At the very center of sexual reproduction is meiosis. During meiosis, the formation of meiotic Double-Strand-Breaks (DBSs) and their repair by homologous recombination are widely conserved events occurring among most eukaryote species. Meiosis-specific DSB formation requires at least nine proteins (Spo11, Ski8, Rec102, Rec104, Mei4, Mer2, Rec114, Mre11/Rad50/Xrs2) in S. cerevisiae, and the resection of the DSB ends requires additional four proteins (Mre11/Rad50/Xrs2, and Sae2). Spo11 has been identified as the catalytic component of this DSB-initiating complex. However, the roles played by the majority of these proteins are not clear. I have purified the recombinant Spo11/Ski8/Rec102/Rec104 complex, characterized its DNA binding ability as well as its cleavage activity on supercoiled plasmid DNA. Sae2 functions in both meiotic and mitotic repair of DNA double-strand breaks (DSBs) in S. cerevisiae. In vivo experiments have shown that Sae2 collaborates with the Mre11/Rad50/Xrs2 (MRX) complex in DNA end processing. Our laboratory previously showed that recombinant Sae2 exhibits endonuclease activity on single-stranded DNA and single-strand/double-strand DNA junctions using purified proteins in vitro. The MRX complex stimulates Sae2 endonuclease activity on single-stranded DNA close to single-strand/double-strand junctions, through its endonucleolytic activity. However, Sae2 contains no conserved typical nuclease domain, and it only shares very limited homology with its human functional counterpart CtIP. To characterize Sae2 and the active sites responsible for its nuclease activity, I used partial proteolysis and site-directed mutagenesis to analyze the protein. Biochemical assays in vitro show that acidic residues in the central domain play an important role in Sae2 endonuclease activity. Sae2 has also been shown to be phosphorylated by CDK (Cyclin-Dependent Kinase) during the S and G2 phases of the cell cycle, as well as by Tel1/Mec1 upon DNA damage. These modifications are essential for the function of Sae2 in DNA repair, but the function of these modifications are not clear. I have demonstrated that, in the presence of MRX, Sae2 (5D/S267E) mimicking constitutive phosphorylation by CDK and Mec1/Tel1 can assist the 5’ to 3’ exonuclease Exo1 significantly in 5’ end resection by suppressing the inhibitory effect of Ku. These results suggest that Sae2 is a critical switching protein which determines the choice between HR and NHEJ in yeast cells upon DNA damage. / text

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is caused by a point mutation in the LMNA gene, resulting in production of a truncated farnesylated-prelamin A protein (progerin).We previously reported that XPAmislocalized to the progerin-inducedDNAdouble-strand break (DSB) sites, blocking DSB repair, which led to DSB accumulation,DNA damage responses, and early replication arrest inHGPS. In this study, the XPA mislocalization to DSBs occurred at stalled or collapsed replication forks, concurrent with a significant loss of PCNA at the forks, whereas PCNA efficiently bound to progerin. This PCNA sequestration likely exposed ds-ssDNA junctions at replication forks for XPA binding. Depletion of XPA or progerin each significantly restored PCNAat replication forks.Our results suggest that although PCNAismuchmore competitive than XPAin binding replication forks, PCNA sequestration by progerin may shift the equilibrium to favor XPA binding. Furthermore, we demonstrated that progerin-induced apoptosis could be rescued by XPA, suggesting that XPAreplication fork binding may prevent apoptosis in HGPS cells. Our results propose a mechanism for progerininduced genome instability and accelerated replicative senescence in HGPS. - Hilton, B. A., Liu, J., Cartwright, B.M.,Liu,Y.,Breitman,M.,Wang,Y., Jones,R.,Tang, H.,Rusinol,A.,Musich,P.R.,Zou,Y.Progerin sequestrationof PCNApromotes replication fork collapse andmislocalization ofXPAin laminopathy-related progeroid syndromes.

No description available.

L’ADN cellulaire qu’il soit nucléaire ou mitochondrial est constamment soumis à l’action de stress d’origine exogène ou même endogène à la base d’altérations plus ou moins profondes de sa structure. Ces modifications chimiques sont très variées et peuvent aller de l’oxydation d’une base aux cassures double-brins de la molécule d’ADN. Ces dernières sont considérées comme les dommages les plus agressifs pour la cellule car peuvent conduire à la perte d’information et donc à la mort cellulaire. Parmi les systèmes de surveillance de la stabilité du génome figure la Poly(ADP-ribosyl)ation (PARylation). Cette modification post-traductionnelle est assurée essentiellement par les protéines PARP et PARG et est caractérisée par l’incorporation des polymères d’ADP ribose (pADPr) sur des protéines cibles. La PARylation constitue un élément clé dans plusieurs voies de maintien de l’intégrité génomique (BER, NHEJ, HR). La PARylation est aussi décrite au niveau de la mitochondrie mais son rôle dans la gestion des DSBs de l’ADNmt n’est pas connu. Le travail, objet de cette thèse, consiste à étudier le rôle de la PARylation dans le cas des DSB au niveau général chez la drosophile et ensuite de comprendre les mécanismes de gestion des DSB mitochondriales et évaluer l’implication de la PARylation dans ce processus. Nos résultats montrent que : (1) le comportement de la PARylation ne varie pas au cours du processus de cassures et de réparation de l’ADN nucléaire, alors que l’expression des ARNm de PARP-I et PARP-II augmente durant la phase de réparation ; (2) les cassures de l’ADN mitochondrial, induites par la bléomycine, entraînent une augmentation du nombre de copies de l’ADNmt. Cette augmentation transitoire de la quantité de l’ADNmt est observée durant la phase des dommages et retourne à la valeur initiale durant la phase de la réparation. Ce comportement semble être régulé par PARP. L’ensemble de ces résultats suggère que la réparation des DSBs est indépendante de la PARylation au niveau nucléaire mais que la présence de PARP est importante. De plus, PARP semble avoir un rôle dans la régulation de la réplication de l’ADNmt en réponse à un stress génotoxique. / Both nuclear and mitochondrial DNA alterationsarise following exposure to environmental and endogenous stresses. These genomic alterations are various, ranging from base oxidation to DNA strand breaks, single- and double-strand breaks. These damages are highly detrimental to the cell because they can lead to loss of genetic information and thus to cell death. However, cells have developed various mechanisms to counteract this biological issue and to lead up to a complex DNA damage response (DDR). The Poly (ADP- ribosyl) ation (PARylation) is among these DDR systems. This post-translational modification is mainly carried out by PARP and PARG proteins and is characterized by the incorporation of polymers of ADP-ribose on target proteins. The majority of the PARylationfunctions are related to cellular stress response, particulary in response to genomic damages where it is implicated in many DNA integrity pathways such as Base Excision Repair, Non Homologous End Joining and Homologous Recombination. In contrast to the nucleus, PARylation is also described in the mitochondria but its role in mtDNA integrityis still a heavily debate issue, particularly in case of mtDNA DSBs.To understand it, we used Drosophila model wherePARP-B isoform (human PARP-1 ortholog) is the only enzymatically active form in Drosophila PARP family. The aim of this thesis is to study the role of PARylation in response to DSBs induction in nucleus and mitochondrial DNAand then to understand the mechanisms involved in mtDNA integrity and to evaluate the role of PARylation in this process. Our results show that PARylation level remains stable during DSBs induction and also during repair process,contrary to what is shown in Human cells.However, PARP-I and PARP-II mRNA expression increase during repair period. In mitochondria compartment,our data show an increase of mtDNA copy number in presence of mtDNA DSBs. This increased level returns to normal during repair period and seems to be dependent on PARP. All these results suggest that DSBs repair is PARylation independent at the nuclear level but that the presence of PARP is important. In addition, PARP appears to have a role in the regulation of mtDNA replication in response to genotoxic stress.

La poly(ADP-ribosyl)ation est une modification post-traductionnelle des protéines par les poly(ADP-ribose) polymérases (PARPs). PARP3 a été identifiée comme un nouvel acteur de la réparation des cassures double-brin (DSBs). Nous avons évalué la contribution de PARP3 dans les différentes voies de réparation (HR, C-NHEJ ou A-EJ). Les résultats obtenus définissent PARP3 comme un modulateur de l’étape de résection d’ADN simple-brin permettant d’engager le choix de la voie de réparation. Nous avons montré que PARP3 favorise le recrutement du complexe Ku70/Ku80 aux sites de cassures et module la balance BRCA1/53BP1. Ces deux événements limitent l’étape de réparation de la voie HR et A-EJ et oriente la réparation vers la voie du C-NHEJ. Par immunoprécipitation de la chromatine, nous avons étudié les conséquences de l’absence de PARP3 sur les modifications d’histones, connues pour moduler la décision entre les différentes voies de réparation. Nos résultats actuels ne nous ont pas permis d’établir de lien entre PARP3 et les modifications d’histones en réponse aux DSBs. Nous avons toutefois observé qu’en absence de dommages, l’absence de PARP3 induit un enrichissement de H3K36me2 une marque d’histone connue pour réguler les gènes transcriptionnellement actifs. Dans un second projet, nous avons étudié l’impact de l’absence de PARP3 sur la viabilité cellulaire et la progression tumorale de cellules cancéreuses mutées en BRCA1. Nous avons montré par des approches in vitro et in vivo que l’absence de PARP3 induit une diminution de la survie et de la prolifération cellulaire plus marquée, une amplification exacerbée des centrosomes, ainsi qu’un ralentissement plus important de la progression tumorale, faisant de PARP3 une cible prometteuse en thérapie du cancer. / Poly(ADP-ribosyl)ation is a post-translational modification of proteins catalyzed by poly(ADPribose) polymerases (PARPs). PARP3 was identified as a novel actor of the double-strand break (DSBs) repair pathway. We evaluated the contribution of PARP3 in these repair pathways(HR, C-NHEJ ou A-EJ). Our results defined PARP3 as a modulator of the single strand DNA resection process which plays a role in driving the repair pathway choice. We showed that PARP3 enhances the recruitement of the Ku70/Ku80 complexe to damaged sites and modulates the BRCA1/53BP1 balance. These two events prevent the DNA end resection step initiating HR and A-EJ and drives the repair towards the C-NHEJ. By chromatin immunoprecipitation, we studied the consequences of the absence of PARP3 on histone modifications, known to modulate the decision of the DSBs repair pathways. Our current results didn’t allow us to establish a link between PARP3 and histone modifications in response to DSBs. However, in absence of DNA damage and PARP3, we observed an accumulation of H3K36me2, a histone mark known to regulate transcriptionally active genes. In a second project, we studied the impact of the absence of PARP3 on cell viability and tumor progression in breast cancer cell lines mutated in BRCA1. By in vitro and in vivo approaches, we showed that the absence of PARP3 induces an important decrease in cell survival and proliferation, an increase in centrosomal amplification and a strong delay in tumor progression. The roles of PARP3 in both cellular response to DNA damage and mitotic progression introduce PARP3 as a possible promising therapeutic target in cancer therapy.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第23090号 / 医博第4717号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 小川 修, 教授 溝脇 尚志 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23114号 / 医科博第125号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 篠原 隆司, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.