_REF][START_REF] Mazumder | Interaction of a cyclin E fragment with Ku70 regulates Bax-mediated apoptosis[END_REF]. Using co-immunoprecipitation we do not observe differences in Bax interaction between Ku70Ser, Ku70Ala and Ku70Glu before and after damage induction (Figure 1.6).

In contrast to the study by Bouley et al. [START_REF] Bouley | A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines[END_REF] we cannot establish any extended cell cycle checkpoint response at 2 or 4 Gy irradiation (Figure 1.7). Checkpoint responses are heavily regulated by the protein p53 and whereas ZR75.1 cell lines have a fully functional p53-protein, the U2OS cell line has an upregulated hMDM2 level, the antagonist of p53. Differences in cell cycle dynamics might thus derive from this cell line specific difference.

Finally, we show that expression of a non-phosphorylable Ku70 protein causes the accumulation of polyploid cells over time after irradiation (Figure 1.8), a characteristic that was already documented for Ku80 haploinsufficient human somatic cells, suggesting that accumulation of genomic instability is a result of defects in DNA repair [START_REF] Li | Ku86 is essential in human somatic cells[END_REF]. The accumulation of hyperploid cells might also point to a checkpoint defect in G2/M-phase in the Ku70Ala cells. This would provide the possibility to drive the cell cycle from G2 to M and interphase in the presence of unrepaired DNA breaks (checkpoint adaption and checkpoint slippage) increasing the amount of aneuploidy and genomic instability, and finally leading to mitotic catastrophe [START_REF] Gudkov | The role of p53 in determining sensitivity to radiotherapy[END_REF]. Interestingly, Shang et al. provide evidence that spindle formation and mitosis are regulated by DNA-PKcs and Ku meditated phosphorylation of Chk2 (T68) [START_REF] Fu Shang | Inactivation of DNAdependent protein kinase leads to spindle disruption and mitotic catastrophe with attenuated checkpoint protein 2 phosphorylation in response to DNA damage[END_REF]. Inhibition of DNA-PK results in aberrant spindle formation during mitosis causing polyploidy and mitotic catastrophe. It might thus be of interest to examine whether spindle formation is affected in Ku70Ala expressing cells.

To summarize, the Ku protein, and its two subunits individually have been associated to the maintenance of radioresistance by their DNA repair function in multiple studies. We show for the first time that phosphorylation on S27 and S33 on Ku70 has significant impact in the maintenance of a radioresistant phenotype.

The phoshomimetic (Ku70Glu) fails to reproduce the results of the phosphorylated Ku70Ser

The results obtained for the phosphomimetic (Ku70Glu) expressing cell line are less pronounced than the phospho-mutant (Ku70Ala) expressing cell line, when compared to the Ku70Ser expressing cells. This is most probably due to the importance of the phosphate group rather than the negative charge in this specific region. The results are in line with the study of Bouley et al. [START_REF] Bouley | A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines[END_REF]. Moreover, we observed that the phosphomimetic (Ku70Glu) shows different nuclear localization compared to Ku70Ala and Ku70Ser cells, when co-expressed with the flourescent protein mEOS (see Figure Appendix B, Figure 7) under untreated conditions. This indicates that the phosphomimetic protein has lost key functions to retain normal localization [START_REF] Dephoure | Mapping and analysis of phosphorylation sites: a quick guide for cell biologists[END_REF]. We strongly believe that the nuclear regions avoided by the phosphomimetic are nucleoli, nuclear regions that are rich in ribosomes and rRNA transcription that now are becoming more and more evident to have an active crosstalk with DNA repair [START_REF] Ogawa | Crosstalk between the nucleolus and the DNA damage response[END_REF]. As a side note, the proteomics approach we performed shows a strong preference for phospho-Ku70 to interact with ribosomal proteins (data not shown).

In summary, the phosphomimetic cell line (Ku70Glu) does not mimic the phosphorylated state of Ku70, most probably due to the importance of the phosphate group in this region. Additionally the phosphomimetic protein did not show similar nuclear localization compared to Ku70Ala and Ku70Ser and its impact in DNA DSB repair was thus no longer pursued.

The phosphorylation of Ku70 is part of the NHEJ complex and impacts on DNA DSB repair

To better situate the phospho-Ku70 protein to the DNA DSB and to underline its function, we ask some easy but yet important questions to localize it to the NHEJ complex and to the DNA DSB. We show that phospho-Ku70 interacts with Part 5 Discussion 152 core factors of NHEJ (Ku80, DNA-PKcs, Ligase 4, XRCC4 and PAXX) using two different approaches (Figures 1.12 A-C). The phosphorylation however does not seem to be a prerequisite for NHEJ-complex formation, as the phospho-mutated Ku70Ala and Ku70Glu equally interact with Ku80, Ligase 4 and PAXX. Moreover, we show that phospho-Ku70 localizes to the damaged DNA after genotoxic stress by phleomycine or microirradiation and co-localizes with γ-H2AX (Figure 1.11 A-C). Altogether these data strongly suggest that phospho-Ku70 is part of the NHEJ-complex and localizes on the DNA DSB.

We then evaluated the functional impact of the phosphorylation on the DNA repair by assessing the persistence of the γ-H2AX signal by immunoflourescence and western blot techniques. We observe a delayed γ-H2AX foci/signal resolution in the phosphomutant (Ku70Ala) cells compared to the phospho-Ku70 expressing cells (Ku70Ser) by both approaches (Figure 1.10 B,C). This is in agreement with the study of Bouley et al. [START_REF] Bouley | A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines[END_REF], who show a delayed γ-H2AX foci resolution in the Ku70Ala expressing ZR75.1 cells thus pointing towards an aberrant DNA DSB repair in these cells.

To better understand the DNA DSB repair defect in the phospho-mutant cell line (Ku70Ala) and as there were no differences in the formation of the core NHEJ complex, we thought that the phosphorylation might have a role in the recruitment and/or dissociation kinetics to/from the DSB. Our experiments using microirradiation induced DNA damage, clearly show that there are no substantial differences in Ku70 recruitment to the DNA damage, however release is hindered up to 240min (4h) in the Ku70Ala cells (Figure 1.13 B), likely due to persisting DNA strand breaks (see γ-H2AX resolution kinetics; Figure 1.10 B,C). A mechanism that was recently described to be important for Ku release is neddylation a precondition for Ku ubiquitination and release [START_REF] Brown | Neddylation Promotes Ubiquitylation and Release of Ku from DNA-Damage Sites[END_REF]. We show here that upon inhibition of neddylation both subunits equally stay and accumulate (approx. 2-fold compared to MLN4924 untreated cells) on the DNA strand break (Figure 1.13 C), this is most likely due to persistent DNA damage (see Figure 1.13 D). Indeed, we observe that neddylation inhibition impacts on the repair kinetics of DNA damage (Figure 1.13 D). This is not due to an intrinsic genotoxic activity of the neddylation inhibitor Part 5 Discussion 153 MLN 4924, as treatment with the inhibitor alone does not induce γ-H2AX (see Figure 1.13 D (NT conditions)). We suppose that phosphorylation is necessary for repair and neddylation dependent ubiquitination as well as release of Ku70 from the repaired DNA. In this context, it is interesting to note that a ubiquitination site is found on aa K31 on Ku70 [START_REF] Phosphosite | PhosphoSite-Ku70[END_REF], which lies in direct proximity to the two phosphorylation sites. It would be interesting to study if this site is ubiquitinated dependent on the phosphorylation of Ku70 at S27,S33 and if this ubiquitination affects the release from the DNA. The possibility that the phosphorylation of Ku70 can impact on its release is not surprising, for example autophosphorylation on DNA-PKcs induces its release from the DNA [START_REF] Chan | The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit[END_REF].

Besides its key function in NHEJ the Ku protein is also an important regulator of HR [START_REF] Sun | Human Ku70/80 protein blocks exonuclease 1-mediated DNA resection in the presence of human Mre11 or Mre11/Rad50 protein complex[END_REF][START_REF] Shao | Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination[END_REF][START_REF] Lee | Phosphorylation of Ku dictates DNA double-strand break ( DSB ) repair pathway choice in S phase[END_REF][START_REF] Hassan | The RNF138 E3 ligase displaces Ku to promote DNA end resection and regulate DNA repair pathway choice[END_REF]. Using a cellular system that enabled us to induce DSB via the endonuclease I-SCEI, we determine the efficiency of NHEJ and HR in Ku70Ala and Ku70Ser cells. We were surprised to see an upregulated NHEJ efficiency for Ku70Ala cells compared to Ku70Ser (Figure 1.14 A), as the γ-H2AX foci suggest a less efficient repair in phospho-mutant cells than Ku70Ser cells. We thus used this cell system and evaluated NHEJ efficiency during G1 after γ-irradiation.

As for the U2OS cells, γ-H2AX foci persisted longer in the Ku70Ala expressing cells than Ku70Ser cells at 4h (Figure 1.14 B). This points to differences in the function of phospho-Ku70, for the repair of complex (irradiation) vs. enzymatic induced (I-SCEI) DSB and it might be of interest to pursue this hypothesis. On the other hand HR efficiency is increased in phospho-Ku70 expressing cells compared to Ku70Ala cells. This is interesting as Lee et al. described a cluster of potential phosphorylation sites in Ku70, that when mutated to alanine inhibit HR by persisting on the DSB [START_REF] Lee | Phosphorylation of Ku dictates DNA double-strand break ( DSB ) repair pathway choice in S phase[END_REF]. It would thus be compelling to further study the mechanisms of phospho-Ku70 in HR regulation.

To conclude, we show that phospho-Ku70 is a member of the NHEJ complex that is found on the DSB site upon damage induction. We further demonstrate that phospho-Ku70 participates in DSB resolution and DNA dissociation after DNA repair. Moreover phospho-Ku70 seems to regulate HR and NHEJ activity, however the exact mechanisms of this observations have still to be determined.

Phosphorylation of Ku70 is important to maintain genomic stability

Whereas it was long thought that mainly alt-NHEJ contributes to chromosomal translocations it now becomes evident that this is the case in murine, rather than in human systems. In their studies Ghezraoui and Biehs et al. [START_REF] Zhang | An essential role for CtIP in chromosomal translocation formation through an alternative end-joining pathway[END_REF][START_REF] Ghezraoui | Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining[END_REF][START_REF] Biehs | DNA double-strand break resection occurs during non-homologous end-joining in G1 but is distinct to resection during homologous recombination in G2[END_REF] provide evidence that it is the classical NHEJ that induces chromosomal aberrations and translocations accompanied by frequent microhomology usage and long deletions on the junctional sites in human cell systems. Especially the study by Biehs et al. presents a model for this phenomenon, raising the possibility of a resection dependent slow-cNHEJ [START_REF] Biehs | DNA double-strand break resection occurs during non-homologous end-joining in G1 but is distinct to resection during homologous recombination in G2[END_REF]. We evaluated two different cell lines (U2OS, HME) and show an increased amount of genomic aberrations and chromosomal translocations in Ku70Ala cells compared to Ku70Ser cells, after γ-irradiation. Together with the results on the delayed DSB repair in Ku70Ala and its persistence on the DNA breakage site it would thus be interesting to evaluate if the slow-cNHEJ pathway is favoured by this mutant. It will be crucial to evaluate the junctional sites in the GC92 cell system on present deletions and microhomologies, to further consider this hypothesis [START_REF] So | Genomic rearrangements induced by unscheduled DNA double strand breaks in somatic mammalian cells[END_REF].

By its function in the regulation of DNA repair, phospho-Ku70 limits the accumulation of chromosomal aberrations and genomic instability upon irradiation.

in vivo study

All the studies performed so far on phospho-Ku70 and its role in DNA repair were performed in vitro. These cell models are quite different from the CLL samples in which phospho-Ku70 was originally identified. The establishment of an in vivo model has the advantage to study phospho-Ku70's role in lymphopoiesis of Bcells, through the study of V(D)J recombination and to approach the original CLL model in the best possible way. That gives the opportunity to evaluate phospho-Ku70's role in the aetiology of R-CLL. Indeed several studies in mice already showed a link between lymphoma development in Ku70 -/-and Ku80 -/- mice [START_REF] Li | Ku70: a candidate tumor suppressor gene for murine T cell lymphoma[END_REF][START_REF] Gu | Growth retardation and leaky SCID phenotype of Ku70-deficient mice[END_REF][START_REF] Lim | Analysis of ku80-Mutant Mice and Cells with Deficient Levels of p53[END_REF][START_REF] Li | Deleting Ku70 is milder than deleting Ku80 in p53-mutant mice and cells[END_REF].

Our preliminary studies focussed on the characterization of the homozygous KI mice. The KI-Ku70Ser KI/KI and KI-Ku70Ala KI/KI mice do not show any growth defects at 3 weeks, a time point at which a growth defect is already visible in Ku70 -/- [START_REF] Gu | Growth retardation and leaky SCID phenotype of Ku70-deficient mice[END_REF] and Ku80 -/- [START_REF] Nussenzweig | Requirement for Ku80 in growth and immunoglobulin V(D)J recombination[END_REF] mice. However we do see some cell growth defects in MEF's isolated from KI-Ku70Ala KI/KI compared to KI-Ku70Ser KI/KI without treatment or after γ-irradiation (2Gy). Additionally, cell death is more pronounced in MEF's from KI-Ku70Ala KI/KI than in KI-Ku70Ser KI/KI without treatment and after genotoxic stress.

In summary these results support the in vitro study using human cell lines. The cells expressing non-phosphorylable Ku70Ala show a growth defect and accelerated cell death. In contrast to the in vitro studies however, KI-Ku70Ala KI/KI show cell growth deficiencies and cell death susceptibility compared to KI-Ku70Ser KI/KI already at untreated conditions. This might be due to higher levels of spontaneous, endogenous damage.

Phosphorylation and their impact on the NHEJ process-impact of phospho-Ku70

Posttranslational modifications such as phosphorylations, are important cellular fine-tuning mechanisms that help the cell to rapidly adapt towards changing environmental or stress conditions, without the need of de novo protein synthesis [START_REF] Oberle | Regulation of the DNA Damage Response to DSBs by Post-Translational Modifications[END_REF]. For instance we have seen that the DDR signalling by ATM and ATR is mainly regulated by coordinated phosphorylation processes.

During DNA repair and more particularly in NHEJ, phosphorylation processes take place. These are mainly due to the PI3-kinases ATM and DNA-PKcs. All members of the core-NHEJ complex have identified phosphorylation sites and even when for some the functional impact still remains elusive, new data on phosphorylation in NHEJ have recently emerged. The most prominent target of phosphorylation during NHEJ is DNA-PKcs with over 40 phosphorylation sites and clusters, from which 16 are autophosphorylation sites [START_REF] Brandi L Mahaney | Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining[END_REF][START_REF] Davis | DNA-PK: A dynamic enzyme in a versatile DSB repair pathway[END_REF]. The phosphorylation of DNA-PKcs (for instance in the Thr2609 or Ser2056 clusters) is important for proper DNA repair. Ablation of phosphorylation by alanine mutations induce impaired DNA repair and radiosensitivity [START_REF] Davis | DNA-PK: A dynamic enzyme in a versatile DSB repair pathway[END_REF]. Additionally, autophosphporylation on DNA-PKcs is required for its proper DNA release [START_REF] Dobbs | A structural model for regulation of NHEJ by DNA-PKcs autophosphorylation[END_REF] and impacts on the recruitment of Artemis [START_REF] Blunt | Unifying the DNA End-processing Roles of the Artemis Nuclease[END_REF].

Moreover, the members of the ligation complex, Ligase 4, XRCC4 and XLF are phosphorylated by DNA-PKcs [START_REF] Gang | Phosphorylation and regulation of DNA ligase IV stability by DNA-dependent protein kinase[END_REF][START_REF] Modesti | DNA binding of Xrcc4 protein is associated with V ( D ) J recombination but not with stimulation of DNA ligase IV activity[END_REF][START_REF] Yu | DNA-PK phosphorylation sites in XRCC4 are not required for survival after radiation or for V(D)J recombination[END_REF][START_REF] Kumar Sharma | In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage[END_REF], the exact role of the phosphorylation of these sites was not clear for a long time. Considerable progress has been made in this field, showing that XRCC4 phosphorylation by CDK2 is implicated in the recruitment of processing factors like PNK, Aprataxin or APLF [START_REF] Cherry | Versatility in phospho-dependent molecular recognition of the XRCC1 and XRCC4 DNA-damage scaffolds by aprataxin-family FHA domains[END_REF][START_REF] Iles | APLF ( C2orf13 ) Is a Novel Human Protein Involved in the Cellular Response to Chromosomal DNA Strand Breaks[END_REF][START_REF] Koch | Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV[END_REF] or that XLF phosphorylation on T181 by AKT delocalizes it into the cytoplasm for degradation after DNA repair [START_REF] Liu | Akt-Mediated Phosphorylation of XLF Impairs Non-Article Akt-Mediated Phosphorylation of XLF Impairs[END_REF]. Recently, Melo et al. broadened our understanding of the possible roles of DNA-PKcs dependent phosphorylation by mutating clusters of 8 or 4 phosphorylation sites in the C-terminal domains of XRCC4 and XLF, respectively and demonstrate that these phosphorylation sites are necessary for DNA end tethering, error-free DNA repair by NHEJ and dismantling from the repaired DNA site [START_REF] Melo | Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF Cterminal tails in modulating DNA bridging during classical non-homologous end joining[END_REF].

Finally, the Ku protein has been shown multiple times to contain phosphorylation sites, however none of them was shown to be necessary for DNA repair [START_REF] Victoria | Ku Regulates Signaling to DNA Damage Response Pathways through the Ku70 von Willebrand A domain[END_REF][START_REF] Jin | Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions[END_REF][START_REF] Doug W Chan | DNA-Dependent Protein Kinase Phosphorylation Sites in Ku 70 / 80 Heterodimer[END_REF][START_REF] Morii | Src acts as an effector for Ku70-dependent suppression of apoptosis through phosphorylation of Ku70 at Tyr-530[END_REF][START_REF] Douglas | DNA-PK-dependent phosphorylation of Ku70/80 is not required for non-homologous end joining[END_REF]. The phosphorylation sites identified in our lab are thus the very first in vitro and in vivo confirmed phosphorylation sites identified in Ku70 that are shown to impact directly on Ku release and DNA DSB repair.

The NHEJ pathway is the major DNA repair process in mammals, active in all cell cycle stages and thus carries a major responsibility to repair a large variety of DNA breaks and end configurations in order to provide genomic stability and prevent carcinogenesis. In this regard the understanding of how post-translational modifications such as phosphorylation regulate the NHEJ pathway is an important parameter not only for the mechanistic understanding of the pathway but also for cancer prevention and treatment.

What impact could phospho-Ku70 have for the clinic?

The identification of phospho-Ku70 is interesting for the clinics in two ways. First, it is a biomarker that distinguishes high risk CLL patients, from patients with an indolent disease progression. The evolution of certain low risk patients towards the high risk group after chemotherapeutic treatment with fludarabine or chlorambucil, point to the development of a resistance phenotype. The measurement of persistent phospho-Ku70 in patients could thus become a potential risk marker to adapt cancer therapy. Second, the mechanistic insights into phospho-Ku70's functions in DNA repair can be important pieces in the characterization of possible drugging strategies. Indeed, the NHEJ pathway and in particular the DNA-PKcs kinase are considered as attractive target for synthetic lethality in HR or ATM deficient tumours [START_REF] Dietlein | Molecular pathways: Exploiting tumor-specific molecular defects in DNA repair pathways for precision cancer therapy[END_REF][START_REF] Knittel | Targeting ATMdeficient CLL through interference with DNA repair pathways[END_REF][START_REF] Setton | Chapter 9 -DNA double-strand repair by nonhomologous end joining and its clinical relevance[END_REF].

However, this study also highlights the complexity of cellular and cancer biology.

Whereas in R-CLL-patients the expression of phospho-Ku70 is associated with an accumulation of genomic instability, this seems not the case for the in vitro studies we performed in U2OS and HME cell lines. In our in vitro studies phospho-Ku70 is an actor in radioresistance that provides less genomic rearrangements compared to the phospho-mutant. This might be due to the different cellular background these studies have been performed in. Lymphocytes are particularly dependent on the NHEJ pathway as they are noncycling cells and thus do mainly use this repair pathway. Additionally these cells are the only to conduct V(D)J recombination and CSR, two molecular pathway that create gene rearrangement and genomic diversity dependent on the NHEJ pathway. Thus, the NHEJ pathway and eventually the phosphorylation of Ku70 might have a peculiar importance in these cells, responsible for these differences. It will be of crucial interest to study the role of phospho-Ku70 in lymphocytes, in order to understand if there exist cell type specific differences.

163

This study characterized the functional role of the newly identified phospho-Ku70 molecule in the dynamics of DNA DSB repair. We show for the first time its importance in radioresistance, characterizing its role in the NHEJ complex and DNA DSB repair. Still, with this work new questions arise.

In direct association to this work it will be of interest to characterize the molecular mechanisms that drive genomic stability in the Ku70Ala cells. We show that the Ku70Ala cells provide a delayed DNA DSB repair, with a longer persistence of Ku70 on the unrepaired DNA end. We speculate that these end extremities ends could become an attractive target for nucleases like CtIP, favouring end resection with revelation of microhomology sites, accounting for genomic rearrangements [START_REF] Zhang | An essential role for CtIP in chromosomal translocation formation through an alternative end-joining pathway[END_REF][START_REF] Biehs | DNA double-strand break resection occurs during non-homologous end-joining in G1 but is distinct to resection during homologous recombination in G2[END_REF][START_REF] So | Genomic rearrangements induced by unscheduled DNA double strand breaks in somatic mammalian cells[END_REF][START_REF] Guirouilh-Barbat | 53BP1 Protects against CtIP-Dependent Capture of Ectopic Chromosomal Sequences at the Junction of Distant Double-Strand Breaks[END_REF]. A first step to investigate this hypothesis would thus be to study the junctional sites on microhomologies and/or deletions in Ku70Ala versus Ku70Ser expressing cells using the NHEJ reporter construct in the GC92 cells.

We further suggest that neddylation dependent ubiquitination of Ku70 is hampered in Ku70Ala expressing cells. Interestingly the phosphorylation site on S27 and S33, surround a ubiquitination site K31 on Ku70. It would be interesting to mutate this ubiquitination site (R31) and to evaluate, if this mutant has similar release kinetics as Ku70Ala. This would suggest that phosphorylation on S27 and S33 is necessary for ubiquitination on K31 and Ku70 release.

Another intriguing question arises through the differences we observe between the usage of HR and NHEJ between Ku70Ser and Ku70Ala expressing cells. Using the microirradiation approach we could explore whether access of HR factors like BRCA1, MRN, RPA or RAD51 are hindered by Ku70Ala, thus diminishing the access and initiation of this repair process. Moreover as stated in the introduction, NHEJ factors and in particular the Ku proteins, Ku70 and Ku80 are absolutely required for the maintenance of telomeres. Given the particular role that Ku has on telomeres acting as a double edged sword it would be compelling to shed light on phospho-Ku's role. This is further underlined by the data that R-CLL patients have severely shortened telomeres.

Part 6 Conclusion & Perspectives 164

In the second part of this study we introduce the in vivo KI mouse model we generated. This model will be of particular interest when studying the role of phospho-Ku70 in V(D)J-recombination and lymphocyte development conducted in a collaboration with L. Deriano (Institut Pasteur). The use of an in vivo model gives us the opportunity to investigate phospho-Ku70's role in lymphocytes in a physiological setting and after p53-KO, a condition that was already shown to favour the generation of lymphomas and cancer in Ku-deficient mice [START_REF] Lim | Analysis of ku80-Mutant Mice and Cells with Deficient Levels of p53[END_REF].

Finally, we started a collaboration with J. Demmers (Proteomics Center Rotterdam) to establish a differential proteomics analysis between R-CLL and S-CLL patient samples based on the interaction with phospho-Ku70 and Ku70. These data should help to establish phospho-Ku70 specific binding partners in R-CLL and S-CLL, thus helping to evaluate critical binding partners and pathways. Additionally we hope that this approach could give further insight into the kinases and phosphatases involved in the (de-)phosphorylation of Ku70.

Part VII Appendix B: Supplementary

Introduction

DNA damages generated by exogenous genotoxic stress must be repaired rapidly and accurately to maintain cell integrity and to prevent genome instability and, to avoid subsequent cancer development. Thus, not only cell's DNA repair capacity but also simultaneous repair fidelity should be orchestrated timely at each DNA lesion. To avoid death and to ensure its legitimate survival, cell developed multiple DNA repair systems providing the specificity of DNA damage 1 and ref. within . DNA DSBs are the most deleterious for cell since only one not-or miss-repaired may be fatal for cell outcome 2-4 . To deal with this kind of damage cell developed the DNA damage response system (DDR) that essentially include damage signaling by three phosphatydilinositol-3-related family kinases ATM, ATR and DNA-PKcs which regulates the activities of proteins involved in non-homologous end joining (NHEJ) and homology-directed recombination (HR) 5-13 and in the engaging cell cycle checkpoints 14 . These last processes are concomitant and inseparable from proper DDR since they impair cell cycle progression with damaged DNA through the DNA synthesis/mitosis (S/G2/M) phase 6,15,16 . One of the main hallmark of cancer cells is a cooperative deregulation of both DDR and cell cycle checkpoints that deserves their clinical targeting 3,5,17-19 . Dogmatically, it is assumed that HR and NHEJ act in a "balanced" cell cycle-dependent competition to efficiently protect cell integrity 9 and ref.within . Two principal "deciders" helped by auxiliary factors, of the engaged pathway are BRCA1 11,20,21 and 53BP1 22, rev. 23, 24 , initiating HR or cNHEJ, respectively.

Generally considered as accurate, HR relies on a homologous sister chromatid strands as identical DNA template available in late-S/G2 of the cell cycle rev9, 25 . By contrast, cNHEJ is considered as error-prone as it is active throughout cell cycle. Ku heterodimer is the key regulator of this type of DNA repair process 10,12 . Displaying high DNA end-binding affinity it forms with DNA-PKcs a trimer referred as DNA-PK holoenzyme, initiating DDR cascade leading to cNHEJ fulfillment 26,27 . Structural studies of the Ku-DNA-end complex and Kutelomeric DNA complex suggested that Ku70 helix DNA-end interaction is specific for NHEJ while Ku80 helix is oriented towards telomeric chromatin; a model that may partly explain telomeric paradox of Ku heterodimer avoiding telomeric fusions 28 . Next step of cNHEJ is DNA-end processing engaging Artemis nuclease, polynucleotide kinase (PNK), DNA polymerases Pol  and Pol µ, Aprataxin and polynucleotide kinase/phosphatase-like factor (APLF), MRN, WRN helicase. The processing step is finally followed by ligation step necessitating DNA ligase IV and XRCC4 as well cofactors XLF (Cernunos) and Paxx rev.12 . Recruitment and activation of cNHEJ proteins is essentially ensured by the Ku-binding motif 29 and DNA-PKcs kinase activity rev. 30 . Following DNA DSBs, DNA-PKcs phosphorylation (mainly autophosphorylation but also ATM-and/or ATR-dependent phosphorylations), occurs on more than 40 sites and this phosphorylation is for instance the only phosphorylation regulating the kinetic of cNHEJ 30 . Recently, a cluster phosphorylation of Ku70 at the junction of its pillar and bridge region (putative S and T residues between T298 and S314 in core domain), has been reported to facilitate the dissociation of the Ku heterodimer from DNA ends and to promote HR in S phase 31 . Another way proposed to remove Ku from the sites of initial DNA damage is the neddylation and subsequent ubiquitylation of Ku 32 . Effectively, ubiquitination sites on both subunits of Ku are at origin of the release of Ku only after successful DNA repair. Moreover, this study uncovers downregulation of Ku70 specific protein interactors after inhibition of neddylation. One of these interactors is the AAA+-type ATPase VCP/p97, shown to extract trapped Ku from DNA 33 .

Finally, DNA ends with 5' or 3' overhangs, may proceed through a search of sequence microhomology before ligation resulting in a nucleotides' loss and repair inaccuracy rev. 8,12,34 . Microhomology-directed DNA end-joining may be achieved by cNHEJ but may also be Kuindependent (alternative NHEJ) and as thus, both of them represent new potential mechanisms of genome instability rev34 . aNHEJ is depending on MRN complex and may be suppressed by the versatile protein WRN (regulating also HR and cNHEJ), through inhibition of Mre11/CtIP-mediated resection and subsequent large DNA deletions 35 . Indeed, of particular importance for genome stability appears to be played by resection-dependent slow kinetics cNHEJ during G1 also sharing initial components of HR. Effectively, this type of end-resection in cNHEJ repair necessitates CtIP/Brca1 interaction and exonuclease Exo1 to be finally achieved by endonuclease Artemis 36 operating in both HR and cNHEJ. This process results also in nucleotides loss and translocation events and as thus, represents a potential source of illegitimate, disordered cancer genome. Hence, depending on the DNA-end configuration at the sites of damage and according to the availability of necessary factors and the cell cycle phase, the step of end processing puts common proteins of different DNA repair pathways into tight dynamic interactions. Their interactions should engage the appropriated pathway to remove DNA DSBs rev 12 and 34, 35-39 . This concept favors thus the interplay and complementarity between different repair pathways in all phases of cell cycle rather than a competition.

The defects in DNA repair and DDR genes allow cancer cells to develop the resistance to radio-and/or chemo-therapies rev. 17, 19 . Recently, this resistance has been "revisited" and provided the new anti-cancer strategy to target specifically malignant cells rev. 40, 41 . Hence, the molecular profiling of tumor samples may improve DNA-damaging based therapy and help a development of personalized treatments as well as the identification of drug resistance mechanisms enabling an appropriated modification of first-line treatments 41,42 . Exploring the mechanisms of the resistance of chronic lymphoid leukaemia (CLL) cells to current clinical treatment, we have recently identified a new stress-inducible form of phospho-S27-S33-Ku70 (pKu70) 43 overexpressed in leukemic cells resistant in vitro and in vivo to DNA damage-induced apoptosis and displaying an upregulated cNHEJ 44,45 . Experimental model of breast cancer cell line with shRNA-controlled repression of endogenous Ku70 simultaneously with the expression of different forms of exogenous Ku70, allowed us to show that this form confers accelerated DNA repair that may deserve basic mechanism through which leukemic cells escape apoptotic death. Here, we used the same shRNA/cDNA vector's approach on different cancer and non-cancer cell lines to further characterize the function of pKu70. We show that pKu70 co-localizes with -H2AX at sites of DNA DSBs and confers rapid repair. Immediately after DNA DSBs induction, S27-S33-Ku70 (ser-Ku70) re-localized from nucleoli to the site of DNA damage and, its dissociation is dependent on phosphorylation/neddylation events that may be crucial in preserving genome stability since alanine-Ku70 form causes chromosomal losses and translocations. Accordingly, DNA damages induced in G1 arrested cells are more rapidly repaired in cells expressing ser-Ku70. Moreover, in cells harboring integrated substrate with unique I-SCE1 restriction enzyme site, ser-Ku70 was shown to stimulate HR opening thus the possibility of a new connection between cNHEJ and HR that deserves further investigations.

Results pKu70 controls cell growth, promotes clonogenic survival and protect cells from apoptosis.

Multiple studies have shown that Ku deficiency leads to a growth defect rev.10,12 . We evaluated the onset of a growth deficiency over several days in untreated and irradiated (2 and 4 Gy) transfected U2OS cells. During untreated conditions no significant effect on growth is observed between the different forms of Ku70 (Fig. 1A). However following irradiation, by the seventh day of culture, cell numbers of ala-Ku70-and glu-Ku70-expressing cells are 37.5% (2Gy), 60% (4Gy) and 25% (2Gy) , 50% (4Gy) lower compared to ser-Ku70expressing cells, respectively (Fig. 1A). Ku70 deficiency was shown to induce a sever radiosensitivity phenotype in mouse model 46 . To determine whether the Ku70 mutations interfere with radiosensitivity, a clonogenic assay of the cell lines expressing different forms of Ku70 was assessed. Unexpectedly, the plating efficiency already differed significantly between untreated ser-Ku70 and ala-Ku70Ala or glu-Ku70, respectively. Whereas about 36% of the plated cells formed colonies in the ser-Ku70S group, only 24% or 28% of ala-Ku70 and glu-Ku70 formed colonies (see Figure 1B,a). Nevertheless, following irradiation, cell survival decreased further in the ala-Ku70-and glu-Ku70-expressing cells particularly after 4 and 6 Gy, compared to ser-Ku70 (Fig. 1B,b). At 2Gy, 4Gy and 6Gy, approximately 15%, 1% and 0.03% of ser-Ku70 transfected cells give clonogenic progeny, compared to 13%, 0.5% and 0.015% of ala-Ku70 and 8%, 0.3%, 0.004% of glu-Ku70 cells, respectively. These data suggest that ala-Ku70 and glu-Ku70 have an initial defect in colony formation and that this deficiency is further enhanced by irradiation.

Based on the observations of a downregulated cell growth and enhanced radiosensitivity in the ala-Ku70-and glu-Ku70-expressing cells compared to ser-Ku70, we further evaluated the sensitivity to cell death induction by irradiation stress. Total cell death and apoptosis were evaluated at day 3 and day 4 following irradiation with a double staining with DiOC6 and propidium iodide (PI) (see Fig. 1C). Cell death increased with time and irradiation dose in all cell lines. Initial cell death in untreated conditions varies between 5 to 7% at day 3 and 6 to 7% at day 4 in all cell lines. At day 3 after irradiation, the percentage of cell death is about 7%, 11% and 8.5% (2Gy) and 12%, 18% and 14% (4Gy) in ser-Ku70-, ala-Ku70-and glu-Ku70expressing cells, respectively. Similarly, at day 4, cell death increases to 12%, 17% and 14% (2Gy) and 19%, 30% and 23% (4Gy ) for ser-Ku70-, ala-Ku70-and glu-Ku70-expressing cells, respectively. Similar to the effects obtained with irradiation, treatment with phleomycine increases cell death in all cell lines compared to the non-treated conditions (Fig. 1C). As all Ku70 construction interacted similarly with Bax (not shown), we concluded that apoptotic death relive on other mechanisms.

Ser-Ku70 preferentially localizes in nucleoli, pKu70 co-localizes with -H2AX at sites of DNA DSBs and interact with core components of NHEJ complex. To explore cell localization of Ku70 at the molecular scale we performed PALM (photoactivated localization microscopy) experiments. We have constructed the vectors enabling an expression of photo-convertible molecule mEos2 fused to different forms of Ku70 (phosphorylable form ser-Ku70, unphosphorylable form ala-Ku70 or phosphomimetic glu-Ku70) to characterize the nuclear repartition of different forms of Ku70 in cells under stress-less conditions. Figure 2A show that ser-Ku70 fully filled nucleolar structure and is densly located to few nuclear subregions while glu-Ku70 was excluded from nucleoli showing a homogenous nuclear localization. ala-Ku70 cells exhibits two different pattern : mainly nucleolar with less dense regions or less frequently, empty nucleoli with homogenous nuclear localization (like glu-Ku70). This was also evidenced by eGFP-Ku70 fusion proteins in laser micro-irradiation approach and life cell imaging (see below).

Before evaluating the role of phospho-Ku70 on the DSBs, we have addressed localization following DNA damage induced by phleomycineand by laser micro-irradiation. Thus, two complementary approaches, a chromatin binding assay and immunofluorescence labelling were applied. Both approaches allowed co-localizing phospho-Ku70 to damaged DNA. After DNA damage induction by micro-irradiation (UV 405nm, with 1µg/ml of Hoechst 33342 and exposing cells to 10% of laser intensity during 2 seconds in 5µ line in nuclear region (on Nikon N1 inverted microscope), phospho-Ku70 co-localizes with the DNA DSB repair marker -H2AX (Fig. 2B). In parallel, this co-localization occurs in DNA damage, dose-dependent manner when cells were treated with the DSB inducer phleomycine (Fig. 2C, a andb). Next, we assessed co-immunopurification assays to validate that pKu70 forms cNHEJ complex in cells. Fig2D,a shows the interactions of phospho-Ku70 with Ku80, Ligase 4 and PAXX, therefore validating that phospho-Ku70 is the part of the cNHEJ complex required to repair a DNA DSB. However, ala-Ku70 and glu-Ku70 equally interact with Ku80, Ligase 4 and PAXX (see Figure 2D,b). Furthermore, a proteomics approach analyzing immunopurified proteins by anit-phospho-S27-Ku70 or anti-Ku70 (clone N3H10, NeoMarkers, Thermo Scientific) antibodies, in a collaborative work with the Proteomic Center, Rotterdam, revealed no significant differences between the binding of total Ku70 or phospho-Ku70 to the NHEJ core factors XRCC5 (Ku80), XRCC6 (Ku70), PRKDC (DNA-PKcs), LIG4 (Ligase 4) or XRCC4 (Fig. 2E). These data may indicate that phospho-Ku70 is not necessary for the formation and recruitment of cNHEJ factors to the break site, but that it is a part of the cNHEJ-complex, interacting with the key players. The fact that the "core" nucleolar factor nucleophosmin (NPM1) interacts less strongly with pKu70 suggests that phosphorylation occurs most likely at the sites of DNA damage induced by laser irradiation (see below). Nevertheless, this interactome approach indicated other factors (most of them already reported in literature to interact with Ku70), that may be specific interactors of unphosphorylated-or phosphorylated-Ku70 such as UBE2M or N4BP1 enzymes involved in NEDD8-or NEDD4conjugation, respectively. Some of ubiquitin-conjugating or -ligase E3 enzymes (UBE2G2 and TRIM33) may interact preferentially with pKu70. In parallel, "auxiliary" factors able to affect cNHEJ were also found as interactors of unphosphorylated-(WRN and DNA-Pol subunit 4), or phosphorylated-Ku70 (CAF1, Fig. 2E).

Kinetic of DSBs repair is accelerated in unsynchronized and G1 arrested cells expressing pKu70. By using -H2AX and comet assay, we have previously shown an accelerated kinetic of DNA repair in cells expressing pKu70 43 . Here, we have validated this in unsynchronized U2OS and G1 arrested GC92 47 cell systems. Following 4Gy irradiation -H2AX foci were assessed by immunofluorescence (illustrated by Fig. 3A) and quantified (automated counting by CellProfiler software, Fig. 3B). Untreated U20S cells displayed similar amount of -H2AX foci in the ser-Ku70-and ala-Ku70A-expressing cell lines (median= 5 foci/cell). 0.5h following irradiation, the amount of -H2AX foci reaches its maximum; both cell lines showed a median of 24 foci/ cell. At 4h after irradiation ala-Ku70 cells displayed significantly higher (p< 0.001) amount of remaining γH2AX foci (median= 13; 25%-percentile= 8; 75%-percentile= 22), compared to the ser-Ku70-expressing cells (median= 11; 25%percentile= 7; 75% percentile= 19). Similarly, Western blot analysis of -H2AX shows a stronger signal at 4 and 6h after irradiation for ala-Ku70-than ser-Ku70-expressing cells (Fig. 3B).

Intrigued by the fact that resection-dependent cNHEJ in G1 may be at origin for the appearance of chromosomal instability 36 , we synchronized GC92 cells over-night by mimosine (300µM) causing G1 arrest. Synchronization of the cells was measured by flow cytometry showing that over 90% of cells were blocked in G1 after overnight treatment (Fig. 3C). One hour after removing mimosine, cells were irradiated at 4Gy and examined for kinetic of -H2AX disappearance. As depicted in Fig. 3D,E, the ala-Ku70-expressing cells displayed higher level of -H2AX foci 2 and 4h, similar to the observations seen in unsynchronized U2OS cells (see Figure 3B). These data thus indicate that rapid repair of DSBs in G1 was conducted by pKu70. pKu70 confers more rapid dissociation from the sites of DNA-damage: inhibition of neddylation sustains both pKu70 release and DSBs following micro-irradiation. Following the demonstration of a delayed resolution of -H2AX foci in the mutated form of Ku70, we have addressed real-time life cell imaging to monitor the dynamic of Ku70 recruitment and release at DSBs. For this purpose we transfected cells with vectors enabling expression of fusion eGFP-Ku70 proteins. DSBs were induced by microirradiation (UV 405nm in presence of Hoechst 33342 1µg/ml), with the same experimental conditions as applied in experiment of phospho-Ku70 co-localization with -H2AX (Fig. 2B). After microirradiation, cells were followed up to 240 min. and pictures were taken every 10 min (Fig 4A). As could be observed, recruitment kinetics are quite similar in eGFP-ser-Ku70-and ala-Ku70-expressing cells, reaching their maximum between 30 and 50min. Quantified intensities seem to indicate that ala-Ku70 gave the stronger signal on DSBs (i.e. 3.1-fold induction for ala-Ku70, whereas the maximal recruitment is 2.6-fold for ser-Ku70, Fig. 4B). Dissociation of eGFP-serKu70 from the DNA damage is much faster and reaches 50% at 150 min. Basal levels for the ser-Ku70-expressing cells were reached between 190 and 200min. In contrast, ala-Ku70 cells reached 50% only after 220min, and did not reached basal levels within the 240 min. These data suggest that the phosphorylation of Ku70 favors Ku70 release from the sites of DNA damage and might thus implicate a faster fulfillment of NHEJ process in these cells, compared to ala-Ku70-expressing cells; these data are in agreement with -H2AX -foci (Fig. 3B) and -protein level (see below).

Neddylation has been recently reported an important prerequisite process for ubiquitination and subsequent Ku release from the DNA 32 . Intrigued by the observation that phosphorylation of Ku70 favours its release, we asked whether an inhibition of neddylation with MLN4924 may impair the release of ser-Ku70 from the sites of DNA damage. Figure 4C shows ser-Ku70 and ala-Ku70 dissociation kinetics after cell treatment with MLN4924 at 3 µM for 1h prior micro-irradiation. Maximal recruitment of ser-Ku70 (6.4-fold) and ala-Ku70 (6.1-fold) after MLN 4924 treatment are approx. 2-fold higher compared to conditions without MLN 4924. Dissociation of ser-Ku70Ser was severely hampered with only 20-25% of Ku70 dissociated at 240min reaching thus the kinetic of ala-Ku70. These data indicate that neddylation of Ku70 acts downstream of Ku70 phosphorylation to conduct Ku70 release. In parallel, -H2AX protein level was sustained in ser-Ku70-expressing cells treated by MLN4924 while it remained the same in cells expressing ala-Ku70 further supporting the necessity of neddylation of pKu70 prior to its release from the DSBs. In agreement with this, proteomic data pointed out NEDD8-conjugating enzyme Ubc12 (UBE2M) and NEDD4-binding protein 1 (N4BP1) as well ubiquitin-conjugating enzyme E2 G2 and ubiquitin-ligase protein TRIM33, as new partners whose affinity may depend on Ku70-phosphorylation status (Fig. 2E). pKu70 stimulates HR activity in absence of exogenous genotoxic stress. To further explore how the general role of pKu70 in DNA repair could be in the absence of exogenous genotoxic stress, we have addressed two specifically dedicated cell models, U2OS 48 and GC92 cells 47 containing a single copy of a HR or NHEJ only reporter substrates, respectively. Substrate contain unique I-SCE1 restriction enzyme site enabling thus to measure the efficacy of DNA break repair after I-SCE1 endonuclease expression. 72h after I-SCE1 transfection, cells were probed for GFP, CD4-and H2Kd-expressions by FACS analysis. NHEJ activity was measured via the expression of CD4 that is induced upon I-SCEI transfection, excision of the internal fragment containing the H2Kd and CD8 genes 47 . H2Kd expression as control was monitored before transfection to be about 97-99% in all cell lines (see Figure 5A and5B). Equal expression of I-SCEI was verified by Western blot (Fig. 5A, c and5B, b). As a negative control an empty transfection plasmid was used. Unexpectedly, an elevated CD4 expression was observed in ala-Ku70 rather than in ser-Ku70-expressing cells (Fig. 5B,c), suggesting that ala-Ku70 cells exhibit higher NHEJ activity toward enzymatically -induced DSB.

HR activity is measured through the expression of GFP, after a recombination event Fig. 5A,a). As for the GC92 cells, equal expression of I-SCEI was evaluated and an empty transfection plasmid was used as negative control (Fig. 5A,c). To exclude any cell cycle dependent events, the cycle was evaluated before and after transfection. The cell cycle was similar in all cell lines (Figure5A, b). The percentage of cells expressing GFP was increased in the ser-Ku70expressing cells as compared to the ala-Ku70-expressing cells. This indicates that ser-Ku70expressing cells have an elevated activity of HR in stress-less conditions and this was not due to a higher proportion of these cells in S/G2. Thus, these data indicate that the phosphorylation of Ku70 can affect the HR and cNHEJ DNA repair pathways. pKu70 promotes genome stability after genotoxic stress. Following impaired release from DNA-damage sites and sustained DSBs in G1 arrested cells expressing the mutated form of Ku70 as well as an increased chromosomal structural aberrations in U2OS expressing ala-Ku70 (not shown), we addressed multi-FISH to assess whether these events may be associated with the appearance of genome instability. Because U2OS and GC92 cell lines are polyploid inherently harboring multiple chromosomal aberrations we have chosen human mammary epithelial (HME) diploid cell line (kind gift from R. Weinberg lab). This approach allowed us to assess more precisely type of chromosomal aberrations that may indicate both chromosomal translocations and lost. Three recurrent aberrations found in this cell line (involving intrachromosomal exchanges short/long arm Chr.1; and interchromosomal exchanges chr. 18:11 and chr. 22:10), were maintained in ser-Ku70-and ala-Ku70-expressing cells. Intriguingly, ser-Ku70-and ala-Ku70-expressing cells' metaphases displayed both additional new aberration der. 8 (amplification possibly affecting C-MYC gene region). Forty % of ala-Ku70 cells metaphases displayed an additional translocation der(13)t(5;13) not observed in ser-Ku70-expressing cells. After irradiation, (4Gy, 24h), ser-Ku70 exhibited significantly lower rate of translocations than ala-Ku70 cells; mean of 1.95 vs 2.72 translocations/metaphase, respectively. In addition, 11 metaphases among 20 analyzed (55%) in ser-Ku70-expressing cells did not shown any additional translocation after irradiation while only 5/21 metaphases (23.8%) of ala-Ku70-expressing cells were without additional translocation events.

Discussion

The main hallmark of cancer cells is the genomic instability that paradoxically, ensures their illegitimate survival and tumor maintenance. Deregulations in DNA repair and DDR genes in cancers have prompted the development of new therapeutic approaches attempting to target underlying DNA repair pathways specifically in malignant, preserving healthy, cells. The concept of synthetic lethality, well-illustrated by the use of PARP inhibitors for the cancers with HR repair defect, is the most promising therapeutic strategy depending on the identifications of DDR targets in each (sub)type of cancer, in order to rationally develop the appropriated drug(s) 40-42 . Aggressive subset of chronic lymphoid leukaemia (CLL) is currently incurable. For the resistant cells from this subset, we have reported several lines of evidences which are indicative of deregulated cNHEJ as well that current front-line therapy based on DNA metabolism-impairing agents may not be beneficial for patients as it may favor disease relapse and progression 49-51 . More recently, we have identified in these resistant CLL cells an overexpression of a new form of ser27-ser33-Ku70 (pKu70) that conferred rapid but inaccurate DNA repair in a breast cancer cell line model 43 . We have speculated that this alteration may be a possible underlying mechanism of CLL cell resistance to treatment and the appearance of multiple chromosomal aberrations. Here, we have used the same EBV-based vectors enabling simultaneous inhibition of endogenous Ku70 and an expression of different forms of exogenous Ku70 both, in cancer-and non-cancer-derived cell lines. Experiments were performed mainly on U2OS osteosarcoma cell line because the unique I-Sce1 substrate, allowing assessing of HR activity was done in this cell line ref48 . By laser micro-irradiation and chromatin binding approaches we first demonstrated pKu70 colocalisation with DNA-damage sites/-H2AX (Fig. 2B). Next, with the assessment of-H2AX foci and protein levels, we have confirmed previously reported data 43 of an accelerated DSBs repair kinetic in cells expressing pKu70 as compared to kinetic in cells expressing ala-Ku70. This was established in unsynchronized U2OS as well in G1-arrested GC92 cells 47 (Fig. 3). Chimeric proteins mEos-Ku70 and eGFP-Ku70 showed that ser-Ku70 is preferentially localized in nucleoli while alanine-form exhibited both nuclear and nucleolar localization. Intriguingly, phosphomimetic Ku70 (glu-Ku70) was excluded from nucleoli and exhibited exclusively nuclear localization (Fig. 2A); this latter giving intermediate result between ser-Ku70 and ala-Ku70 in all experiments was abandoned during the course of some kinetic studies. Ser-Ku70 nucleolar localization is in agreement with the proteomic data of pKu70/Ku70 interactome pointing out nucleophosmin and the proteins involved in ribosomal biogenesis as the main partners of Ku70 (Fig. 2E and unpublished data). Immediately after micro-irradiation ser-Ku70 and ala-Ku70 delocalized from nucleoli to the sites of DSBs. Similar nucleolar delocalization has already been described for Rad52 involved in HR 52 . Other factors (such as WRN, p97/VCP, ABH2 and TRF2) 53-55 , involved in DNA damage repair have already been localized in nucleoli. Thus, these subnuclear membranes-less structures dealing rDNA and rRNA transcription and processing, respectively, and ribosomal assembly rev. 56 , appeared thus as the essential storage organelles of the factors involved in the maintenance of genome stability 57 .

Real-time kinetics in the laser micro-irradiation approach allowed us to establish accelerated release of pKu70 at the sites of DNA damage as compared to ala-Ku70 (Fig. 4A,B). This is also in agreement with the kinetics of -H2AX foci/protein levels showing time-dependent decrease in -H2AX content (Fig. 4D,a). Ubiquitin-like protein NEDD8 and subsequent ubiquitylation have been recently shown to promote the release of Ku and Ku-associated proteins from DSBs following repair 32 . Accordingly, we have treated cells with MLN4924 that specifically inhibits NEDD8 conjugation to Ku heterodimer. This treatment clearly sustained eGFP-ser-Ku70 signal at the sites of DNA damage (Fig. 4C) and -H2AX content in cells expressing phosphorylable form of Ku70 with no effect on ala-Ku70 expressing cells (Fig. 4D,b)). Clearly, Ku70 phosphorylation precedes its dissociation from DNA-damage sites. Early nucleoli empting following laser microirradiation and delocalization of both, ser-Ku70 and ala-Ku70 suggests that Ku70 phosphorylation occurred at the sites of DNA damage even if the basal level of pKu70 in ser-Ku70 expressing cells was significant without any treatment. Further, phospho-S2056-DNA-PKcs and phospho-S1981-ATM, both were shown to be implicated in Ku70 phosphorylation 43 , co-localize in -H2AX foci indicating that its phosphorylation most probably take place at the DSBs sites. Further, an increased early signal in ala-Ku70-expressing cells may indicate that the repair and Ku70 dynamics at DNAdamage sites was slowed-down (i.e. signal at 30min post-irradiation in Fig. 4B). Based on these and previously reported data concerning the accuracy of DNA repair 43 , we have next transfected U2OS cells harboring a substrate with a unique I-Sce1 site 48 allowing an assessing of HR activity. In parallel, we have transfected the GC92 human fibroblast cell line harboring unique NHEJ substrate cleavable by the I-Sce1 enzyme 47 . As shown in Fig. 5A andB, ser-Ku70 expressing cells stimulated HR upon I-Sce1 induction while, unexpectedly, ala-Ku70expressing cells exhibited significantly higher NHEJ activity. Chen and coll. reported similar results where cluster phosphorylation of Ku70 stimulates HR in S phase and that the ablating phosphorylation gave rise to the sustained retention of at DSBs with a decrease in HR in S phase 58 . However, in that case this cluster phosphorylation in junction/bridge region of Ku70 modulating DNA end resection and HR, was not required for NHEJ activity. Ser27-ser33-Ku70 phosphorylation appears to control both NHEJ and HR activities opening the possibility that like in DNA-PKcs rev. 10, 59 , Ku70 molecule has several phosphorylation sites with variable effect on NHEJ/HR activities. An increased NHEJ activity observed in GC92 cells expressing ala-Ku70 72h following I-Sce1 induction, in absence of stress conditions, appears in a contradiction with other experiments with irradiations/drugs-induced complex DSBs in the same cell type where ser-Ku70 accelerated NHEJ. Since DNA-end binding activity remained the same for ser-Ku70 and ala-Ku70 43 and since eGFP-ala-Ku70 and eGFP-ser-Ku70 are recruited with similar kinetic to DNA-damage sites, this may suggest that in enzymatically-induced DSBs, there is no need for pKu70 where a repair complex may be slightly different to joint blunt ends without any need of end-processing. Effectively, in absence of stress, phosphorylation of Ku70 by cyclinB1/CDK1 in S-M cell cycle phase disrupted Ku dimer formation and subsequent interaction with the origin of DNA replication 60 . At the end of mitosis and in G1, Ku70 became dephosphorylated allowing replication complex formation illustrating thus a cell cycle-dependent function of non-phosphorylated Ku70. Upon DNA damage, cyclin A1/CDK2-dependent phosphorylation of Ku70 stimulated DNA repair with an inhibition of apoptosis 61 . Otherwise, if sustained DSBs in cells expressing ala-Ku70 were not completely repaired and when they maintained ala-Ku70, then repeated systemic activation of NHEJ may be explained by the model proposed by E. Rosenberg and coll 62 .

Ser27-ser33-Ku70 phosphorylation seems to be critical in end resection-dependent process in G1 36 . Effectively, G1 arrested GC92 cells expressing ala-Ku70 displayed a significantly higher excess of -H2AX foci at 2 and 4 hrs post-irradiation; meaning not only that Ku70 was not released but also that NHEJ repair was not achieved. This may suggest that neddylation/ubiquitylation enzymatic complex make the part of DNA repair process. These results (as well as the observed polyploidy in ala-Ku70-expressing cells starting from day 3 post-irradiation, not shown), prompted us to address genome (in)stability in two subsets of Ku70-expressing cells. While U2OS cells are polyploid cells (up to 80 chromosomes) with high level of chromosomal abnormalities, we used non-cancerous mammary epithelium-derived HME cell line 63 . This cell line immortalized by the hTert activation has quasi-normal diploid karyotype enabling thus a relevant assessment of exogenously-induced chromosomal rearrangements. In a multi-FISH approach, ala-Ku70-expressing HME cells irradiated at 4Gy and following first cell division, displayed high number of chromosomal translocations events (Fig6) indicating thus the essential role of pKu70 in maintaining genome stability. Even in absence of exogenous stress ala-Ku70-expressing cells displayed der(13)t(5;13) translocation that was not observed in ser-Ku70-expressing cells. This finding may be suggestive of the role of pKu70 in endogenous-(replication?)-stress-induced DSBs. These results appears to contradict the results previously published 43 where after an estimation of chromosomal structural aberrations in breast cancer cell line ZR75.1, ser-Ku70-expressing but not ala-Ku70-expressing, cells displayed higher level of chromosomal structural aberrations. This discrepancy deserves now further characterizations but it may be due to the differences/relevance in these two methodologies as well as in cell models (i.e. noncancer vs cancer cell lines with different origin/defect). Effectively, in case of cancer cell lines, we observed significant differences between irradiation-induced features of breast cancer and osteosarcoma cell lines (no major cell cycle checkpoint activation in U2OS cells while ZR75.1 exhibited late S/G2 arrest; significantly higher number of DSBs in ser-Ku70expressing ZR75.1 untreated cells while this was not observed in U2OS cells, ect.). More recently, we observed an instability in ala-Ku70 ZR75.1 cells but appearing after several (5) days following irradiation opening the possibility of a delayed appearance of instability. To resolve, at least partly, these at first sight inconsistencies, we started a study of pKu70/Ku70 interactome where anti-Ku70 (clone N3H10) or anti-pKu70 43 antibodies were used to immunopurify the partners preferentially interacting with pKu70. First results obtained with HME and two subsets of CLL cells pointed out already identified partners of Ku70 but also the new cell-type specific candidates (Fig2E and unpublished data). In parallel, mouse KI model expressing human ser-Ku70 or ala-Ku70 has been created. For instance, first results did not reveal a significantly differential radiosensitive phenotype but ala-Ku70-dervied embryonic fibroblasts were defective in growth and underwent high apoptosis rate following irradiation (data not shown). Full characterization of these mice is currently underway. In summary, this work establishes pKu70 as an active effector of cNHEJ, regulating dynamics of its release from the sites of DNA-damage; a process that proved to be critical in the maintaining genome stability. Stimulating HR activity, Ku70 appears as versatile molecule in directing precise DNA repair across cell cycle phases like its HR-specified homolog BRCA1 which is able to modulate resection-dependent cNHEJ in G1 36,64,rev.65 that may be impaired by pKu70 which, in parallel may favor HR in S/G2.

Methods

Cell culture. The cancer cell lines U2OS (osteosarcoma) have been purchased at the ATCC. The U2OS cell line containing reporter system to measure HR 47 efficiency and human fibroblast cell line GC92 containing a reporter system to measure NHEJ 48 were cultured in Dulbecco's modified Eagle's medium (DMEM GlutaMAX, Life Technologies, Thermo Scientific) supplemented with 10% (v/v) fetal bovine serum, 1x non-essential amino acids (Life Technologies, Thermo Scientific). The non-cancerous mammary epithelium-derived HME cell line was a kind gift from R.A. Weinberg lab (Whitehead Institute for Biomedical Research, MIT). HME cell line was additionally supplemented with insulin, EGF and hydrocortisone (all purchased at Sigma-Aldrich). For GC92 cell arrest in G1, cells were treated by mimosine 300µM (Sigma-Aldrich), over-night followed 2h of mimosine-free cell culture before irradiation.

Clonogenic assay and apoptosis. Cells were grown to 70-80% confluency were trypsinized, counted and suspended at cell concentrations of ranging 5 to 40x10 3 cells/mL. Cell suspensions were either non irradiated (0Gy) or irradiated at 2, 4 or 6 Gy. Cells were seeded into 10cm2 cell dishes and left to grow 14 days. For cell death assessing, cells were seeded in duplicate in 24-well plates at 0.03x10 6 (IR) and 0.02x10 6 (NT) cell density. Approximately 4-6h after seeding, cells were irradiated at 2Gy, 4Gy or treated 1h with 100 µM phleomycine (InvivoGen). Cell medium was exchanged immediately after treatment and cells left to grow. At day 3 and 4 cells were trypsinized and cell suspension added to FACS-tubes. After centrifugation, supernatant was removed and cell pellet resuspended in medium containing propidium iodide (1:1000) (Sigma-Aldrich) and DiOC6 (3,3'-Dihexyloxacarbocyanine Iodide) (1:2000) (Thermo Scientific). Data were acquired using a cytometer (FACScalibur, BD Biosciences) Ku70 shRNA/cDNA vectors and transfections. EBV-based vectors were generated as previously described 43 to generate stable cell lines that either express: S27-S33-Ku70, A27-A33-Ku70 or E27-E33-Ku70 (phospho-mimetic). Endogenous Ku70 is repressed using shRNA encoded on the same pEBV-vector (exogenously expressed Ku70 was resistant to shRNA repression). The same vectors were used to generate eGFP-Ku70 (pcDNA-eGFP, Addgene, UK) and mEos-Ku70 (mEos-2 pcDNA, Addgene, UK), fusion proteins. Cell transfection for all vectors was performed the same day, using the same JetPrime DNA transfection kit (Polyplus,-Transfection SA, France) and antibiotic selection conditions (hygromycine or puromycine). As a control for antibiotic selection, non-transfected cells were antibiotictreated and cell death was monitored over time by light microscopy. Successful transfection for the different Ku70 mutants was monitored using SDS PAGE and Western Blot analysis. All stably transfected cell lines were frozen and stored in liquid nitrogen. eGFP-Ku70 and mEos-Ku70 expressing cells were freshly transfected before each live cell acquisition. All cell lines were kept under constant antibiotic selection pressure.

-Irradiation, laser micro-irradiation and real-time life cell imaging. Gamma irradiation was performed using the -irradiator IBL-637 (Cs137). Dose rate used was 4.96 Gy/min for all experiments. For laser micro-irradiation and life cell imaging experiments, U2OS cells expressing eGFP-Ku70 and mEos2-Ku70 fusion proteins (ser-Ku70Ser, ala-Ku70), were transfected a week before each experiment. To stain the nucleus, Hoescht33342 (Sigma-Aldrich) at 1µg/ml was added 30min before laser irradiation by 405nm laser beam (2 seconds of 10% of laser intensity) in a line of 5µ in nuclear region (avoiding nucleoli). Background eGFP-fluorescence intensity was measured before irradiation for each cell. Cells were followed in intervals of 10min up to 240 min after irradiation. Data were acquired using the video-confocal inverted microscope Nikon A1 with integrated chamber assuring 5% CO2 atmosphere and 37°C temperature. Data analysis was conducted using the NIS Elements software (Nikon). For the cell localization analysis of Ku70, PALM acquisitions were performed with the N-STORM (Nikon) system and a SR Apo TIRF 100x (1.49 numerical aperture) oil objective. Images were taken using a TIRF inverted Nikon Eclipse Ti-E microscope, equipped with a quad band emission filter (450/60-525/50-605/50-730/120, Chroma), and coupled to an EMCCD camera (iXon DU897, Andor). Fixed cells expressing mEos2 constructs were used with a sequential activation at 405nm (Cube laser, 100mW, Coherent) and then excitation at 561 nm (Sapphir laser, 150mW, Coherent). NIS-elements AR software (Nikon, v. 4.30.01) and the STORM analysis module (Nikon) was used to control the system and to perform molecule detection. To prevent axial drift we used the perfect focus system (Nikon), and to correct any lateral or axial drift during the acquisition, we apply the auto-correlation algorithm of the NIS-elements AR software (Nikon, v. 4.30.01). Local density analysis was performed using SR-Tesseler software. The local density of the mEos2 molecules were calculated using Voronoi tessellation and then displayed according to a color scale normalized by the mean molecule density of each nucleus.

-H2AX foci/protein levels assessment. Cells were grown on 8-well cover slides (Millicell EZ Slides, Merck-Millipore) for 2 days until 70-80% confluency. Slides were irradiated at 4Gy and left in the incubator for the indicated time points (0.5h, 4h or 24h). After anti-g-H2AX labeling, data acquisition was performed using a microscope (Zeiss, Imager Z2) as previously described 43 . Data analysis was performed using CellProfiler (version 2.2.0, Carpenter lab, free-ware). Graphics and statistical analysis was performed with GraphPad Prism Software (Inc. La Jola, USA). Western blot analysis was performed as described 43 .

Chromatin binding assay. Cells were seeded at until approx. 70% of confluency. For protein extraction the Pre-extraction (PEB) buffer (PIPES 10mM (pH 7.0) , NaCl 100mM, Sucrose 300mM, MgCl 3mM, Triton X-100 0.7%, H2O) was prepared on ice. Cells were washed 3 x with PBS w/o Ca 2+/ Mg 2+ , then PEB-R buffer (PEB buffer, RNAse A 7%) was incubated for 3 min. The supernatant was collected as Fraction 1. The same procedure was repeated once again, to obtain Fraction 2. After twice PBS washing Laemmli-buffer (2x) (100mM Tris HCl (pH 6.8), 10% SDS, 20% Glycerol, 100mM DTT, 1% bromophenol blue, H2O) was added to obtain Fraction 3 (chromatin fraction). DNA was then digested by Benzonase (Merck Millipore). To prepare samples for SDS-PAGE, Laemmli 2x were added to Fraction 1 and 2 and all samples were heated for 5min. at 95•C.

Anti-pKu70/anti-Ku70 protein immunopurifications. Proteins from at least 15x10 6 cells were extracted on ice for 1h in hypertonic buffer containing NaF 50mM, NaCl 450mM, HEPES 20mM, EDTA 0.2mM, DTT 0.5mM, Iodoacetamide 5mM, "Complete" cocktail (Roche Diagnostic) inhibitor of phosphatases (100µL extraction buffer per 10 7 cells). Cell lysates were centrifuged at 67.000rpm for 1h, 4•C. 10% Glycerol and β-Mercaptoethanol (1:1250) were added to the supernatant and divided in 3 parts (two parts for immunoprecipitations and one for input control). To equilibrate salt content, equilibration buffer (EQ-buffer) (KCl 10mM, MgCl 10mM, HEPES 20mM, EDTA 2.5mM, DTT 0.5mM, Iodoacetamide 5mM, Complete cocktail 1x, Glycerol 10%,) was added in a ratio of 1:3 and protein dosage was performed (Bradford method). Dynabeads (BE-M01/03, EMD Millipore) ( 1µL per 10µg total protein) were washed with PBS w/o Ca 2+ /Ca 2+ twice and cell lysate was added to the beads. To obtain a total volume of 500µL EQ-buffer was added. Antibodies for Ku70 (NeoMarkers, Thermo-Fisher) and p-Ku70 43 were mixed to the lysate/Dynabeads mixture at a concentration of 1% of the total protein amount, incubated under agitation overnight at 4°C. Supernatants were discarded and beads washed three times in PBS, before adding EQbuffer. Laemmli-buffer (5x) (0.255M Tris HCl (pH6.8), 50% Gycerol, 5% SDS, 0.05% bromophenol blue, 0.1 M DTT), was added and samples were heated at 95•C for 10 min. Immunoprecipitated proteins were resolved on SDS-PAGE for orbital-based shotgun mass spectrometry according to standardized protocol (Proteomic Center, Rotterdam).

HR and NHEJ activities. The NHEJ-HR efficiency was performed as previously described by using transfected (ser-Ku70, ala-Ku70 or glu-Ku70) U2OS 47 and GC92 cell models 48 . These cells contain a single copy of a HR and NHEJ or NHEJ only reporter substrates, respectively, to measure the efficacy of DNA break repair after I-SCE1 endonuclease expression. Briefly, 72h after transfection with a plasmid coding for I-SCEI, cells were probed for GFP, CD4-and H2Kd-expressions by FACS analysis (FACScalibur, BD Biosciences). Data analysis was performed using FlowJo 5.7. NHEJ and HR efficiency is obtained by subtracting the signal of the control plasmid expressing samples from the I-SCEI expressing samples. H2Kd was used as a control for reporter expression in all transfected cells.

Multi-FISH analysis of chromosomal translocations.

HME mammary epithelial cells (used also in proteomic approach for the analysis of Ku70 interactome), were used to assess chromosomal anomalies following irradiation. Cells were irradiated at 4Gy and and metaphases prepared after 24h. Slides were pre-treated with RNAse A (120µL per slide in 2xSSC buffer (AM9763, Thermo Scientific)) during 45min, 37 °C to eliminate RNA. To remove remaining cytoplasm a pretreatment with pepsine (P6887, Sigma Aldrich) was performed. Slides were incubated for 30sec-1min, 37 °C in 0.01M HCl, pH 2; 2µg/mL pepsin. Slides were washed with PBS w/o Ca2 + /Mg2 + (2x), then with PBS with Ca2 + /Mg2 + for 5min. To fix pepsin treated metaphases, slides were incubated in 1% formaldehyde/ PBS with Ca2 + /Mg2 + for 10min. at RT. Slides were washed with PBS w/o Ca2 + /Mg2 + , 5min. Dehydration was performed in 3 steps, at 70%, 90% and 100% ethanol at RT for 3min. and left to dry for 2-3min. mFISH probe denaturation and hybridization were performed according to the manufacturer's protocol (24XCyte, D-0125-120-DI, MetaSystems). Metaphases were mounted with Flouromount-G aqueous (F4680, Sigmal Aldrich). Data were acquired using the Zeiss Imager Z2 microscope. Data analysis was performed with ISIS software (Metasystem). Whole cell protein extracts of transfected HME cells expressing ser-Ku70 were coimmunoprecipitated, resolved by SDS-PAGE and proteolysed with trypsin. Peptides were analyzed through a Label-free-quantification (LFQ)-approach using an orbitrap-bazed mass spectroscopy analyzer. Corresponding genes symbolized by grey squares are considered with non-significant differences in their affinity towards Ku70 and/or pKu70 whereas these in red squares are considered as more specific partners of total Ku70 or pKu70. after cell treatment with MLN4924 (3µM, 1h) followed by irradiation (4Gy). Actin is used as a loading control.

Figure5

Ser-Ku70 stimulates HR activity in absence of exogenous genotoxic stress. (A) HR activity was measured using the U2OS cell line that expresses the integrated reporter construct pDR-GFP. This cell line was transfected by ser-, ala-or glu-Ku70 vectors as described in Methods section. The reporter is composed of two inactive eGFP genes. The upstream GFP gene is truncated; the gene downstream of the promoter has an integrated I-SCEI cleavage site and is therefore inactive. Upon I-SCEI expression the cleaved GFP gene recombines with the truncated GFP gene on the sister chromatid, resulting in the expression of GFP that was measured by flow cytometry. Results represent values that were calculated as: (I-SCEItransfection events) -(control-transfection events). n= 6. Unpaired t-test, p < 0.01 = *. (B) NHEJ activity was measured using the human fibroblast cell line GC92 (SV40-transformed) that has the pCOH-CD4 (cohesive ends) reporter construct integrated intrachromosomally.. Upon cleavage by I-SCEI a fragment containing the H2Kd and CD8 genes is excised. Rejoining of the flanking ends by cNHEJ brings the pCMV promoter closer to the CD4 gene, which promotes its expression. The CD4 expression on the cell surface was quantified by flow cytometry. Results represent values that were calculated as: (I-SCEI-transfection events) -(control-transfection events). n= 4. Unpaired t-test, p <0.01 = *.

4. 1 V 1

 11 (D)J-Recombination . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Other cellular roles of Ku . . . . . . . . . . . . . . . . . . . . . . . 7.1 Radiosensitivity of LLC samples and classification in R-CLL and S-CLL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Comparative survival proportion of patients diagnosed with R-CLL and S-CLL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 R-CLL derived cells have faster DNA repair kinetics and show chromosomal aberrations compared to S-CLL derived cells . . . . . . . . 7.4 NHEJ activity in R-CLL and S-CLL derived lymphocytes . . . . . . 7.5 Telomere dysfunction with telomere loss is higher in R-CLL than in S-CLL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6 Identification of pS27,S33-Ku70 . . . . . . . . . . . . . . . . . . . . 7.7 Induction of p-Ku70 by genotoxic stress . . . . . . . . . . . . . . . . 7.8 Identified phosphorylation sites of Ku70 . . . . . . . . . . . . . . . Establishment of a working model with the U2OS cell line . . . . . 1.2 Induction of p-Ku70 by different genotoxic stressors . . . . . . . . . 1.3 Cell growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Clonogenic Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 Cell death by apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 Interaction between (phospho-)Ku70 and Bax . . . . . . . . . . . . 1.7 Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.8 Hyperploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.9 Phenotypes of Ku70 mutants . . . . . . . . . . . . . . . . . . . . . . 1.10 γ-H2AX expression . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.11 Co-localization of p-Ku70 to damaged DNA . . . . . . . . . . . . . 1.12 Interaction of phospho-Ku70 in the NHEJ-complex . . . . . . . . . 1.13 Ku70 DNA DSB dissociation kinetics . . . . . . . . . . . . . . . . . 1.14 NHEJ and HR efficiency . . . . . . . . . . . . . . . . . . . . . . . . 1.15 Chromosomal aberrations (U2OS) . . . . . . . . . . . . . . . . . . . 1.16 Chromosomal translocations (HME) . . . . . . . . . . . . . . . . . . 2.1 Humanized KI mouse strategy . . . . . . . . . . . . . . . . . . . . . 2.2 Homozygous KI-Ku70Ser and KI-Ku70Ala mice . . . . . . . . . . . 2.3 Phenotypic characterization of MEF's generated from KI-Ku70Ser and KI-Ku70Ala mouse lines . . . . . . . . . . . . . . . . . . . . . .

  a Ku heterodimer bound to a DNA molecule by Walker et al.[14] in 2001. Walker et al. reveal that Ku70 and Ku80 share a three-domain topology comprising a N-terminal α/β -domain that lies at the periphery of the heterodimer; a central β -barrel core domain forming a cradle for the DNA-binding groove; and a helical C-terminal arm, containing the DNA-PKcs binding element in Ku80 and a DNA-binding SAP domain in Ku70. The three domain topology of the Ku proteins is depicted in Figure 2.1A.

5 - 4 .

 54 2.1B). Using information gained by the crystal structure, the high affinity but sequence non specific binding of Ku to DNA (K d -values: 1.0 x 10 -10 M [19, 20]) is explained by two means: first, polarization of positive electrostatic charge at the inner surface of the Ku heterodimer interacts with the 1.Introduction 8 negatively charged sugar-phosphate backbone of the DNA, and second, the preformation of the heterodimeric ring before DNA is bound.

Figure 2 . 1 :

 21 Figure 2.1: Ku functional domains and interaction with DNA (A) Schematic view of Ku70 and Ku80 functional domains, showing their three-domain topology. (B) Ribbon diagram of the Ku heterodimer formed around DNA (PDB 1JEQ)

Figure 2 . 2 :

 22 Figure 2.2: Functions of Ku70 and Ku80 C-terminal domains (adapted from[24]) (A)Upon DNA breakage Ku gets recruited to the break site, the movable linker of Ku70 extends the SAP domain to the continuous strand of DNA stabilizing the heterodimeric complex, (B) and upon DNA-PKcs recruitment Ku80 extends its Cterminal domain to interact with DNA-PKcs thereby initiating further steps of the repair process

  Figure 3.1).

Figure 3 . 1 :

 31 Figure 3.1: DNA repair mechanisms ( modified from [58]) Different DNA repair mechanisms cope with a plethora of different DNA damages threatening our genome daily. The graph gives a non-exhaustive list of repair factors involved in the particular DNA repair pathways. Highlighted in blue are the so far known double strand break repair pathways. Highlighted in yellow are the single strand damage, repair pathways.

) 62 ]

 62 deals with more bulky lesions, that can distort the DNA structure as a whole and include alterations like pyrimidine dimers (T-T) after UV exposure. The pathway is separated in two sub-pathways, either operating on the whole genome or in transcribed regions. The bulky lesion is excised due to the endonuclease duo XPG and ERCC1/XPF and creates a open stretch of single breaks, which is then filled by DNA replication polymerases [57, Mismatch repair (MMR) deals with base-base mismatches, insertions or deletions of DNA nucleotides that arise during DNA replication or DNA recombination of repetitive sequences. The Mismatch repair homologs MutS and MutL identifythe newly synthesized strand to determine the wrong nucleotide, followed by a exonuclease-induced degradation and DNA resynthesis[START_REF] Hoeijmakers | Genome maintenance for Preventing Cancer[END_REF].Interstrand cross-link (ICL) repair is a complex repair system that relies on different repair factors, typically found in NER, HR or translesion synthesis (a damage tolerance pathway), as well as the fanconi anemia proteins. During the G1-cell cycle phase, some ICLs are processed by the NER. However some crosslinks pass into S-phase where they constitute a replication block, that triggers 1.Introduction 16 replication fork stalling or collapse, generating an one ended DSBs. These are recognized by the Fanconi-anemia proteins, that induce HR mediated replication fork stabilization. Incision on both sides of the ICL by endonucleases unhooks the lesion, and allows strand extension past the damage. Subsequently the unhooked ICL is hydrolysed or excised by NER factors [63]. Double strand breaks (DSBs) Double strand breaks are far less frequent than single strand breaks but can have severe consequences if non-or misrepaired. Non-repaired the cell can lose parts of entire chromosomes during the next cell cycle, most certainly leading to cell death; and if misrepaired they can become a source of genetic mutations, deletions, inversions or translocations threatening genomic integrity and favouring oncogenic transformation. Double strand breaks are triggered from exogenous sources like irradiation and chemotherapeutics, or endogenously by metabolic ROS and replication fork collapse during DNA replication. However, cells also induce double strand breaks intentionally to maintain genetic variety during meiotic recombination or receptor rearrangement during V(D)J-recombination [60, 64-66].No matter if a DSB is desired or not, it can be repaired by a multitude of different repair options. The principal repair strategies being classical-non-homologousend -joining (c-NHEJ) and homologous recombination (HR)[START_REF] Jackson | The DNA-damage response in human biology and disease[END_REF][START_REF] Bhattacharjee | Choices have consequences: the nexus between DNA repair pathways and genomic instability in cancer[END_REF][START_REF] Bétermier | Is non-homologous end-joining really an inherently error-prone process?[END_REF], we now know that two other pathways can act in DNA DSB repair, alternative-NHEJ (a-NHEJ) and single strand annealing (SSA)[START_REF] Brown | Targeting DNA repair in cancer: Beyond PARP inhibitors[END_REF]. Unlike single-strand damages, where the chemical structure of the damage decides what kind of repair pathway is activated, DSB repair relies on the overall cellular situation in which the DSB occurs e.g. cell cycle, meiosis, V(D)J recombination, genomic localization or the availability of necessary repair factors. Homologous Recombination (HR) Homologous recombination is the most conservative double strand break repair pathway, because it uses a homologous sister chromatid for repair. Upon DNA breakage the DNA is resected on both strands leaving 3' overhangs, which can invade the sister chromatid and replicate the aggrieved area of the DNA. HR is highly dependent on the cell cycle and 1.Introduction acts in S/G2, with highest activity in mid S-phase [65, 68] (more details on this pathway in Chapter 3.1.3).

  corresponding ends especially when several DNA DSBs occur in near proximity, thus preventing erroneous joining and translocations. Several factors like Ku, DNA-PKcs and XRCC4/XLF where shown to take part in this process.

5 )

 5 Ku removal from the repaired DNAOne of the unresolved mysteries in c-NHEJ is the removal of Ku from ligated double stranded DNA. Unlike other DNA binding proteins like PNCA, which can be released from DNA due to conformational changes separating its two subunits, both Ku subunits encircle DNA and form a protein-protein interdigitation, which gets trapped on the repaired DNA strand[START_REF] Postow | Ku80 removal from DNA through double strand break-induced ubiquitylation[END_REF]. It seems evident that Ku has to be removed after DNA repair to make place for other DNA repair pathways, and to give access to transcriptional and replicative activity[START_REF] Birthe B Kragelund | The Ku70/80 ring in Non-Homologous End-Joining: easy to slip on, hard to remove[END_REF]. Microirradiation experiments show disappearing Ku signals several hours after DSB induction, but how is it removed, considering the ring shaped structure of both subunits cradling the DNA? Ubiquitination is a post-translational modification associated with proteasomal degradation and protein regulatory function. In studies using Xenopus leavis as a model, Postow et al. discovered that the Skp1-Cul1-F box (SCF) E3 ubiquitin ligase complex accumulates on broken DNA in a Ku dependent manner, where Ku80 becomes highly ubiquitinated (poly K48-ub-chains) leading to its release from the DNA [125, 127]. Interestingly, conjugation of NEDD8 (called neddylation) to the Cullin subunits in the SCF complexes stimulate the SCF ubiquitin-protein ligase activity and Brown et al. show that neddylation is an important step in Ku ubiquitination [126, 128]. However, in contrast to the Postow studies that demonstrate that Ku80 is ubiquitinated independently of accomplished repair, Brown et al. report ubiquitination sites on both subunits of Ku and insist on the release of Ku only after successful DNA repair [125, 127, 128]. Moreover the study of Brown et al. uncovers downregulation of Ku70 specific protein interactors after inhibition of neddylation. One of these interactors is the AAA+-type ATPase VCP/p97, shown to extract trapped Ku from DNA [128, 129]. Another E3-ligase, RNF8, ubiquitinates Ku80 to regulate its release from DNA and successful repair by NHEJ, however Brown et al. could not reproduce retention times of Ku80 on DNA breaks after RNF8 depletion [128, 130]. The above mentioned studies give first insights of the removal of the Ku protein from DNA, but need to be further explored, as it is not clear what mechanism 1.Introduction 23 activates ubiquitination activity on Ku. It has been proposed that Ku changes conformation upon binding to the DNA and that these might unravel Lysine sites

Figure 3 . 2 : 3 . 1 . 2

 32312 Figure 3.2: c-NHEJ: Scheme of the five steps necessary for c-NHEJ

  describes the generation of long 3' ssDNA stretches on both sides of the DSB, which are covered by Rad51 filaments. Initial 5' to 3' end resection is performed by endo-and exonucleases such as MRN (MRE11-NBS1-Rad50)/ CtIP. A more extensive resection is assured due to helicases such as BLM (Bloom) and exonucleases such as Exo1 (Exonuclease 1). The free 3'overhangs are recognized and covered by RPA-filaments, preventing secondary DNA structure generation. Later RPA is displaced by RAD51. Several mediators are known to support RPA exchange by RAD51 such as RAD51B, RAD51C, RAD51D, XRCC3, XRCC2 and BRCA2. Especially BRCA2 with its ssDNA and dsDNA binding motifs and RAD51 binding sites has a prominent role in RAD51 filament nucleation to the 3' filament covered 3' overhang performs invasion and homology search, called synapsis, on the sister chromatid. RAD51 forms a displacement loop (Dloop) stabilized by RAD54, which then displaces RAD51 from the heteroduplex DNA to prime DNA synthesis by the invading strand.The postsynaptic phase summarizes several sub-pathways of HR based on their D-loop resolution and recombination outcomes via either (non-) or crossover mechanisms. These include BIR (Break induced repair), SDSA (synthesisdependent strand annealing) or double Holliday Junction (dHJ) resolution[START_REF] Jasin | Repair of Strand Breaks by Homologous Recombination[END_REF][START_REF] Heyer | Regulation of homologous recombination in eukaryotes[END_REF]. Organisms control the appropriate outcome based on the cellular context, in meiosis for example a cross-over mechanism will be favoured to obtain gene conversion outcomes, whereas in mitosis such mechanisms can lead to loss of heterozygosity (LOH)[START_REF] Ross Chapman | Playing the end game: DNA double-strand break repair pathway choice[END_REF].

Figure 3 . 3 :

 33 Figure 3.3: Homologous recombination The three main phases and the main actors of HR are depicted[START_REF] Heyer | Regulation of homologous recombination in eukaryotes[END_REF][START_REF] Iliakis | Alternative endjoining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: Implications for the formation of chromosome translocations[END_REF]

Figure 3 . 4 :

 34 Figure 3.4: alt-NHEJ Schematic overview of the 5 steps in alt-NHEJ

  cell models after chromosomal breaks induction. Loss of components of the alt-NHEJ however does not reduce chromosomal translocations, underlining the role of c-NHEJ in chromosomal aberrations in humans compared to mice. Findings in murine and human cell systems thus stand in direct opposition to each other, perhaps due to the higher c-NHEJ activity in human compared to murine systems[START_REF] Ghezraoui | Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining[END_REF]. But wasn't c-NHEJ considered as an error-free mechanism? So, how could the first choice repair pathway in humans account for such a dangerous outcome like chromosomal aberrations? Biehs et al.[START_REF] Biehs | DNA double-strand break resection occurs during non-homologous end-joining in G1 but is distinct to resection during homologous recombination in G2[END_REF] provide evidence for a resection dependent (slow) c-NHEJ and a resection independent (fast) c-NHEJ. They argue that Polo-like-kinase3 (Plk3) activated CtIP and Artemis are important factors in resection dependent slow c-NHEJ in G1 and that this type of repair induces MH-mediated repair and chromosomal translocations by c-NHEJ instead of a-NHEJ in human models.

  and genomic alterations. A central role in DNA repair pathway choice is the cell cycle phase, regulating the availability/activity of HR-factors and the sister chromatid during S/G2, as well as Cdk-(cyclin dependent kinase)

Figure 3 . 5 :

 35 Figure 3.5: DNA DSB repair mechanisms and end resection as a central decision point(modified from [65]) Available DNA DSB repair mechanisms in the cell. The main repair strategies consist of c-NHEJ and HR which operate during different cell cycle stages, both are considered as accurate. a-NHEJ represents the most recently discovered repair pathway. It is highly mutagenic and it's role as a dedicated repair pathway is still under debate. End resection stands at the crossroads of these repair pathways.

Figure 3 . 6 :

 36 Figure 3.6: End Resection Coordination of the short and long range end resection by MRN/CtIP and BLM/Exo1, respectively to create long stretches of ssDNA 3' overhangs to initiate repair by HR.

  role in this dynamic interplay between HR and NHEJ.

  Ku as a part of the DDR, having functions in DNA damage signalling, cell cycle, senescence and apoptosis will be evaluated. A graphical outline of the DDR, depicting the central steps and some of its central players is shown in Figure3.7.

Figure 3 . 7 :

 37 Figure 3.7: DNA damage response Scheme of the general outline of the DDR (adapted from [206, 207])

Figure 3 . 8 :

 38 Figure 3.8: ATM as a central player in the DDR ATM activation on the DSB and the coordination of downstream effectors during the DNA damage response coordinate cell fate by activating DNA repair, cell cycle senescence or apoptosis

  cell cycle phase is driven by a particular Cdk/cyclin complex (see Figure 3.9). Cdk's are inhibited by phosphorylation through the kinases Wee1 and Myt1, and activated by the phosphatase-family Cdc25 proteins [226]. The transition between the cell cycle phases is highly controlled and, among others, depends on intact DNA integrity. DNA damage triggers, a transient, reversible block of the cell cycle, termed cell cycle checkpoint, that generates a time window for the repair machinery to fix the broken DNA. Three checkpoints are distinguished, based on the moment between which phases they occur: G1/S-, intra-S-and G2/Mcheckpoint.

Figure 3 . 9 :

 39 Figure 3.9: Cell cycle Oscillating activity of cyclins in complex with Cdks drive the cell cycle (adapted from [227])

Cdk1/cyclinBFigure 3 .

 3 Figure 3.10 C).

Figure 3 . 10 :

 310 Figure 3.10: Cell cycle checkpoints (A) G1/S-checkpoint, (B) intra-S-checkpoint, (C) G2/M-checkpoint (according to [228, 230])

(

  SASP), to attract immune cells for phagocytosis, on the other hand help the organism to prevent malignant transformation and tissue regeneration. Paradoxically senescence is also associated with increased carcinogenesis on an organismal 1.Introduction 45 level, with the accumulation of senescent cells during aging. The constant secretion of inflammatory cytokines, extracellular-matrix degrading proteases and growth factors by senescent cells, disrupts normal tissue structure and stimulates growth and angiogenic activity in potentially premalignant cells in close proximity [241, 242].

Figure 3 . 11 :

 311 Figure 3.11: Damage induced senescence is activated through the DDR and the p53-and p16/RB pathways[START_REF] Rebecca | Cellular Senescence -its role in cancer and the response to ionizing radiation[END_REF]

  cell numbers in multicellular organisms. Inappropriate apoptosis is associated with neurodegenerative diseases, ischemic damage, autoimmune disease or different types of cancers[START_REF] Elmore | Apoptosis: A Review of programmed Cell Death[END_REF]. Therefore it is of no surprise that upon an overwhelming amount of DNA damage apoptosis is considered the prevailing defence mechanism to protect the organism from malignant transformation[START_REF] Elmore | Apoptosis: A Review of programmed Cell Death[END_REF][START_REF] Matt | The DNA damage-induced cell death response: a roadmap to kill cancer cells[END_REF].Morphological changes like chromatin condensation, nuclear fragmentation and collapse of the cytoskeleton, leading to cell blebbing, a phenomenon observable by microscopic examination, accompany apoptosis. During these processes the cell membrane stays intact, but undergoes inner-outer inversion exposing phosphatidylserine to trigger phagocytic clearance by the immune system[START_REF] Bruce | Molecular biology of the cell[END_REF], thus making apoptosis a "silent" death executer.The molecular drivers of apoptosis are called caspases, as they show a cystein in their active site and cleave proteins on specific Asp-residues. Functionally caspases can be divided into initiator caspases (caspase 2, 8, 9, 10) and effector caspases (caspase 3, 7). They are synthesized as non-active pro-caspases (zymogens) that are activated upon cleavage by active caspases, amplifying a proteolytic cascade[START_REF] Matt | The DNA damage-induced cell death response: a roadmap to kill cancer cells[END_REF][START_REF] Bruce | Molecular biology of the cell[END_REF].Two apoptotic pathways are generally distinguished (see Fig 3.12): intrinsic (induced by the depolarisation of the mitochondrial potential) and extrinsic apoptosis (induced by cell death receptors on the cell membrane). Both pathways are activated by DNA damage, they cross communicate and finally lead to the activation of effector caspases that induce cellular decomposition[START_REF] Wynand P Roos | DNA damage and the balance between survival and death in cancer biology[END_REF].The extrinsinc pathway is induced through activation of death receptors (DR) which are part of the tumour necrosis factor receptor (TNFR) super family. The best known death receptors are FASR, TNFR-1, TNF-R2, TRAIL-R1, TRAIL-R2, DR3 and DR6[START_REF] Matt | The DNA damage-induced cell death response: a roadmap to kill cancer cells[END_REF]. All of them contain a cytoplasmic death domain (DD) that upon binding by their cognate/specific ligand initiate the apoptotic cascade.Upon DNA damage, p53 activates the expression of FASR (CD95, DR5) and its ligand FASL (CD95L). Extracellular binding of trimerized FASL clusters FASR 1.Introduction 48 on the cell membrane and their intracellular death domains. Intracellularly this recruits the adaptor protein FADD (Fas-associated death domain), which binds cross-linked CD95-DD, forming the death inducing signalling complex (DISC).

  is driven by the loss of mitochondrial potential after pore formation and results in the release of apoptotic factors. The status quo of the mitochondrial membrane is dependent on the action of the BCL-2 (B cell lymphoma-2) family members that can have either proapoptotic (BAX, BAK, BOK, BID, BAM, BIM, BIK, BMF, NOXA, PUMA and HRK) or anti-apoptotic (BCL-2, BCL-X L and BCL-W) actions. Pro-and antiapoptotic BCL-2 members keep each other in check by mutual sequestration.After exposure to stress the induction of pro-apoptotic factors, for example due to p53 dependent upregulation, results in pore formation of the mitochondrial outer membrane by BAX and BAK, as they can no longer be neutralized by BCL-2[START_REF] Elmore | Apoptosis: A Review of programmed Cell Death[END_REF][START_REF] Matt | The DNA damage-induced cell death response: a roadmap to kill cancer cells[END_REF][START_REF] Wynand | DNA damage-induced cell death: From specific DNA lesions to the DNA damage response and apoptosis[END_REF]. Mitochondrial outer membrane permeabilization (MOMP) enables the release of cytochrome c, SMAC/DIABOLO, AIF, endonuclease G and CAD. Cytochrome c and SMAC/DIABOLO, on one hand, help activate caspase 9, which in turn cleaves procaspases 3 and 7; AIF, CAD and endonuclease G, on the other hand, induce nuclear fragmentation[START_REF] Elmore | Apoptosis: A Review of programmed Cell Death[END_REF].The intrinsic and extrinsic pathways cross-talk through the pro-apoptotic protein BID, which is cleaved to tBID (truncated BID) by caspase 8 and translocates to mitochondria inducing cytochrome c release[START_REF] Li | Cleavage of BID by Caspase 8 Mediates the Mitochondrial Damage in the Fas Pathway of Apoptosis[END_REF]. Additionally caspase 6 activated downstream of mitochondria, may feed back on the extrinsic pathway by cleavage of procaspase 8[START_REF] Fulda | Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy[END_REF]. Caspase 2 is activated through extrinsic and intrinsic signals and is the only caspase that is found in the nucleus in response to DNA damage. 1.Introduction 49 It interacts and gets activated by ATM, ATR and DNA-Pkcs. Beyond its role in apoptosis, caspase 2 is also shown to affect cell cycle checkpoint regulation, NHEJ and genomic stability [261-264].

Figure 3 . 12 :

 312 Figure 3.12: Extrinsic and intrinsic apoptosis pathways and the caspases involved[START_REF] Wynand P Roos | DNA damage and the balance between survival and death in cancer biology[END_REF]

  kinase, an activator of autophagy. Second, nucleation is driven by different phosphatidylinositol 3-kinases (Vps 34,Vps15/p150, Vps14, and Vps30/Atg6 (Beclin1)), that produce the lipid phosphatidylinositol-3-phosphate, the building unit of the autophagosome. Subsequent, vesicle elongation represents the third step and allows the engulfment of cytoplasmic cargo. It is driven by the covalent binding/ decoration of the autophagosome with Atg12, Atg5 and lipidated LC3 (called LC3-II). Following the completion of the autophagic vesicle, it matures through the fusion with lysosomes, leading to the formation of the degradative organelle, called autolysosome[START_REF] Aristides | DNA Damage Response and Autophagy: A Meaningful Partnership[END_REF][START_REF] Hewitt | Repair, Reuse, Recycle: The Expanding Role of Autophagy in Genome Maintenance[END_REF].DNA damage initiates autophagy by several molecular pathways that include the activation of AMPK through ATM and PARP1, transcriptional activation of PTEN, DAPK or DRAM by p53 and p63, and the activation of Beclin1 by

  tophagy protects genomic stability. In first place, this is due to the removal and neutralisation of impaired cellular components like mitochondria, micronuclei or cytoplasmic chromatin fragments, but also the regulation of DNA repair pathways. Autophagy impairs repair by HR by p62-dependent mechanisms that lead to blocked access of HR-proteins to the DNA damage site or the degradation of 1.Introduction 53 RAD51 or filamin A. Additionally the inactivation of autophagy by Atg7 knock out leads to an increased proteasomal degradation of Chk1 and decreased HR activity, making cells hyper-reliant on NHEJ [277].

Figure 3 . 13 : 4 V

 3134 Figure 3.13: Summary of DDR cell fate decisions after irradiation The different alternatives of DNA damage response pathways are depicted. Upon DNA damage cells adapt a determined hierarchy of decision points. Successful repair can resume in normal proliferation. If repair was unsuccessful the cell can either succumb to senescence or apoptosis or resume proliferation.If the acquired damage is lethal, cells undergo mitotic catastrophe, however if it is not lethal cell can continue aberrant cycles of proliferation and increase genomic instability and carcinogenesis. Ku was shown to have multiple roles in the control of these decision points, first of all as a repair protein, but also as a factor regulating proper cell cycle, cell cycle arrest, senescence and apoptosis[START_REF] Gudkov | The role of p53 in determining sensitivity to radiotherapy[END_REF].

  controlled manner, accomplished by the existence of recombination signal sequences (RSS), flanking each of the V, D and J gene segments[START_REF] Schatz | V(D)J Recombination: Mechanisms of Initiation[END_REF] (summarized in Figure4.1 B)). The whole recombination process can be divided into two steps, the cleavage phase and the cNHEJ dependent joining phase[START_REF] Schatz | V(D)J Recombination: Mechanisms of Initiation[END_REF]. During the cleavage phase, two RSS are brought into close proximity, by the RAG (recombination activating gene) 1/2 protein-complex, forming a multi-subunit synaptic complex, called paired complex. RAG1/2 nicks both RSS to induce DNA-SSB and catalyse the coupled cleavage of the RSS by direct transesterification. This ultimately generates a pair of coding ends that terminate with a covalently sealed hairpin structure and a pair of blunt 5' signal ends, representing an excised part of the genomic DNA. The four DNA ends first remain associated to the RAG1/2 protein in a post-cleavage complex (PCC). At the end of the cleavage phase the coding ends are thought to dissociate first from the RAG protein, leaving the signal ends in a RAG associated signal end complex (SEC). This initiates the joining phase, which consists of the DNA DSB recognition, processing and ligation of the rearranged gene fragments. The Ku heterodimer is the first sensor, that recognizes the RAG cleaved DNA structures, thereby displacing the RAG proteins on the signal joint. Ku, also recruits DNA-PKcs and the endonuclease 1.Introduction 57

Figure 4 . 1 : 2

 412 Figure 4.1: V(D)J-recombination mechanism (A) General overview of the combination sequence of the different gene segments V, D and J in gene receptor assembly. (B) Detailed overview of the formation of the recognition of the gene segments by RSS sequences by RAG1/2 and the formation of coding and signal joints[START_REF] Villartay | Congenital defects in V(D)J recombination[END_REF][START_REF] Schatz | V(D)J Recombination: Mechanisms of Initiation[END_REF]

5

 5 ]. XLF and PAXX have redundant functions in V(D)J recombination, however these functions are accomplished through different mechanisms. Finally the study of V(D)J recombination is a elegant model to study the orchestration of cNHEJ factors in the repair of DNA double strand breaks. The knowledge gained by studying this process can certainly, to some extent, be extrapolated to the classical NHEJ reaction during unanticipated DSB induction. Moreover deficiencies and mutations in the core NHEJ factors have clinical importance and have severe consequences for the individual. These patients are predisposed to carcinogenesis, have immunodeficiencies and a high sensitivity to genotoxic agents, which undercuts whatsoever treatment with chemotherapeutics or irradiation. The lifetime expectancy for these patients are generally low, and does often not exceed adolescence. Chapter Other cellular roles of Ku Given the biochemical characteristic of Ku, to bind DNA in a sequence non specific manner with high affinity, it is certainly of no surprise that the DNA-related functions of Ku go beyond DNA DSB repair. Its function thus reaches from replication, to transcription, over mobile genetic element transposition and telomere maintenance. More unexpected are its roles in the DDR where Ku participates in cell cycle regulation, apoptosis and senescence (discussed in Chapter 3.3.3, 3.3.4.1 and 3.3.2). Additionally several enzymatic activities are attributed to Ku, such

Figure 5 . 1 :

 51 Figure 5.1: The many roles of the Ku protein adapted from[START_REF] Downs | A means to a DNA end: the many roles of Ku[END_REF]

  . The association of Ku to the origins of replication is regulated by the periodic phosphorlyation of Ku70 by cyclin-Cdk complexes during S,G2 and M-phase. Phosphorylation of Ku70 on Thr residues 401, 428, 455 and perhaps 58 inhibits the association of the heterodimer to origins during these cell cycle phases. With the 1.Introduction 64 decreasing levels of cyclin B at the end of mitosis the inhibitory phosphorylation

1

 1 Chronic lymphocytic leukaemia (CLL)-an overviewLeukaemia is a type of blood cancer that affects the white blood cells, which accumulate as incompletely matured leukocytes in the bone marrow, lymph nodes and blood stream. Having not passed through the entire maturing process, these white blood cells have restrained ability to defend the organism against pathogens and patients are thus immunodeficient. The accumulation of leukaemia cells furthermore restrains the development of red blood cells and platelets, thus provoking anemia (loss of red blood cells) and thrompocytopenia (loss of platelets). Different types of leukaemia are distinguished based on the involved cell type (lymphoids vs. myeloids) and their aetiopathology (chronic vs. acute). Four major forms of leukaemia are distinguished: acute myeloid leukaemia (AML), acute lymphocytic leukaemia (ALL), chronic myeloid leukemia (CML) and chronic lymphocytic leukaemia (CLL).

  Three different classes of inhibitors are distinguished: BTK-(Burton's Tyrosine kinase), PI3K-(Phsohphatidyl-3-kinase) and SYK-(spleen tyrosine kinase) inhibitors. 1.Introduction 76 The inhibitors ibrutinib (BTK inhibitor) and idealisib (PI3K inhibitor) are approved by the FDA as front line treatment (ibrutinib) and in relapsed disease (idealisib) in combination with ofatumumab or rituximab, respectively. Moreover ibrutinib shows significant improvements of PFS and overall survival in patients with > 65 years of age and del[17p] compared to a treatment with chlorambucil. For the moment SYK-inhibtors show considerable toxicities in phase I and II studies. Side effects for ibrutinib include fatigue, diarrhoea, bleeding ecchymoses, rash, arthralgia, myalgia, increased blood pressure and atrial fibrillation. Adverse effects for idealisib include transaminitis, pneumonitis and colitis.BCL-2 inhibitorThe BH3-mimetic venetoclax inhibits the antiapoptotic BCL-2 protein and thus triggers apoptosis in CLL cells. Its efficiency is proved in patients with relapsed or refractory disease and del[17p] and authorized by the FDA.

  of cell death) at 24h and after up to 10 Gy post-γ-irradiation or are radiosensitive (up to 80-90% of cell death) in vitro[START_REF] Masdehors | Increased sensitivity of CLL-derived lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin[END_REF][START_REF] Delic | The proteasome inhibitor lactacystin induces apoptosis and sensitizes chemo-and radioresistant human chronic lymphocytic leukaemia lymphocytes to TNF-alpha-initiated apoptosis[END_REF] (Figure7.1).

Figure 7 . 1 :

 71 Figure 7.1: LLC samples can be classified based on their response to γ-IR in resistant (R) and sensitive (S) The graph represents data from n=79 samples and n=13 for S-and R-CLL, respectively. (Graph taken from [361])

  Figure 3.8). However, no strict correlation between the R-CLL phenotype and

Figure 7 . 2 :

 72 Figure 7.2: Comparative survival proportion of patients diagnosed with R-CLL and S-CLL The graph represents data from n=11 samples and n=39 for S-and R-CLL, respectively. Bad prognostic markers are defined as: ZAP70, CD38 or sCD23 positivity, unmutated IgV h , cytogenic abnormalities, aberrant karyotype and ATM or p53 mutations (Graph taken from [362])

  Figure 7.3 D clearly depict an increased proportion of chromosome lesions in the R-CLL compared to the S-CLL subpopulation.

Figure 7 . 3 :Figure 7 . 4 :Introduction 81 Figure 7 . 5 :

 73748175 Figure 7.3: R-CLL derived cells have faster DNA repair kinetics and show chromosomal aberrations compared to S-CLL derived cells (A) An example of a comet, with an elongated tail, representing non-repaired SSB and DSB; (B) DNA repair kinetics after irradiation of 5Gy (24h) expressed as the residual damage measured by comet assay;(C) An example of a metaphase spread (D) Number of chromosomal aberrations per 50 metaphase spreads at 48h post-irradiation (Graph taken from [366])

Figure 7 .Introduction 83 Figure 7 . 6 :

 78376 Figure 7.7A,B). Additionally, phosphorylation is enhanced in vivo after classical chemotherapy with fludarabine (cf Figure 7.7C).

1 .Introduction 84 Figure 7 . 7 :

 18477 Figure 7.7: Induction of phospho-Ku70 by genotoxic stress (A) The phosphorylation of Ku is enhanced after irradiation and NCS treatment as soon as after 30min and persists several hours in S-CLL cells. Spots in the non-treated (NT) cells are less strong, indicating less phosphorylation events. (B) Induction of p-Ku70 in healthy B-cells (C) A R-CLL sample before and after treatment with the chemotherapeutic fludarabine, shows induction of p-Ku70 in vivo (Graph taken from [369])

Figure 7 . 8 :

 78 Figure 7.8: Identified phosphorylation sites of Ku70 Five phosphorylation sites on serine residues of the Ku70 protein have been identified and confirmed by site specific methods.

  transfection reagent was prepared as follows: 75µL of JetPrime transfection buffer, 0,75µg of plasmid-DNA and 1,5µL of JetPrime reagent were mixed, vortexed and left at RT for approx. 15 min. During the incubation time the culture medium was changed. After incubation the transfection reagent was quickly vortexed and 75µL of the solution were added dropwise to the cells. As a transfection control a GFP-pEBV vector was used. Transfection efficacy was monitored by GFP flourescence (Zeiss Axiovert 200) and generally reached 40% after 48h. Cells were then trypsinized and 50% re-seeded into a 6-well plate containing medium and appropriate concentration of antibiotics (Puromycine, A1113802, Thermo Scientific, GIBCO or Hygromycine B, 10687010, Thermo Scientific, invitrogen). As a control for antibiotic selection, non-transfected cells and the pEBV-GFP transfected cells were set under antibiotic selection and cell death was monitored over time by light microscopy (Zeiss, Axiovert A1). Generally after several days under antibiotic selection, control cells died, and the antibiotic concentration was decreased to the final concentration in the target cell lines. Successful transfection for the different Ku70 mutants was monitored using SDS PAGE and Western Blot. All stably transfected cell lines were frozen and stocked in liquid nitrogen. GFP-Ku70

3. Materials and Methods 100 1. 4 1 . 5 1 . 6 6 ( 3 .

 1004151663 Cell growthCell growth was assessed for transfected U2OS cells. The cells were grown to[START_REF] Fell | The Ku heterodimer: Function in DNA repair and beyond[END_REF]-80% confluence, trypsinized, counted in a Malassez chamber and seeded in duplicate for non-treated (NT) and irradiated (IR) conditions into 25cm 2 flasks at 0.2*10 6 density. Approximately 4-6h after seeding, cells were irradiated at 2Gy or 4Gy. Cells were left to grow over 7 days. From day 1 to day 7 cell numbers were counted with a Malassez chamber under the light microscope (Leitz, Dialux 20EB). Clonogenic assay Cells grown to 70-80% confluence were trypsinized, counted in a Malassez chamber and suspended at cell concentrations of 5*10 3 cells/mL (NT), 10*10 3 cells/mL (2Gy), 20*10 3 cells/mL (4Gy) and 40*10 3 cells/mL (6Gy). Cell suspensions were either non irradiated (0Gy) or irradiated at 2, 4 or 6 Gy. Cells were seeded in duplicates into 10cm 2 cell dishes. Cells were left to grow 14 days. Cell death and apoptosis using DiOC 6 -IP Cells were grown to 70-80% confluence, trypsinized and counted in a Malassez chamber and seeded in duplicate to 24-well plates at 0.03*10 6 (IR) and 0.02*10 NT) cell density. Approximately 4-6h after seeding, cells were irradiated at 2Gy, 4Gy or treated 1h with 100 µM phleomycine (ant-ph-2p, invivoGen). Cell medium was changed immediately after treatment and cells left to grow. At day 3 and 4 cells were trypsinized and cell suspension added to FACS-tubes. After centrifugation (1200rpm, 5min)(Jouan GR422), supernatant was aspired and the cell pellet resuspended in medium containing propidium iodide (1:1000) (P 4170, Thermo Scientific) and DiOC 6 (3,3'-Dihexyloxacarbocyanine Iodide) (1:2000) (D 273, Thermo Materials and Methods 101 Scientific). Data were acquired using a cytometer (FACScalibur, BD Biosciences).

1

 1 Establishment of a cell model and induction of phospho-Ku70 by different genotoxic stressesTo study the role of the Ku70 phosphorylation on S27, and S33, we generated a working model in the osteosarcoma derived U2OS cell line. Therefore a specific vector plasmid (pEBV) is transfected, that simultaneously represses endogenous Ku70 and expresses a customized exogenous Ku70 (S27-,S33-Ku70 (Ku70Ser); A27-,A33-Ku70 (Ku70Ala); E27-,E33-Ku70 (Ku70Glu) (Figure1.1 A). Phosphorylation of Ku70 is detected using a specifically raised antibody against the phosphorylation containing epitope. As expected the antibody recognizes the phosphorylated form of Ku70 in the U2OS Ku70Ser cell line, whereas no expression is detected in the U2OS Ku70Ala and U2OS Ku70Glu expressing cell lines (Figure1.1 B). Total Ku70 is however expressed in all of the transfected cell lines. We can conclude that all three cell lines express exogenous Ku70 and that endogenous Ku70 is repressed. Expression of phospho-Ku70 is enhanced after irradiation stress (10Gy) after 24h.

Figure 1 . 1 :.Results 115 Figure 1 . 2 :

 1111512 Figure 1.1: Establishment of a working model with the U2OS cell line (A)pEBV vector used to transfect the U2OS cell line. This vector contains a shRNA against endogenous Ku70 and expresses exogenous Ku70 protein with either S27-,S33-Ku70 (Ku70Ser), phosphomutant A27-,A33-Ku70 (Ku70Ala) or phosphomimetic E27-,E33-Ku70 (Ku70Glu). (B) Western Blot after transfection with the pEBV vector shows phosphorylated Ku70 in the Ku70Ser expressing cell line, but not in the phosphomutant or phosphomimetic cell line. However total Ku70 is expressed in all three cell lines. NT: non-treated; IR: irradiated, 10Gy, 24h

Figure 1 . 3 :

 13 Figure 1.3: Cell growth of Ku70-transfected cell lines up to 7 days after irradiation. (A) No growth defects are observed in non-treated cells (B) After 2Gy irradition a growth defect is noted after 4 days, which propagates until day 7 (C) After 4Gy the growth defect is most pronounced. Whereas Ku70Ala and Ku70Glu expressing cells remain at constant cell numbers between day 3 and 7, Ku70Ser expressing cells resume cell growth after day 5. Data represent total cell numbers. Error bars indicate SEM, n=4. Two-way ANOVA, Bonferroni-Test; p < 0.05 = *; p < 0.01 = **; p < 0.001 = ***.

Figure 1 . 4 A

 14 Figure 1.4 A). Nevertheless following irradiation treatment, cell survival decreased further in the Ku70Ala and Ku70Glu expressing cells particularly after 4 and 6 Gy, compared to Ku70Ser (Figure 1.4 B). At 2Gy, 4Gy and 6Gy, approximately 15%,

4 .Results 117 Figure 1 . 4 :

 411714 Figure 1.4: Clonogenic Survival (A) Plating efficiency (PE) for the Ku70 mutants. Error bars indicate SEM, n = 4. Two-way ANOVA, Bonferroni-Test; p < 0.001 = *** (B) Survival fraction of Ku70Ser, Ku70Ala and Ku70Glu cells. PE is standardized to 1 at 0Gy, for each cell line. Data are fit to the linear-quadratic model. Error bars indicate SEM, n = 4. Two-way ANOVA, Bonferroni-Test; p < 0.05 = *; p < 0.001 = ***.

6

 6 and propidium iodide (PI) (see Figure1.5 A). Cell death increased with time and dosis in all cell lines. Initial cell death under NT conditions fluctuates between 5 to 7% at day 3 and 6 to 7% at day 4 in all cell lines. At day 3 after irradiation, the percentage of cell death is about 7%, 11% and 8.5% (2Gy) and 12%, 18% and 14% (4Gy) in Ku70Ser, Ku70Ala and Ku70Glu expressing cells, respectively (Figure1.5 B). Similarly after day 4, cell death increases to 12%, 17% and 14% (2Gy) and 19%, 30% and 23% (4Gy )Ku70Ser, Ku70Ala and Ku70Glu expressing cells, respectively (Figure1.5 D). Similar to the effects seen with irradiation, treatment with phleomycine (100µM, 1h) increases cell death in all cell lines compared to the non-treated conditions. The percentage of cell death is about 15%, 22% and 17% (J3) and 26%, 42% and 31% (J4) for the Ku70Ser, Ku70Ala and Ku70Glu cells, respectively (Figure1.5 C, E).Compared to Ku70Ser expressing cells, Ku70Ala cells are about 40% and Ku70Glu 4.Results 118 cells are about 20% more sensitive to cell death induced by genotoxic agents. About 2/3 of total cell death are due to apoptosis in all cell lines and treatment conditions.

Figure 1 . 5 : 1 . 2 . 4

 15124 Figure 1.5: Cell death by apoptosis (A) A representative plot of cell death and living cells after DiOC 6 -IP staining. The lower right quadrant shows cells that are alive. When losing the mitochondiral outer membrane potential (MOMP) due to apoptosis, cells lose the staining for DiOC 6 (lower left). Dead cells lose their cellular membrane integrity and thus propidim iodide (PI) can accumulate in these cells (upper left). (B) Percent of apoptotic and dead cells on day 3 after NT conditions, 2Gy or 4Gy irradiation. (C) Percent of apoptotic and dead cells on day 3 after Phleomycine treatment (1h). (D) Percent of apoptotic and dead cells on day 4 after NT conditions, 2Gy or 4Gy irradiation. (E) Percent of apoptotic and dead cells on day 4 after Phleomycine treatment (1h). Data represent percent of apoptotic and dead cells. Error bars indicate SEM, n > 6. Two-way ANOVA, Bonferroni-Test; p < 0.05 = *; p < 0.01 = **; p < 0.001 = ***

Figure 1 . 6 :

 16 Figure 1.6: Interaction between (phospho-)Ku70 and Bax Coimmunoprecipitation with Bax, reveals an interaction of Bax with phospho-Ku70, nevertheless the phosphorylation is not specific for the phospho-Ku70 as all Ku70 mutants do interact with Bax similarly.

Figure 1 . 7 :

 17 Figure 1.7: Cell cycle after irradiation (A)Cell cycle profiles for the Ku70Ser, Ku70Ala and Ku70Glu at the indicated time points before or after γ-irradiation (2Gy).(B) Comparative analysis of the percentage of cells in the G1-, S-and G2-phase for Ku70Ser, Ku70Ala and Ku70Glu. Error bars indicate SEM, n = 4. Two-way ANOVA, Bonferroni-Test; non-significant. (C) Cell cycle profiles for the Ku70Ser, Ku70Ala and Ku70Glu at the indicated time points before or after γ-irradiation (4Gy). (D) Comparative analysis of the percentage of cells in the G1-, S-and G2-phase for Ku70Ser, Ku70Ala and Ku70Glu. Error bars indicate SEM, n = 3. Two-way ANOVA, Bonferroni-Test; nonsignificant. (E) Time-dependent accumulation of hyperploid cells in Ku70Ser, Ku70Ala and Ku70Glu cells.

ure 1 .

 1 7 E).After irradiation, cells have a limited time window to repair the acquired damage before resuming the cell cycle. Misrepaired or persistent DSB can be a source of aneuploidy (hyperploidy) and genomic instability during transition from interphase to mitosis[START_REF] Hoeijmakers | Genome maintenance for Preventing Cancer[END_REF]. As a consequence, these cells can either develop a mutator phenotype and further drive genomic instability or undergo cell death via mitotic catastrophe[START_REF] Gudkov | The role of p53 in determining sensitivity to radiotherapy[END_REF].To further pursue this observation, we assessed the cell cycle over 7 days after 4 Gy γ-irradiation and measured the amount of cells that accumulate with n>4 (see Figure1.8 A). As depicted in Figure1.8 B the amount of hyperploidy in nontreated cells is stable at 0.5-2%. After γ-irradiation the amount of hyperploid cells increases in all cell lines. Nevertheless the Ku70Ala cells accumulate significantly more (60-80%) hyperploid cells than the Ku70Ser expressing cells (12% vs. 7% at day 7). Ku70Glu expressing cells accumulate approximately 20% more hyperploid cells (9% vs. 7% at day 7). At the same time the mortality in the Ku70Ala expressing cells increases, suggesting that these cells are genomically unstable and undergo mitotic catastrophe (Figure1.8 C).

4 .Results 122 Figure 1 . 8 :

 412218 Figure 1.8: Hyperploidy (A)Cell cycle profiles that show the accumulation of hyperploid cells (n>4) from day 3 to 7, after γ-irradiation (4Gy) (B) Percentage of hyperploid cells before and after irradiation (C) Percentage of cell survival before and after irradiation. Error bars indicate SEM, n = 2. Two-way ANOVA, Bonferroni-Test; p < 0.05 = *; p < 0.01 = **; p < 0.001 = ***.

Figure 1 . 9 :

 19 Figure 1.9: Phenotypes of Ku70 mutants Summary of the cell line characteristics after irradiation at 2 and 4Gy.

4 .Results 126 Figure 1 .

 41261 Figure 1.10: γ-H2AX expression (A) Representative pictures of γ-H2AX foci in nontreated and irradiated cells (4Gy) at several time points (0.5h, 4h, 24h).Nuclei are stained with Hoechst 33342 (blue), γ-H2AX with AlexaFlour 488 (green). Scale bar: 10µM.(B) Results after automated quantification of γ-H2AX foci. Cells were scored positive, when at least 3 foci where detected. Per cell line at least 800 cells have been evaluated. Graphs represent data as boxplots with median, 1st and 3rd quartile, outliners are shown as dots, n= 4. Unpaired t-test, p < 0.001 = ***. (C) Western Blot of γ-H2AX after irradiation (4Gy). Actin is used as a loading control. p53 is stabilized in both cell lines after irradiation. One representative western blot is depicted. n= 2.

(see Figure 1 .Results 127 Figure 1 . 11 :

 1127111 Figure 1.11: Co-localization of p-Ku70 to damaged DNA (A)Representative western blot of a chromatin binding assay (B) Quantification of the chromatin binding assay. n = 4. One-way ANOVA, p < 0.0001 = ****. (C) Immunoflourescence after microirradiation. Red, γ-H2AX. Green, phospho-Ku70. Blue, Hoechst 3342, scale bar: 10µM.

4 .Results 128 Figure 1 . 12 :

 4128112 Figure 1.12: Interaction of phospho-Ku70 in the NHEJ-complex (A) Coimmunoprecipitation in U2OS cells shows that phospho-Ku70 interacts with Ku80, Ligase 4 and PAXX. (B) Similarly Ku70 mutants show interaction with Ku80, Ligase 4 and PAXX. (C) Whole cell protein extracts of transfected HME Ku70Ser (HME 2464) cells were co-immunoprecipitated, resolved by SDS-PAGE and digested with trypsin. Peptides were analysed through a label-free-quantification (LFQ) -approach using an orbitrap mass analyzer.

  Figure 1.13 C depicts Ku70Ser and Ku70Ala dissociation kinetics after treatment with 3 µM MLN 4924 (1h). Maximal recruitment of Ku70Ser (6.4-fold) and Ku70Ala (6.1-fold) after MLN 4924 treatment are approximately 2-fold compared to conditions without MLN 4924. Dissociation of Ku70Ser and Ku70Ala is severely hampered in both cell lines, with only 20-25% of Ku70 dissociated at 240min. Inhibition of neddylation dependent ubiquitination thus blocks the release of Ku70Ser and Ku70Ala in a similar manner. Moreover the DNA repair of DNA DSB, measured by γ-H2AX resolution is equally inhibited in Ku70Ser and Ku70Ala cells after inhibition of neddylation (see Figure 1.13 D) compared to non-treated conditions where γ-H2AX disappears faster in Ku70Ser cells (see Figure 1.10 C).

Figure 1 . 13 :

 113 Figure 1.13: Ku70 DNA DSB dissociation kinetics (A) Representative pictures of dissociation kinetics in GFP-Ku70Ser and GFP-Ku70Ala cells. Pictures depict cells before(NT), shortly after (t0) and at three time points after microirradiation (405nm). White arrows point to the damage site. (B) Relative intensity fold change based on the initial fluorescence intensity in NT cells. n=20 (Ku70Ser); n=27 (Ku70Ala). Unpaired t-test, p < 0.05 = *. Scale bar: 10µM. (C) Relative intensity fold change based on the initial fluorescence (NT) after treatment of the cells with the NEDD8 inhibitor MLN 4924 (3µM, 1h before microirradiation). n= 19 (Ku70Ser); n= 11 (Ku70Ala), Unpaired t-test, non-significant. (D) Representative Western Blot depicting γ-H2AX after pre-treatment with neddylation inhibitor MLN4924 (3µM, 1h) and irradiation (4Gy). Actin was used as a loading control.

  us to further characterize the quota of genomic instability at the chromosome level. Irradiation can induce chromosomal aberrations like dicentrics, acentrics, ring structures or chromosomal translocations when DSB are left unrepaired and/or rejoined erroneously.

4 .Results 135 Figure 1 . 14 :

 4135114 Figure 1.14: NHEJ and HR efficiency (A) NHEJ activity was measured using the human fibroblast cell line GC92 (SV40-transformed) that has the pCOH-CD4 (cohesive ends) reporter construct integrated intrachromosomal Upon cleavage by I-SCEI a fragment containing the H2Kd and CD8 genes is excised. Rejoining of the flanking ends by cNHEJ brings the pCMV promoter closer to the CD4 gene, which promotes its expression. The CD4 expression on the cell surface can be detected via a fluorescence labelled CD4-antibody and flow cytometry. Results represent values that were calculated as: (I-SCEI-transfection events) -(control-transfection events). n= 4. Unpaired t-test, p < 0.01 = *. (B) Left: Representative images of γ-H2AX foci in Ku70Ser and Ku70Ala expressing cells after synchronization in G1. Right: Results after automated quantification of γ-H2AX foci. Cells were scored positive, when at least 3 foci where detected. Per cell line at least 50 cells have been evaluated. Graphs represent data as boxplots with median, 1st and 3rd quartile, upper and lower extreme whiskers at 95 and 5 percentiles, outliners are shown as dots, n= 2. Unpaired t-test, p < 0.001 = **. Scale bar: 10 µM (C) HR activity was measured using the U2OS HREJ cell line that expresses the integrated reporter construct pDR-GFP. The reporter is composed of two inactive EGFP genes. The upstream GFP gene is truncated, the gene downstream of the promoter has an integrated I-SCEI cleavage site and is therefore inactive. Upon I-SCEI expression the cleaved GFP gene recombines with the truncated GFP gene on the sister chromatid, resulting in the expression of GFP that can be measured via flow cytometry. Results represent values that were calculated as: (I-SCEI-transfection events) -(control-transfection events). n= 6. Unpaired t-test, p < 0.01 = *.

136 Figure 1 . 15 :

 136115 Figure 1.15: Chromosomal aberrations (A) Representative picture of a metaphase in non-treated conditions. (B) Representative metaphase of γ-irradiated (2Gy) cells (48h). Arrow indicates a dicentric chromosome.(C) Numbers of unrepaired chromosomal DSB during the first metaphase (48h) after irradiation (2Gy). (D) Numbers of counted chromosomal aberrations and extrapolation to 100 metaphases for each cell line.

Figure 1 . 16 :

 116 Figure 1.16: Chromosomal translocations (A) Western Blot showing the HME cell lines after transfection with pEBV vector, and verification of phospho-Ku70 expression.(B) The number of chromosomal translocations/metaphase is higher in Ku70Ala compared to Ku70Ser cells. (C) Examples of 4 metaphases analyzed. NT Ku70Ser: 46, XX, ins(1), ins(8), -10, del[START_REF] Reeves | Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen[END_REF], der(18)t(11;18), +20, der(22)t(10;22)[START_REF] Yaneva | Interaction of DNA-dependent protein kinase with DNA and with Ku-biochemical ans atomic force microscopy studied.pdf[END_REF] ; NT Ku70Ala: 46,XX, ins(1), ins(8), -10, del[START_REF] Reeves | Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen[END_REF], der(18)t(11;18), +20, der(22)t(10;22), der(13)t(5;13;13)[START_REF] Downs | A means to a DNA end: the many roles of Ku[END_REF]; after irradiation only those translocations that are distinct compared to the nontreated condition have been counted. Other markers of genomic instability: dmin, double minute; del, deletion; ace, acentric fragment; C-Frag, chromosomal fragment; mar, marker

Figure 2 . 1 :

 21 Figure 2.1: Humanized KI mouse strategy (A) Comparison of the protein sequences (aa 1-60/58) in human and mice for the Ku70 protein. Yellow underlined aa are the serine residues 27 and 33 in human and the corresponding aa 25 (glycine) and 31(serine)in mice. The residues underlined in pink are the additionally mutated residues in mice to obtain a humanized protein sequence, surrounding the phosphorylation sites. The sequences in blue (Ku70Ser) and red (Ku70Ala), correspond to the final aa sequence of the Ku70 protein in each of the KI models, respectively. (B) Generation of a KI model. With the help of a targeting vector, containing the nucleotide sequence corresponding to the substituted aa, mouse embryonic stem cells were transfected. Selection with a antibiotic resistance and subsequent PCR analysis revealed successfully recombined ES cells. These could be transferred into an empty donor blastocyst and carried out by a surrogate mother mouse. The born chimeras are composed of cells, from the recombined ES cells and from the donor blastocyst. To select mice that have integrated the KI in their reproductive organs, they were backcrossed with the wt-mouse to give heterozygous progeny (F1). Crossing of the F1 generation can ultimately produce homozygous mice that express KI-Ku70Ser or KI-Ku70Ala on both gene alleles.

Figure 2 . 2 :

 22 Figure 2.2: Homozygous KI-Ku70Ser and KI-Ku70Ala mice (A) Picture of the established humanized KI-Ku70Ser and KI-Ku70Ala KI mouse lines. (B) Comparison of weight [gram] of male and female homozygous KI-Ku70Ser and KI-Ku70Ala mice; n=3.

143 Figure 2 . 3 :

 14323 Figure 2.3: Phenotypic characterization of MEF's generated from KI-Ku70Ser KI/KI and KI-Ku70Ala KI/KI mouse lines (A) Cell growth is impaired in KI-Ku70Ala KI/KI compared to KI-Ku70Ser KI/KI MEFs, independent of genotoxic stress. One representative experiment is depicted. (B) Cell death is impaired for KI-Ku70Ala KI/KI compared to KI-Ku70Ser KI/KI expressing cells in non-treated conditions and at 2Gy. n=3.Unpaired t-test, p < 0.01 = *, p < 0.001 = **, p < 0.0001 = ***.

Figure 6 :

 6 Figure 6: Synchronization of GC92 cells in G1 Cells were synchronized ON with 300µM of mimosine.

Figure 7 :

 7 Figure 7: Nuclear localization (A) U2OS-mEos-Ku70Ser cells show a clear compartmentalization (B) U2OS-mEos-Ku70Ala cells show compartmentalization (C) U2OS-mEos-Ku70Ser cells show no compartmentalization

Figure legends

 legends Figure legends

Figure2Ser-

  Figure2Ser-Ku70 preferentially localizes in nucleoli, co-localizes with -H2AX at sites of DNA DSBs and interact with core components of NHEJ complex. (A). (Upper panel), Cells were transfected with mEos2-ser-Ku70, mEos2-ala-Ku70 or mEos2-glu-Ku70 vectors and 48h after the florescence was examined by photo-activated localization microscopy (PALM). Scale bar =5µ. (Lower panel), Local density analysis was also performed on the PALM data to characterize the different patterns of Ku70 nuclear distribution. (B) Cells were laser microirradiated (as described in Method section). Anti-pKu70 (b, green) and anti--H2AX (c, red) immunostaining was performed as described. Hoecst 33342 was used for chromatin-DNA labelling (a), overlay (d). (C) (a) Representative Western blot of a chromatin binding assay U2OS cells expressing ser-Ku70 were untreated (NT) or treated with indicated concentrations of DSBs-inducer Phleomycine. Two soluble fractions (F1 and F2) and chromatin fraction (F3, see Methods section) were probed for pKu70, Ku70 and -H2AX contents. (b) Quantification of the chromatin binding assay. n = 4. One-way ANOVA, p < 0.0001 = ****. (D) (a) Co-immunoprecipitation shows that pKu70 interacts with Ku80, Ligase

4

  and PAXX. (b) Similarly Ku70 mutants show interaction with Ku80, Ligase 4 and PAXX. (E)

Figure3

  Figure3Kinetic of DSBs repair is accelerated in unsynchronized and G1 arrested cells expressing pKu70. (A panel) Representative images of -H2AX foci in untreated and irradiated cells (4Gy) at several time points (0.5h, 4h, 24h) were shown. Nuclei are stained with Hoechst 33342 (blue), γ-H2AX with AlexaFlour 488 (green). Scale bar=5µm. (B) Results after automated quantification of -H2AX foci (CellProfiler). Cells were scored positive, when at least 3 foci where detected. At least 800 cells per cell line have been evaluated. Graphs represent data as boxplots with median, 1st and 3rd quartile, outliners are shown as dots, n= 4. Unpaired t-test, p < 0.001 = ***. (C) GC92 cells were untreated (dotted blue line) or treated by mimosine (300µM overnight (red line), fixed and the cell cycle verified by flow cytometry. (D) -H2AX persistence in G1 after mimosine treatment. Representative images of -H2AX foci in ser-Ku70 and ala-Ku70A-expressing cells before (NT) and 2 and 4h after 4Gy-irradiation were shown.(E) -H2AX foci were scored as in (B). Graphs represent data as boxplots with median, 1st and 3rd quartile, upper and lower extreme whiskers at 95 and 5 percentiles, outliners are shown as dots, n= 2. Unpaired t-test, p < 0.001 = **. Scale bar: 10 µm.

Figure4 pKu70 confers more

  Figure4pKu70 confers more rapid dissociation from the sites of DNA-damage: inhibition of neddylation sustains both pKu70 release and DSBs following micro-irradiation. Ku70 DNA DSB dissociation kinetics (A) Representative images of dissociation kinetics in GFP-Ku70Ser and GFP-Ku70Ala cells. Pictures depict cells before (NT), shortly after (t0) and at three time points after micro-irradiation (405nm). White arrows point to the damage site. (B) Relative intensity folds change based on the initial fluorescence intensity in NT cells. n=20 (ser-Ku70); n=27 (ala-Ku70). Unpaired t-test, p < 0.01 = *. Scale bar: 10µM. Western Blot of γ-H2AX after cell treatment with MLN4924 (3µM, 1h) followed by irradiation (4Gy). Actin is used as a loading control.

Figure6 pKu70 promotes genome

  Figure6 pKu70 promotes genome stability after genotoxic stress. HME cells expressing ser-Ku70 or ala-Ku70 forms were irradiated at 4Gy and 24h after metaphases were probed for chromosomal aberrations by multi-FISH approach. Examples of 4 representative metaphases are shown. (Upper panel, left) untreated (NT) ser-Ku70 cells showing ins(1), ins(8), -10, del(11), der(18)t(11:18), +20, der(22)t(10;22); (Upper panel, right) ala-Ku70 cells showing the same aberrations as in left panel with an additional der(13)t((5;13) translocation. After irradiation (lower panels) only new translocation events (compared to untreated conditions), were taken into account. Other markers of genome instability : dmin (double minute); del (deletion); ace (acentric fragment); C-Frag (chromosomal fragment); mar (marker), are shown.

Fig 1 A

 1 Fig 1 A

Fig. 1B

  Fig. 1B

Fig. 1C

  Fig. 1C

  2.1 Ku functional domains and interaction with DNA . . . . . . . . . . 2.2 Functions of Ku70 and Ku80 C-terminal domains . . . . . . . . . .

3.1 DNA repair mechanisms . . . . . . . . . . . . . . . . . . . . . . . . 3.2 c-NHEJ: 5 steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Homologous Recombination . . . . . . . . . . . . . . . . . . . . . . 3.4 alt-NHEJ: 5 steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 DNA DSB repair mechanisms . . . . . . . . . . . . . . . . . . . . . 3.6 End Resection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 DNA damage response . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 ATM in the DDR . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 Cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.10 Cell cycle checkpoints . . . . . . . . . . . . . . . . . . . . . . . . . . 3.11 Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.12 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13 DDR cell fate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Table 2 .1: X-ray cross complementation (XRCC) gene

 2

	names and DNA repair

DNA is the essential molecule of live carrying genetic information in all kinds of living organisms. It is transmitted from one generation to another and encodes

Table 6 . 1 :

 61 Ku expression in cancer tissue samples

	Phenotype

  These include genetic, molecular and biochemical characteristics of the CLL cell, such as: molecular cytogenetics, mutation status of the Ig heavy chain locus (IgV h ), several serum markers and bone marrow infiltration [355].

	1.Introduction	73
	[353, 355]. • Molecular cytogenetics: Approximately 80% of CLL patients carry at
	least one of the following chromosomal aberrations: [del(13q)] in 55% of CLL;
	[del(17p)] in 7%; [del(11q)] in 18% and trisomy 12 in 16% [353]. [del(11q)]
	or [del(17p)] are frequently associated with the loss of one allele of the tu-
	mour suppressor genes ATM or TP53 (encoding p53), respectively and poor
	medical response to chemotherapeutics [356, 357]. These patients have an
	inferior prognosis and overall survival compared to [del(13q)] or B-CLL with-
	out chromosomal abnormalities [355].

• Mutation status of the Ig heavy chain locus (IgV h ): CLL patients with unmutated IgV h have an inferior prognosis than mutated IgV h . However usage of the V h -gene 3.21 is an unfavourable prognostic marker independent of the IgV h mutation status. The expression of the surface markers ZAP70 and CD38 is correlated with an unmutated IgV h and thus is associated with unfavourable prognosis

[355]

.

Table 7 . 1 :

 71 Rai and Binet staging system

	System Risk group	Clinical features	Median life ex-
					pectancy
		Low risk (Rai	Lymphocytosis without cytope-	13 years
		stage 0/I)		nia and without lymphadenopa-
				thy and/or splenomegaly
		Intermediate	Lymphocytosis without cytope-	8 years
	Rai	risk (Rai stage II)	nia, but with lymphadenopa-thy and/or splenomegaly and/or
				hepatomegaly
		High risk (Rai	Lymphocytosis and cytopenia	2 years
		stage III/IV)	(Hb level of ≤ 11 g/dL and/ or a
				platelet count of ≤ 10x10 5 cells/
				µL )
		Low risk (Binet	Less than three palpably en-	13 years
		stage A)		larged sites 1 without cytopenia
		Intermediate	Three or more palpably enlarged	8 years
	Binet	stage B) risk	(Binet	sites without cytopenia
		High risk (Binet	Cytopenia (Hb level of ≤ 10	2 years
		stage C)		g/dL and/ or a platelet count of
				≤ 10x10 5 cells/ µL ))

Table 1 . 1 :

 11 Cell Culture Conditions

	Cell line	Culture Media	Culture Condi-
			tions
	U2OS	DMEM Glutamax, 10 % Fetal bovine serum, 1x	37 • , 5 % CO 2
		Antibiotic-Antimycotic
	U2OS	DMEM Glutamax, 10 % Fetal bovine serum, 1x	37 • , 5 % CO 2
	HREJ	Antibiotic-Antimycotic
	GC92	DMEM Glutamax, 10 % Fetal bovine serum, 1x	37 • , 5 % CO 2
		Antibiotic-Antimycotic
	ZR75.1	DMEM, 5 % Fetal bovine Serum, 1x Antibiotic-	37 • , 5 % CO 2
		Antimycotic
	HME	DMEM/F12+ Glutamax, 5 % Fetal bovine serum,	37 • , 5 % CO 2
		1x Antibiotic-Antimycotic, Insulin 10µg, Hydro-
		cortisone 0.5µg/mL, EGF 10 ng/mL,

Table 1 .

 1

	2: pEBV transfection conditions
	Cell line	Vector (Vector No.)	Antibiotic	Selection	Final con-
				concentra-	centration
				tion
	U2OS	Ku70Ser (2103)	Hygromycine 125µg/mL 125µg/mL
	U2OS	Ku70Ala (2104)	Hygromycine 125µg/mL 125µg/mL
	U2OS	Ku70Glu (2106)	Hygromycine 125µg/mL 125µg/mL
	U2OS	GFP-Ku70Ser(3452)	Hygromycine 125µg/mL 125µg/mL
	U2OS	GFP-Ku70Ala (3453) Hygromycine 125µg/mL 125µg/mL
	U2OS	GFP-Ku70Glu (3455) Hygromycine 125µg/mL 125µg/mL
	U2OS HREJ	Ku70Ser (2103)	Hygromycine 200µg/mL 125µg/mL
	U2OS HREJ	Ku70Ala (2104)	Hygromycine 200µg/mL 125µg/mL
	U2OS HREJ	Ku70Glu (2106)	Hygromycine 200µg/mL 125µg/mL
	GC92	Ku70Ser (2464)	Puromycine	0.4µg/mL	0.4µg/mL
	GC92	Ku70Ala (2467)	Puromycine	0.4µg/mL	0.4µg/mL
	GC92	Ku70Glu (2468)	Puromycine	0.4µg/mL	0.4µg/mL
	ZR75.1	Ku70Ser (2103)	Hygromycine 200µg/mL 125µg/mL
	ZR75.1	Ku70Ala (2104)	Hygromycine 200µg/mL 125µg/mL
	ZR75.1	Ku70Glu (2106)	Hygromycine 200µg/mL 125µg/mL
	HME	Ku70Ser (2464)	Puromycine	0.4µg/mL	0.2µg/mL
	HME	Ku70Ala (2467)	Puromycine	0.4µg/mL	0.2µg/mL
	HME	Ku70Glu (2468)	Puromycine	0.4µg/mL	0.2µg/mL
	Control	GFP-control (1486)	Blasticidine
	Control	GFP-control (1675)	Hygromycine

Table 1 .3: Antibodies

 1

	Type	Target	Species	Dilution	Supplier (Reference)
		(clone)
		phospho-	mouse	1:1000 (5%BSA	BioGenes GmbH
		Ku70		/TBS-T)
		(p-Ku70)
		Ku70	mouse	1:1000 (5%BSA	Thermo	Scientific
		(N3H10)		/TBS-T)	(MA5-13110)
		Ku80	mouse	1:1000 (5%BSA	Thermo	Scientific
				/TBS-T)	(MS-285)
	Primary
		Ligase 4	rabbit	1:1000 (5%BSA	Proteintech (12695-1-
	Antibodies
				/TBS-T)	AP)
		Bax	rabbit	1:1000 (5% milk	Millipore (06-499)
				/TBS-T)

Table 1 .3: Antibodies

 1

	Type	Target	Species	Dilution	Supplier (Reference)
		(clone)
		PAXX		1:2000 (5% milk	abcam (ab126353)
				/TBS-T)
		γ-H2Ax	rabbit	1:1000 (5% milk	Cell Signalling (9718)
		(Ser139)		/TBS-T)
		(20E3)
		anti-HA-	mouse	1:1000 (5% BSA	Biolegend (901513)
		Tag (clone		/TBS-T)
		16B12)
		β-Actin	mouse	1:3000 (5% BSA	Sigma	Aldrich
				/TBS-T)	(A2228)
		α-Tubulin mouse	1:2000 (5% BSA	Sigma	Aldrich
				/TBS-T)	(T6199)
	Secondary	anti-rabbit goat	1:30000	Thermo Scientific (A-
	Antibodies				11001)
		anti-mouse goat	1:30000	Thermo Scientific (A-
					11034)

Table 1 .4: Software

 1

	Application	Name, Version	Company
	Western blot quantification imageJ, Version	freeware
		1.45 S
	DNA quantification	Nanodrop2000	Thermo Scientific
	Plasmid digestion	Sequencher 5.1	Gene Codes Coopera-
		Demo		tion
	Live microscopy quantifica-	NIS Elements,	Nikon
	tion	Version 4.30
	Flow cytometry	FlowJo, Version	FlowJo, LLC
		7.6.5
	Cell death assay (DiOC 6 -	R,Version 3.3.1	freeware, script: Allan
	IP)			Sauvat
	Shotgun Proteomics	Perseus, Version	Max-Planck Institute
		1.5.6.0		of Biochemistry, free-
				ware, script: H.W.J
				Rijkers
	Metaphase analysis, multi-	ISIS,	Version	MetaSystems
	FISH	5.7.1
	γ-H2AX foci count	CellProfiler,	Carpenter Lab, free-
		Version 2.2.0	ware, script: Vincent
				Paget
	1.23 Statistics
	Statistical analysis was performed with GraphPad Prism Software. Statistical
	tests are specified underneath each Figure.

Light Microcopy Facility, Imagerie-Gif, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198, Gif-sur-Yvette cedex, France

DSBs : functional link to genome stability (En préparation)

Acknowledgements

Acknowledgements AS benefited fellowship from French Ministary of Research and Technology (MRT) and CEA. This work was supported by Fondation de France, CEA and EdF.

The present work has benefited from the light microscopy facility of Imagerie-Gif, (http://www.i2bc.paris-saclay.fr), member of IBiSA (http://www.ibisa.net), supported by "France-BioImaging" (ANR-10-INBS-04-01), and the Labex "Saclay Plant Science" (ANR-11-IDEX-0003-02).

Part IV

Results

Part V

Discussion

Part VI Conclusion & Perspectives