No description available.

<p>DNA double-strand breaks pose a serious threat to genomic integrity. Double-strand breaks can cause chromosomal rearrangement, leading to uncontrolled cell proliferation, or even cell death. However, mammalian systems have in place the non-homologous end-joining pathway for repair of DNA double-strand breaks, which requires a core group of proteins to function: Ku70/80, DNA-PKcs, and Artemis for recognition, protection, and processing of the DNA ends, and XLF, XRCC4, and DNA LigaseIV for ligation of the DNA break. The work presented here focuses on the specific roles of XLF and XRCC4 within non-homologous end-joining. Initially, the structure of the N-terminal 224 residues of XLF was determined and found to consist of a head and tail domain, structurally homologous to XRCC4. Furthermore, L115 of XLF and K63, K65 and K99 of XRCC4 were identified as essential for an interaction between both proteins. This interaction was then shown to be required for stimulating ligation of mismatched DNA ends. To further understand how XRCC4 and XLF enhance LigaseIV activity, an XRCC4-XLF complex was crystallized. Truncated XRCC4 (1-157) was co-crystallized with truncated XLF (1-224), grown under conditions of decreasing temperature and increasing dehydration. The resulting structure at 3.94Å confirmed the necessity of L115 (XLF) and K63, K65 and K99 (XRCC4) to the XRCC4-XLF interaction, but also illustrated that XRCC4-XLF exists as an extended helical filament. DNA binding regions in both XRCC4 and XLF were also identified and used to construct a structural XRCC4-XLF-DNA binding model. Interestingly, XRCC4-DNA binding occurs in the same region of XRCC4 required for homo-tetramerization and binding to LigaseIV. These results culminate in a proposed model of non-homologous end-joining where XRCC4-XLF is involved not only in ligation of the double-strand break, but also in initial protection of the DNA ends.</p> / Doctor of Philosophy (PhD)

Les cassures double brin de l'ADN altèrent l'intégrité physique du chromosome et constituent l'un des types les plus sévères de dommages à l'ADN. Pour préserver l'intégrité du génome contre les effets potentiellement néfastes des cassures double brin de l'ADN, les cellules humaines ont développé plusieurs mécanismes de réparation, dont la réparation par recombinaison de l'ADN et la jonction d'extrémités non-homologues (NHEJ), catalysés par des enzymes spécifiques. Pendant ma thèse, nous avons caractérisé la dynamique de certaines des interactions protéines/ADN impliquées dans ces mécanismes au niveau de la molécule unique. Dans ce but, nous avons combiné des pinces optiques et de la micro-fluidique avec de la microscopie de fluorescence à champ large afin de manipuler une ou deux molécules d'ADN individuelles et d'observer directement les protéines de la réparation marquées par fluorescence agissant sur l'ADN. Nous avons concentré notre analyse sur trois protéines/complexes essentiels impliqués dans la réparation de l'ADN: (i) la protéine humaine d’appariement de brin RAD52, (ii) les protéines humaines XRCC4, XLF et le complexe XRCC4/Ligase IV de la NHEJ et (iii) le complexe humain MRE11/RAD50/NBS1. / DNA double-strand breaks disrupt the physical continuity of the chromosome and are one of the most severe types of DNA damage. To preserve genome integrity against the potentially deleterious effects of DNA double-strand breaks, human cells have evolved several repair mechanisms including DNA recombinational repair and Non-Homologous End Joining (NHEJ), each catalyzed by specific enzymes. In this thesis we aimed at unraveling the dynamics of protein/DNA transactions involved in DNA double-strand break repair mechanisms at single molecule level. To do this, we combined optical tweezers and microfluidics with wide-field fluorescence microscopy, which allowed us to manipulate individual DNA molecules while directly visualize fluorescently-labeled DNA repair proteins acting on them. We focused the study on three crucial proteins/complexes involved in DNA repair: (i) the human DNA annealing protein RAD52, (ii) the non-homologous end joining human proteins XRCC4 and XLF and the complex XRCC4/Ligase IV, and (iii) the human MRE11/RAD50/NBS1 complex.

No description available.

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.

Les défauts dans la réparation des cassures double-brin de l'ADN (DSBs) peuvent avoir d'importantes conséquences pouvant entrainer une instabilité génomique et conduire à la mort cellulaire ou au développement de cancers. Dans la plupart des cellules mammifères, le mécanisme de Jonction des Extrémités Non Homologues (NHEJ) est le principal mécanisme de réparation des DSBs. L'ADN Ligase IV (LigIV) est une protéine unique dans sa capacité à promouvoir la NHEJ classique. Elle s'associe avec deux autres protéines structuralement similaires, XRCC4 et XLF (ou Cernunnos). LigIV interagit directement avec XRCC4 pour former un complexe stable, tandis que l'interaction entre XLF et ce complexe est médiée par XRCC4. XLF stimule fortement l'activité de ligation du complexe LigIV/XRCC4 par un mécanisme encore indéterminé. Récemment, un rôle structurel non catalytique a été attribué à LigIV (Cottarel et al., 2013). Dans le travail de thèse présenté ici, nous avons reconstitué l'étape de ligation de la NHEJ en utilisant des protéines recombinantes produites dans des bactéries afin d’une part, d'explorer les bases moléculaires du rôle structural de LigIV, d’autre part de comprendre le mécanisme par lequel XLF stimule le complexe de ligation, et enfin de mieux comprendre comment ces trois protéines coopèrent au cours de la NHEJ. Nos analyses biochimiques suggèrent que XLF via son interaction avec XRCC4 lié à LigIV, pourrait induire un changement conformationnel dans la LigIV. Ce réarrangement de la ligase exposerait son interface de liaison à l'ADN ce qui lui permettrait alors de ponter deux molécules indépendantes d'ADN, une capacité indépendante de l'activité catalytique de LigIV. / Failure to repair DNA double-strand breaks (DSBs) may have deleterious consequences inducing genomic instability and even cell death. In most mammalian cells, Non-Homologous End Joining (NHEJ) is a prominent DSB repair pathway. DNA ligase IV (LigIV) is unique in its ability to promote classical NHEJ. It associates with two structurally related proteins called XRCC4 and XLF (aka Cernunnos). LigIV directly interacts with XRCC4 forming a stable complex while the XLF interaction with this complex is mediated by XRCC4. XLF strongly stimulates the ligation activity of the LigIV/XRCC4 complex by an unknown mechanism. Recently, a structural noncatalytic role of LigIV has been uncovered (Cottarel et al., 2013). Here, we have reconstituted the end joining ligation step using recombinant proteins produced in bacteria to explore not only the molecular basis for the structural role of LigIV, but also to understand the mechanism by which XLF stimulates the ligation complex, and how these three proteins work together during NHEJ. Our biochemical analysis suggests that XLF, through interactions with LigIV/XRCC4 complex, could induce a conformational change in LigIV. Rearrangement of the LigIV would expose its DNA binding interface that is able to bridge two independent DNA molecules. This bridging ability is fully independent of LigIV’s catalytic activity. We have mutated this interface in order to attempt to disrupt the newly identified DNA bridging ability. In vitro analysis of this LigIV mutant will be presented as well as a preliminary in vivo analysis.