L'augmentation des taux cellulaires de p53 en réponse aux dommages à l'ADN a été largement attribuée à une augmentation de la demi-vie de la protéine. Il est maintenant bien établi que la régulation traductionnelle de l'ARNm de p53 est également critique à la fois pour la répression de l'accumulation de p53 dans des conditions normales, et l'induction de la protéine en réponse aux dommages de l'ADN. Nos travaux se sont accès sur l'étude du rôle de facteur de réparation de l'ADN Ku dans la régulation traductionnelle de l'ARNm P53. Nous avons montré que Ku réprime la synthèse de la protéine p53 et l'apoptose p53-dépendante via la liaison à une structure en tige-boucle dans la 5'UTR de l'ARNm. Cependant, la répression traductionnelle exercée par Ku est levée après un stress génotoxique. Le mécanisme sous-jacent implique l'acétylation de Ku qui perturbe les interactions Ku-ARNm p53. Ces résultats suggèrent que la répression traductionnelle de p53 par Ku constitue un nouveau mécanisme cytoprotectif liant la réparation de l'ADN et la traduction des ARNm. / Increases in p53 protein levels after DNA damage have largely been attributed to an increase in the half-life of the p53 protein. It is now well accepted that translational regulation of p53 mRNA is also critical for both repression of p53 accumulation in unstressed conditions and induction of the p53 protein in response to DNA damage. Our work focused on studying the role of DNA repair factor Ku in the regulation of P53 mRNA translation. We showed that Ku represses p53 protein synthesis and p53-mediated apoptosis by binding to a stem-loop structure within the p53 5'UTR. However, Ku-mediated translational repression is relieved after genotoxic stress. The underlying mechanism involves Ku acetylation which disrupts Ku-p53 mRNA interactions. These results suggest that Ku-mediated repression of p53 mRNA translation constitutes a novel cytoprotective mechanism linking DNA repair and mRNA translation.

Le cancer colorectal (CCR) représente la 2ème cause de mortalité par cancer dans les pays industrialisés. De nouveaux traitements permettant de bloquer l’évolution de la maladie sont nécessaires. Il est donc important de mieux connaitre les acteurs impliqués dans la cancérogenèse. Lors du développement du cancer, des gènes suppresseurs de tumeur sont inhibés et des oncogènes sont activés, perturbant ainsi l’équilibre des cellules et les transformant. Au cours de ma thèse, je me suis intéressée à deux gènes homéotiques qui possèdent des rôles opposés dans les CCR. Cdx2 exerce un rôle suppresseur de tumeur, alors que HoxB7 agit comme un oncogène. Mon travail de thèse a permis (i) de mettre en évidence une nouvelle fonction non-transcriptionnelle de Cdx2 : inhibiteur de la réparation des cassures de l'ADN spécifiquement dans le côlon, (ii) et de révéler que le niveau d'expression des gènes Cdx2 et Hoxb7 au sein de la tumeur peut avoir une importance pour le choix du traitement des CCR. / Colorectal cancer is the 2nd cause of mortality by cancer in industrialized countries. New treatments allowing to prevent the evolution of the disease are needed. It is important to better understand the actors implicated in carcinogenesis. During cancer development, tumor suppressor genes are inhibited and oncogenes are activated, thus disrupting the homeostasis of the tissue and transforming the cells. During my thesis, I have been interested in two genes having two opposite functions in CCR : Cdx2 is a tumor suppressor while Hoxb7 acts as an oncogene. My work allows to highlight (i) a new non-transcriptional function of Cdx2 : inhibitor of the reparation of DNA breaks specifically in the colon, (ii) and that the expression level of Cdx2 and Hoxb7 genes inside the tumor can have an importance in the choice of the CCR treatment.

Homologous recombination (HR) repair is critical for the maintenance of genomic stability, as it is involved in the precise repair of DNA double-strand breaks (DSBs) using an intact homologous template for repair. The initiation of 5' strand resection of DNA ends is a critical determinant in this process, which commits cells to HR repair and prevents repair by non-homologous end joining (NHEJ). The human single-stranded DNA (ssDNA) binding complexes, RPA and SOSS1, are involved in regulating DSB signaling and HR repair. In this study, I demonstrate a novel function of SOSS1 in HR repair, in which SOSS1 stimulates hExo1-dependent resection. Despite its poor activity in binding duplex DNA, SOSS1 facilitates hExo1 recruitment to duplex DNA ends and promotes its activity in resection independently of MRN in vitro. MRN(X) is a highly conserved complex that is involved in the early steps of HR repair by regulating DSB resection. MRN interacts with CtIP and constitutes resection machinery that can perform limiting processing on DNA ends. In this study, I also examine the biochemical activities of MRN and CtIP in DSB resection through reconstituted in vitro assays. I show that the ATP-dependent DNA unwinding activity of MRN is responsible for overcoming Ku inhibition of hExo1- and Dna2/BLM-dependent resection activity in vitro. I propose that this unwinding step displaces Ku away from the DNA ends and facilitates the recruitment of hExo1 to the DNA ends for efficient resection. In addition, I show that CtIP can promote overcoming the inhibitory effect of Ku in resection together with MRN. Further, I demonstrate that MRN nuclease activity is required for efficient processing of covalent adducts from DNA ends in vitro, suggesting that the nucleolytic removal of covalent adducts by MRN generates free ends for hExo1- and Dna2/BLM binding. Overall, this study provides mechanistic insight into the regulation of DSB resection in human cells. / text

Indiana University-Purdue University Indianapolis (IUPUI) / In mammalian cells DNA double strand breaks (DSBs) are highly variable with respect to sequence and structure all of which are recognized by the DNA- dependent protein kinase (DNA-PK), a critical component for the resolution of these breaks. Previously studies have shown that DNA-PK does not respond the same way to all DSBs but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanism by which DNA-PK is activated by various DNA substrates. We provide evidence that recognition of DNA structural variations occur through distinct protein-protein interactions between the carboxy terminal (C-terminal) region of Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Discrimination of terminal DNA sequences, on the other hand, occurs independently of Ku 80 C-terminal interactions and results exclusively from DNA-PKcs interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. Our results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately Non-homologous End Joining (NHEJ) efficiency.