Eukaryotic cells repair DNA double-strand breaks (DSBs) through both non-homologous and homologous recombination pathways. The initiation of homologous recombination requires the generation of 3' overhangs, which are essential for the formation of Rad51 protein-DNA filaments that catalyze subsequent steps of strand invasion. Experiments in budding yeast show that resection of the 5' strand at a DSB is delayed in strains lacking any components of the Mre11/Rad50/Xrs2 (MRX) complex¹ . In meiosis, a specific class of hypomorphic mutants of mre11 and rad50 (Rad50S) are completely deficient in 5' resection and leave Spo11 covalently attached to the 5' strands of DNA breaks². Similar to mre11S and rad50S mutants, sae2 deletion strains fail to resect 5' strands at meiotic DSBs and accumulate covalent Spo11 adducts³;⁴. In addition, Sae2 and MRX were also found to function cooperatively to process hairpin-capped DNA ends in vivo in yeast. sae2 and mrx null strains show a severe defect in processing these structures and accumulate hairpin-capped DNA ends⁵;⁶. The Longhese laboratory has also shown that Sae2 deletion strains show a delay in 5' strand resection, similar to rad50S strains⁷. Recently, Bettina Lengsfeld in our laboratory demonstrated that Sae2 itself possesses nuclease activity and that MRX and Sae2 act cooperatively to cleave single-stranded DNA adjacent to DNA hairpin structures⁸. In vitro characterization of Sae2 showed that the central and N-terminal domains are required for MRX-independent nuclease activity and that the C-terminus is required for cooperative activities with MRX. Sae2 also acts independently of MRX as a 5' flap endonuclease on branched structures in vitro. Our studies investigate whether MRX, Sae2, and Exo1 function cooperatively in DNA resection using recombinant, purified proteins in vitro. We developed assays utilizing strand-specific Southern blot analysis to visualize DNA end processing of model DNA substrates using recombinant proteins in vitro. Our results demonstrate that MRX and Sae2 cooperatively resect the 5' end of a DNA duplex together with the Exo1 enzyme, supporting a role for these factors in the early stages of homologous recombination and repair. / text

Les modifications post-traductionnelles des protéines par des polymères d’ADP-ribose (PAR) ou par phosphorylation permet l’assemblage des complexes de la réparation de l’ADN à la chromatine endommagée dont les fonctions sont essentielles pour assurer le maintien de la stabilité du génome. En réponse aux lésions de l’ADN, l’activité de synthèse de PAR des protéines PARP1 et PARP2 est fortement stimulée. Les PAR servent de signalisation pour le recrutement de multiples protéines, dont la protéine plateforme XRCC1.Les études menées au cours de cette thèse ont porté sur l’étude de la régulation des fonctions des protéines PARP1, PARP2 dans la réparation des cassures double brins (CDB) et l’étude des modifications de XRCC1 par phosphorylation en réponse à des dommages de l’ADN. En utilisant des substrats permettant de mesurer l’efficacité des différentes voies de réparation des CDB, nous avons démontré que PARP2, et non PARP1, est impliqué dans la régulation du choix des voies de la réparation des CDB. Plus spécifiquement, nous avons montré que PARP2 stimule l’initiation de la résection des extrémités des CDB dépendante de CtIP, indépendamment de son activité catalytique. Par des approches de vidéo-microscopie, nous avons pu déterminer que PARP2 limite l’accumulation de 53BP1 aux sites de dommages induits par micro-irradiation laser. Nous proposons que la protéine PARP2, en limitant le recrutement de la protéine 53BP1 aux sites de dommages, favorise la réparation des CDB dépendante de la résection des extrémités d’ADN, au détriment de la voie canonique de jonction des extrémités. Ces résultats sont les premiers démontrant un rôle de PARP2 dans le choix des voies de réparation des CDB.En parallèle, nous avons analysé comment la phosphorylation régule les fonctions de la protéine XRCC1. Par des approches in vitro et in vivo, nous avons pu déterminer que l’interdomaine 1 de XRCC1 est phosphorylé par la kinase CDK5. En réponse aux dommages induits par un agent alkylant, XRCC1 est activement déphosphorylé in vivo. De plus, nous avons observé que lorsque l’interdomaine 1 ne peut pas être phosphorylé in vitro, l’interaction de XRCC1 avec les PAR synthétisés par PARP1 et PARP2 augmente, et le recrutement de XRCC1 aux sites de dommages de l’ADN est accru. Ces résultats indiquent pour la première fois que la déphosphorylation de XRCC1 en réponse à un stress génotoxique participe activement à son recrutement aux sites de dommages.Dans leur ensemble, ces travaux ont contribué à améliorer nos connaissances fondamentales des réseaux de protéines impliquées dans la prise en charge des dommages de l’ADN. La compréhension de ces mécanismes est essentielle non seulement car ils participent au maintien de la stabilité du génome mais aussi du fait du développement exponentiel de nouvelles stratégies anti-tumorales qui visent à inhiber les voies de la réparation dans la but de cibler spécifiquement les cellules cancéreuses. / Post-translational modifications of proteins by polymers of ADP-ribose (PAR) or by phosphorylation allow the assembly of DNA repair protein complexes at damaged chromatin and are crucial to ensure genome stability. In response to DNA insults, the synthesis of PAR by the PARP1 and PARP2 proteins is strongly induced. PAR act as a signaling platform for the recruitment of multiples proteins at the sites of DNA damages, including the scaffold protein XRCC1. Research conducted during this PhD have been focused on studying the regulation of PARP1 and PARP2 functions in double-strands break repair (DSBR), and in investigating the role of XRCC1 modifications by phosphorylation in response to DNA damage.Using DNA repair assay allowing us to assess the accuracy of the different DSBR pathways, we demonstrated that PARP2, and not PARP1, is involved in the regulation of DNA double-strands break repair pathway choice. More precisely, we showed that PARP2 stimulates CtIP dependent initiation of end-resection at DSB, independently of its catalytic activity. By live cell imaging, we were able to determine that PARP2 limit 53BP1 accumulation at DNA damage sites induced by laser-microirradiation. We propose that by limiting 53BP1 accumulation at DNA damage sites, PARP2 stimulate DSB repair pathway that depend on DNA end-resection, thus counteracting the canonical end-joining pathway. These results are the first demonstrating a role for PARP2 in DNA DBSR pathway choice.In addition, we analyzed how the functions of XRCC1 are regulated by phosphorylation. Using in vitro and in vivo approaches, we were able to demonstrate that the linker 1 region of XRCC1 is phosphorylated by the CDK5 kinase. XRCC1 is actively dephosphorylated in response to DNA damage induced by an alkylating agent in vivo. We also observed that when the linker 1 cannot be phosphorylated, the XRCC1 interaction between the PAR synthetized by PARP1 and PARP2 is stimulated, and XRCC1 recruitement at the sites of DNA damage is far more efficient. These evidences indicate for the first time that the dephosphorylation of XRCC1 actively participate in its recruitment at the site of DNA damage. Put together, this work contributed to strengthen our fundamental knowledge of the protein network involved in the DNA damage response. Knowledge of those mechanisms is crucial since they participate in maintaining genome stability, and because new antitumoral drugs targeting DNA repair pathways in the attempt to specifically killed tumor cells are exponentially released.

RAD18 is an E3 ubiquitin ligase that prevents the replication forks from collapsing caused by damaged DNA. As an important factor controlling replication, its dysregulation was shown to be associated with some human tumours. However, the clinical relevance of this finding is unknown. The aim of the thesis was evaluation of selected RAD18 variants that had been identified in breast and ovarian cancer patients. This work revealed functional defects of RAD18 variants not only in replication fork protection but also in repair of DNA double-strand breaks. This unconventional role of RAD18 is known to be dependent on upstream ubiquitination events, however, its contribution to the repair per se is not understood. This work aimed to elucidate the function of RAD18 in DNA double-strand break repair by homologous recombination focusing especially on its relationship with 53BP1. Data presented here show that RAD18 effectively disrupts 53BP1 accumulation in the repair foci by competition for the same binding partner and thus promotes resection of DNA ends. This antagonistic function of RAD18 is restricted both spatially (to the vicinity of the repair centre) and temporarily (to S phase). Moreover, it seems to be regulated by existence of RAD18 in two distinct complexes. Potential models for this regulation...