<p>Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase &#950; through the Rev7 subunit and with polymerases &#954;, &#953; and &#951; in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. </p><p>In chapter 1, the solution structures of the mouse Rev1 CTD and its complex with the Pol &#954; RIR are reported, revealing an atypical four-helix bundle. Yeast two-hybrid assays were used to identify a Rev7-binding surface centered at the &#945;2-&#945;3 loop and N-terminal half of &#945;3 of the Rev1 CTD. Binding of the mouse Pol &#954; RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn &#945;-helix, with the helix stabilized by an N-terminal cap. RIR-binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a &#946;-hairpin that projects over the shallow &#945;1-&#945;2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. The combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes.</p><p>The multifaceted abilities of the Rev1 CTD are further explicated in chapter 2 where the purification and structure determination of a quaternary translesion polymerase complex consisting of the Rev1 CTD, the heterodimeric Pol &#950; complex, and the Pol &#954; RIR is reported. Yeast two-hybrid assays were employed to identify important interface residues of the translesion polymerase complex. The structural elucidation of such a quaternary translesion polymerase complex encompassing both insertion and extension polymerases bridged by the Rev1 CTD provides the first molecular explanation of the essential scaffolding function of Rev1 and highlights the Rev1 CTD as a promising target for developing novel cancer therapeutics to suppress translesion synthesis. Our studies support the notion that vertebrate insertion and extension polymerases could structurally cooperate within a mega translesion polymerase complex (translesionsome) nucleated by Rev1 to achieve efficient lesion bypass without incurring an additional switching mechanism.</p><p>Chapter 3 explores the ubiquitin-binding capacity of the FAAP20 UBZ in an effort to begin understanding its requirement for recruitment of the Fanconi anemia complex to interstrand DNA crosslink sites and for interaction with the translesion synthesis machinery through recognition of monoubiquitinated Rev1. FAAP20 is an integral component of the Fanconi anemia core complex that mediates the repair of DNA interstrand crosslinks. Although the UBZ-ubiquitin interaction is thought to be exclusively encapsulated within the &#946;&#946;&#945; module of UBZ, it is revealed that the FAAP20-ubiquitin interaction extends beyond such a canonical zinc-finger motif. Instead, ubiquitin-binding by FAAP20 is accompanied by transforming a disordered tail C-terminal to the UBZ of FAAP20 into a rigid, extended &#946;-loop that latches onto the complex interface of the FAAP20 UBZ and ubiquitin, with the invariant C-terminal tryptophan emanating toward I44Ub for enhanced binding specificity and affinity. Substitution of the C-terminal tryptophan with alanine in FAAP20 not only abolishes FAAP20-ubiquitin binding in vitro, but also causes profound cellular hypersensitivity to DNA interstrand crosslink lesions in vivo, highlighting the indispensable role of the C-terminal tail of FAAP20, beyond the compact zinc finger module, toward ubiquitin recognition and Fanconi anemia complex-mediated DNA interstrand crosslink repair.</p><p>Having structurally elucidated the molecular basis of the essential scaffolding function of the Rev1 CTD, the search for small molecule inhibitors of the Rev1-Rev7 interaction has been initiated toward the goal of developing novel adjuvants to DNA targeting chemotherapeutics. Screening efforts have led to the discovery of a lead compound, JH-RE-06*NaOH, that specifically targets the Rev7-binding hydrophobic pocket of the Rev1 CTD with low micromolar affinity, effectively inhibiting the Rev1-Rev7 interaction in an in vitro ELISA assay developed for high-throughput screening of small molecule libraries. With the potential for positive outcomes in future in vivo assays, we hope to develop JH-RE-06*NaOH into the first potent inhibitor of translesion synthesis in cancer patients being treated with DNA-targetng chemotherapeutics to aid in sensitization and prevention of chemoresistance development in malignancies.</p> / Dissertation

La réplication de l'ADN est un évènement majeur pour la cellule car elle permet la duplication fidèle du matériel génétique. Il s'agit d'une étape critique du cycle cellulaire, car les fourches de réplication rencontrent fréquemment des barrières naturelles ou des lésions d'origine endogènes (lésions oxydatives) ou exogènes (agents physiques ou chimiques), sources de cassures chromosomiques et donc d'instabilité génétique. Une des réponses à ces fourches bloquées est l'activation du point de contrôle (checkpoint) de la phase S du cycle cellulaire. Nous avons montré que l'ADN polymérase Kappa (pol Kappa), polymérase dite translesionnelle en raison de ses capacité à franchir des lésions sur l'ADN, s'avère être aussi un acteur du point de contrôle de phase S. En effet, la déplétion de pol Kappa par ARN interférence dans différentes lignées cellulaires ou par immunodépletion d'un extrait de Xénope, entraîne un défaut de phosphorylation de Chk1. Pol Kappa est impliquée dans la synthèse de brins naissant d'ADN au niveau des fourches bloquées, ce qui facilite le recrutement du complexe 9-1-1 composé des protéines Rad9, Rad1 et Hus1et permet alors, une activation correcte du checkpoint de phase S. Afin de décrypter le rôle de pol kappa, nous avons construits différents mutants et nous avons analysé leur capacité à former des foyers, à être recrutés à la chromatine et à interagir avec différents partenaires dans des conditions d'activation du point de contrôle de phase S. Nous avons pu constater que le mutant du domaine d'interaction à PCNA était incapable de former des foci foyers ?. Nous avons ensuite observé, qu'en condition de stress réplicatif, pol Kappa était recruté à la chromatine grâce à son domaine d'interaction à PCNA et par différentes approches biochimiques, nous avons pu voir que pol kappa interagissait avec Rad9 et Chk1. Nous avons également mis en évidence que le défaut d'activation de Chk1 en l'absence de pol kappa reflétait d'une diminution de son taux dans le noyau, suggérant une régulation commune entre Chk1 et pol Kappa. En effet, nous avons observé que pol Kappa, comme Chk1, était régulés par l'ubiquitine hydrolase USP7. En effet, l'interaction entre pol Kappa et USP7 est augmentée en condition de stress. Nous avons pu voir, qu'à l'instar de Chk1, l'absence de USP7 entrainait une baisse du niveau de pol kappa dans le noyau. Ainsi nous proposons qu'en réponse à un stress réplicatif, pol Kappa et Chk1 soient stabilisés via leur dé-ubiquitination par USP7, permettant leur recrutement à la chromatine et une activation correcte du checkpoint de phase S. Parallèlement à ces travaux, des publications récentes montrent une implication possible de pol Kappa au niveau des séquences répétées. Nous avons pu mettre en évidence une interaction entre pol Kappa et Cenpb, protéine centromérique reconnaissant une séquence de 17 paires de bases dans l'ADN a-satellite. Ces résultats préliminaires suggèrent que le rôle de pol Kappa dans le checkpoint de phase S s'adresse notamment aux régions d'hétérochromatine. L'ensemble des résultats obtenus montre l'importance de pol Kappa dans le maintien de la stabilité génomique, par son rôle dans le checkpoint de phase S, et par son implication dans la régulation de Chk1 en condition de stress réplicatif. / DNA replication is a major event for cells which allow the faithful duplication of genetic material. It is a critical step of cell cycle, because replication forks encounters frequently naturals barriers (non B-DNA structures), exogenous barriers (chemicals agents), or endogenous barriers (oxydatives lesions). These different barriers can be at the origin of chromosomes breaks and lead to genetic instability. To overcome the stalled forks, cells have evolved two major mechanisms: the induction of ATR replication checkpoint pathway and the recruitment of specialized DNA polymerase to perform the translesion synthesis. This two pathways are essential to maintain genomic stability. Human DNA polymerase Kappa (pol Kappa), the most conserved specialized DNA polymerase, is best known to participate to translesion synthesis. Recently, we have shown that pol kappa has a crucial role in the S-phase checkpoint activation. Indeed, pol Kappa is implicated in the synthesis of short DNA intermediates at the stalled forks, facilitating the recruitment of 9-1-1 clamp, and is required for an optimal phosphorylation of Chk1, the main effector of the S-phase checkpoint. Durant my PhD thesis, I explored the molecular mechanisms underlying this newly identified role. We have constructed several pol kappa mutants, and we have observed that for the mutation in the PCNA binding domain impeded pol kappa to form foci in response to replication stress. We also showed the requirement of this domain for pol Kappa recruitment on chromatin. By different experimental approaches, we have described a complex in which pol Kappa interacts with Rad9 and Chk1, two proteins required for the S-phase checkpoint activation. The Chk1 phosphorylation defect observed in absence of Kappa could also be the consequence of the Chk1 protein level decreased in the nucleus, meaning a potential common regulation between pol Kappa and Chk1. Based on this observation, we have studied how pol Kappa is regulated upon a replication stress and like Chk1, pol Kappa seems to be regulated by ubiquitination. We focused our attention on USP7 an ubiquitin hydrolase known to regulate Chk1. We have demonstrated an interaction between pol Kappa and USP7, which is stimulated after replication stress. Moreover, USP7 depletion leads to a decrease of pol Kappa level in the nucleus, suggesting that de-ubiquination of pol Kappa could to be a prerequisite for its checkpoint function and its stability.