In eukaryotes, the repair of DSBs is accomplished through two broadly defined processes: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). The central step of HR is pairing and exchange of strands between two homologous DNA molecules, which is catalyzed by the conserved Rad51/RecA family of proteins. Prior to this step, an essential step in all HR pathways i.e. 5'→3' resection of broken DNA ends to generate 3' single stranded DNA tails. At the molecular level, initiation of DNA end resection is accomplished through the concerted action of MRX complex (Mre11, Rad50 and Xrs2) and Sae2 protein. To elucidate the molecular basis underlying DSB end resection in S. cerevisiae mre11 nuclease deficient mutants, we have performed a comprehensive analysis of the role of S. cerevisiae Mre11 (henceforth called as ScMre11) in the processing of DSB ends using a variety of DNA substrates. We observed that S. cerevisiae Mre11(ScMre11) exhibits higher binding affinity for single- over double-stranded DNA and intermediates of recombination and repair and catalyzes robust unwinding of substrates possessing a3' single-stranded DNA overhang but not of 5' overhangs or blunt-ended DNA fragments. Furthermore, reconstitution of DSB end resection network in-vitro revealed that Rad50, Xrs2, and Sae2 potentiated the DNA unwinding activity of Mre11. Since the exonuclease activity of Mre11 is of the opposite polarity to that expected for resection of DSBs, unwinding activity of Mre11 in conjunction with Rad50, Xrs2, and Sae2 might provide an alternate mechanism for the generation of ssDNA intermediates for DSB end repair and HR. Additionally, ScMre11 displays strong homotypic as well as heterotypic interaction with Sae2. In summary, our results revealed important insights into the mechanism of DSB end processing and support a model in which Sae2, Rad50, and Xrs2 positively regulate the ScMre11-mediated DNA unwinding activity via their direct interactions or through allosteric effects on the DNA or cofactors. Prompted by the closer association of MRX and Sae2 during DSB end processing, we asked whether Sae2 and its endonuclease activity is required for cellular response to replication stress caused by DNA damage. Toward this end, we examined the sensitivity of S. cerevisiae wild type, sae2Δ and various SAE2 mutant strains defective in phosphorylation and nuclease activity in the presence of different genotoxic agents, which directly or indirectly generate DSBs during replication. We found that S. cerevisiae lacking SAE2 show decreased cell viability, altered cell cycle dynamics after DNA damage, and more specifically, that Sae2 endonuclease activity is essential for these biological functions. To corroborate the genetic evidences for role of SAE2 during replicative stress, we investigated SAE2 functions in-vitro. For this, we purified native Sae2 protein and nuclease dead mutant of Sae2 i.e. sae2G270D. Our studies revealed dimeric forms of both the wild type and mutant forms of Sae2. Furthermore, Sae2 displays higher binding affinity and catalytic activity with branched DNA structures, such as Holliday junction and replication forks. By using nuclease dead Sae2 protein i.e. sae2G270D, we confirmed that the endonuclease activity is not fortuitous and is intrinsic to Sae2 polypeptide. Furthermore, nuclease-defective Mre11 stimulates Sae2endonuclease activity. Mapping of the cleavage sites of Sae2 revealed a distinct preference for cleavage on the 5' end of the Holliday junction, suggesting the importance of Sae2 nuclease during recombination mediated restart of the reversed replication fork. In summary, our data clearly demonstrate a previously uncharacterized role for Sae2 nuclease activity in resection of DSB ends, processing of intermediates of DNA replication/repair and attenuation of DNA replication stress-related defects in S. cerevisiae.

My research was focused on understanding the importance of mammalian RAD51 paralogs in genome maintenance and suppression of tumorigenesis. The investigation carried out during this study has been addressed toward gaining more insights into the involvement of RAD51 paralogs in DNA damage signalling, repair of various types of lesions including double stranded breaks (DSBs), daughter strand gaps (DSGs), interstrand crosslinks (ICLs), and in the protection of stalled replication forks. My study highlights the molecular functions of RAD51 paralogs in Fanconi anemia (FA) pathway of ICL repair, in the ATM and ATR mediated DNA damage responses, in homologous recombination (HR), and in the recovery from replication associated lesions. My research also focused on the development of a novel photoinducible ICL agent for targeted cancer therapy. The thesis has been divided into following sections as follows: Chapter I: General introduction that describes about DNA damage responses and the known functions of RAD51 paralogs across species in DNA repair and checkpoint The genome of every living organism is susceptible to various types of DNA damage and mammalian cells are evolved with various DNA damage surveillance mechanisms in response to DNA damages. In response to DNA damage, activated checkpoints arrest the cell cycle progression transiently and allow the repair of damaged DNA. Upon completion of DNA repair, checkpoints are deactivated to resume the normal cell cycle progression. Defective DNA damage responses may lead to chromosome instability and tumorigenesis. Indeed, genome instability is associated with several genetic disorders, premature ageing and various types of cancer in humans. The major cause of chromosome instability is the formation of DSBs and DSGs. Both DSBs and DSGs are the most dangerous type of DNA lesions that arise endogenously as well as through exogenous sources such as radiations and chemicals. Spontaneous DNA damage is due to generation of reactive oxygen species (ROS) through normal cellular metabolism. Replication across ROS induced modified bases and single strand breaks (SSBs) leads to DSGs and DSBs, respectively. Such DNA lesions need to be accurately repaired to maintain the integrity of the genome. To understand the various cellular responses that are triggered after different types of DNA damage and the possible roles of RAD51 paralogs in these processes, chapter I of the thesis has been distributed in to multiple sections as follows: Briefly, the initial portion of the chapter provides a glimpse of various types of DNA damage responses and repair pathways to deal with the lesions arising from both endogenous as well as exogenous sources. Owing to the vast range of cellular responses and pathways, the following section provides the detailed description and mechanisms of various pathways involved in taking care of wide range of DNA lesions from SSBs to DSBs. Subsequent section of chapter I provides a comprehensive description of maintenance of genome stability at the replication fork and telomeres. Germline mutations in the genes that regulate genome integrity cause various genetic disorders and cancer. Mutations in ATM, ATR, MRE11, NBS1, BLM and FANC (1-16), BRCA1 and BRCA2 that are known to regulate DNA damage signaling, DNA repair and genome integrity lead to chromosome instability disorders such as ataxia-telangiectasia, ATR-Seckel syndrome, AT-like disorder, Nijmegen breakage syndrome, Bloom syndrome, FA, and breast and ovarian cancers respectively. Interestingly, RAD51 paralog mutations are reported in patients with FA-like disorder and various types of cancers including breast and ovarian cancers. Mono-allelic germline mutations in all RAD51 paralogs are reported to cause cancer in addition to the reported cases of FA-like disorder with bi-allelic germline mutations in RAD51C and XRCC2. In accordance, the last section of the chapter has been dedicated to describe the genetics of breast and ovarian cancers and the known functions of tumor suppressors such as BRCA1, BRCA2 and RAD51 paralogs in the protection of genome. Despite the identification of five RAD51 paralogs nearly two decades ago, the molecular mechanism(s) by which RAD51 paralogs regulate HR and genome maintenance remain obscure. To gain insights into the molecular mechanisms of RAD51 paralogs in DNA damage responses and their link with genetic diseases and cancer, the following objectives were laid for my PhD thesis: 1) To understand the functional role of RAD51 paralog RAD51C in FA pathway of ICL repair and DNA damage signalling. 2) To dissect the ATM/ATR mediated targeting of RAD51 paralog XRCC3 in the repair of DSBs and intra S-phase checkpoint. 3) To uncover the replication restart pathway after transient replication pause and the involvement of distinct complexes of RAD51 paralogs in the protection of replication forks. 4) To design photoinducible ICL agent that can be activated by visible light for targeted cancer therapy. Chapter II: Distinct roles of FANCO/RAD51C protein in DNA damage signaling and repair: Implications for Fanconi anemia and breast cancer susceptibility RAD51C, a RAD51 paralog has been implicated in HR. However, the underlying mechanism by which RAD51C regulates HR mediated DNA repair is elusive. In 2010, a study identified biallelic mutation in RAD51C leading to FA-like disorder, whereas a second study reported monoallelic mutations in RAD51C associated with increased risk of breast and ovarian cancers. However, the role of RAD51C in the FA pathway of DNA cross-link repair and as a tumor suppressor remained obscure. To understand the role of RAD51C in FA pathway of ICL repair and DNA damage response, we employed genetic, biochemical and cell biological approaches to dissect out the functions of RAD51C in genome maintenance. In our study, we observed that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G2/M accumulation, which are hallmarks of the FA phenotype. We found that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrated that RAD51C plays a vital role in the HR-mediated repair of DSBs associated with replication. Finally, we showed that RAD51C participates in ICL and DSB induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C displayed that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor. Chapter III: ATM-and ATR-mediated phosphorylation of XRCC3 regulates DNA double-strand break-induced checkpoint activation and repair The RAD51 paralogs XRCC3 and RAD51C have been implicated in HR and DNA damage responses, but the molecular mechanism of their participation in these pathways remained obscured. In our study, we showed that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We found that RAD51C in CX3 complex but not in BCDX2 complex is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G2 phases. XRCC3 phosphorylation was found to be required for chromatin loading and stabilization of RAD51 and HR-mediated repair of DSBs. Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G2/M checkpoint independently of its phosphorylation. Strikingly, we found that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation was required for the HR-mediated recovery of collapsed replication forks but is dispensable for the recovery of stalled replication forks. Together, our findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair. Chapter IV: RAD51 paralogs protect stalled forks and mediate replication restart in an FA-BRCA independent manner Mammalian RAD51 paralogs RAD51 B, C, D, XRCC2 and XRCC3 are critical for genome maintenance. To understand the crucial roles of RAD51 paralogs during spontaneously arising DNA damage, we have studied the RAD51 paralogs assembly during replication and examined the replication fork stability and its restart. We found that RAD51 paralogs are enriched onto the S-phase chromatin spontaneously. Interestingly, the number of 53BP1 nuclear bodies in G1-phase and micro-nucleation which serve as markers for under replicated lesions increases after genetic ablation of RAD51C, XRCC2 and XRCC3. Furthermore, we showed that RAD51 paralogs are specifically enriched at two major fragile sites FRA3B and FRA16D after replication fork stalling. We found that all five RAD51 paralogs bind to nascent DNA strands after replication fork stalling and protect the fork. Nascent replication tracts created before fork stalling with hydroxyurea degrade in the absence of RAD51 paralogs but remain stable in wild-type cells. This function was dependent on ATP binding at the walker A motif of RAD51 paralogs. Our results also suggested that RAD51 paralogs assemble into BCDX2 complex to prevent generation of DSBs at stalled replication forks, thereby safeguarding the pre-assembled replisome from the action of nucleases. Strikingly, we showed that RAD51C and XRCC3 in complex with FANCM promote the restart of stalled replication forks in an ATP hydrolysis dependent manner. Moreover, RAD51C R258H mutation that was identified in FA-like disorder abrogates the interaction of RAD51C with FANCM and XRCC3, and prevents fork restart. Thus, assembly of RAD51 paralogs in different complexes prevents nucleolytic degradation of stalled replication forks and promotes restart to maintain genomic integrity. Chapter V: Trans-dichlorooxovandium(IV) complex as a potent photoinducible DNA interstrand crosslinker for targeted cancer therapy Although DNA ICL agents such as MMC, cisplatin and psoralen are known to serve as anticancer drugs, these agents affect normal cells as well. Moreover, tumor resistance to these agents has been reported. We have designed and synthesized a novel photoinducible DNA crosslinking agent (ICL-2) which is a derivative of oxovanadiumterpyridine complex with two chlorides in trans position. We found that ICL-2 can be activated by UV-A and visible light to enable DNA ICLs. ICL-2 efficiently activated FA pathway of ICL repair. Strikingly, photoinduction of ICL-2 induces prolonged activation of cell cycle checkpoint and high degree of cell death in FA pathway defective cells. Moreover, we showed that ICL-2 specifically targets cells that express pathological RAD51C mutants. Our findings suggest that ICL-2 can be potentially used for targeted cancer therapy in patients with gene mutations in FA and HR pathway.

Nous avons identifié une nouvelle forme de phospho-S27-S33-Ku70 constitutivement surexprimée dans des cellules issues de la leucémie lymphocytaire chronique résistante à la chimiothérapie basée sur des agents alkylants de l’ADN et/ou analogues nucléotidiques. La protéine Ku70 est une protéine essentielle du maintien de la stabilité génomique par son rôle dans la réparation non-homologue (système NHEJ) des cassures double brin de l’ADN (CDB) et par sa fonction télomérique. Le laboratoire d’accueil a déjà démontré, in vitro et in vivo, dans les cellules LLC résistantes une altération de la réparation par le système NHEJ et un dysfonctionnement télomérique. Le travail de thèse a porté sur la caractérisation fonctionnelle de cette nouvelle forme phospho-S27-S33-Ku70. Pour ceci, nous avons utilisé des vecteurs d’expression permettant simultanément d’inhiber l’expression du Ku70 endogène (shRNA) et d’exprimer de façon épisomale différentes formes de Ku70 exogène. Ainsi, nous avons démontré : i) une stricte colocalisation de pS27-pS33-Ku70 avec les foyers γ-H2AX; ii) des cassures double brin (DSB) induisent la phosphorylation de S27-S33-Ku70 sous forme hétérodimère avec Ku80. Cette phosphorylation a lieu quelques minutes après le stress génotoxique et implique l'activité et l'interaction physique avec pS2056-DNA-PKcs, reliant ainsi pS27-pS33-Ku70 au système NHEJ ; iii) les cellules exprimant la forme sauvage exogène S27-S33-Ku70 ou la forme phosphomimétique E27-E33-Ku70 présentent une cinétique de réparation de l’ADN plus rapide que celle des cellules exprimant la forme mutée A27-A33-Ku70. Cependant, iv) la forme sauvage de Ku70 contribue à un niveau plus élevé d'aberrations structurales chromosomiques après la première division cellulaire suite à un stress génotoxique indiquant une infidélité lors de la réparation des dommages de l’ADN. En outre, les cellules exprimant A27-A33-Ku70 possèdent un index cellulaire plus élevé qui est corrélé avec une activation de la voie β-caténine. En adéquation avec sa surexpression dans la forme résistante de la LLC, l’ensemble de ces résultats suggère un rôle oncogénique de la forme phosphorylée de Ku70. Nous avons ensuite testé l’effet des nanodiamants hydrogénés (ND-H) dans des lignées exprimant différentes formes de Ku70. Grâce à leurs propriétés physico-chimiques les ND-H sont capables de potentialiser sous irradiation la production intracellulaire des espèces réactives de l’oxygène (ROS) et ainsi augmenter le taux des cassures (simple et double brin de l’ADN) et solliciter d’avantage le système de réparation de l’ADN. Nous observons que indépendamment de la forme exprimée de Ku70, ce double traitement induisait la sénescence cellulaire ; une découverte d’un intérêt à la fois fondamental (compréhension des voies apoptotiques vs senescence) et d’utilité pharmacologique potentielle. / We have identified a new form of phospho-S27-S33-Ku70 constitutively overexpressed in a subset of chronic lymphocytic leukemia (CLL) B cells resistant to apoptosis induced by DNA double strand breaks (DSB). Ku70 is one of the essential proteins involved in the maintenance of genomic stability through its role in DNA double strand break repair (non-homologous end-joining, NHEJ) and in telomeric protection.Laboratory previously established that resistant CLL cells disclose an upregulated NHEJ DNA repair and an impaired structure of telomeres. The goal of this thesis was to characterize the biological function(s) of this new form of Ku70. For this purpose we have constructed specific EBV-based vectors (siRNA / cDNA) enabling a simultaneous inhibition of endogenous Ku70 and an expression of different forms (mutated, wild, phosphomimetic at ser27-33) of Ku70 resistant to siRNA. Thus, we showed: i) a strict colocalisation of phospho-Ku70 with γ-H2AX foci; ii) that DSB induces the phosphorylation of Ku70 within minutes after genotoxic stress in heterodimer complex Ku70/Ku80. This phosphorylation necessitates both the physical interaction and the activity of pS2056-DNA-PKcs and/or ATM, linking phospho-Ku70 to NHEJ-mediated DNA DSB repair; iii) cells expressing mutated A27-A33-Ku70 exhibit a delayed G2/M cell cycle arrest, slower kinetic of DNA repair, lower level of genotoxic stress-induced chromosomal aberrations, and a higher cellular impedance correlated with translocation of transcriptional factor β-catenin from cytoplasmic membrane to the nucleus. Together, these data unveil an involvement of phospho-Ku70 in fast and inaccurate DNA repair; new paradigm for NHEJ regulation and to the control of resistance and maintenance of malignant cells.In parallel, we have initiated experimental approaches to explore other potential roles of phospho-Ku70. Especially, we were interested to determine whether it could play a role in an initiation of cell senescence induced by combined cells’ treatment by hydrogenated nanodiamonds (H-NDs) particles and ionizing irradiation. H-NDs exhibit positive surface charge in aqueous solutions allowing, when irradiated by photons, electrons’ emission and the release of reactive oxygen species (ROS) causing DNA damage. Effectively, we have established an intracellular increase of ROS that drive cell cycle arrest in G1/S in addition to the G2 arrest activated by irradiation alone. Finally, cells underwent the senescence process characterized byγ-galactosidaze activity, persistent large γ-H2AX foci and senescence-associated heterochromatinisation. Noteworthy, the senescence induced in this way occurred independently of Ku70 (ser27-ser33) status and irrespectively of cell resistance to genotoxic agents administrated alone; a finding of potential use in clinical trials.

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