DNA double strand breaks can occur from various sources and the timely and accurate repair of these breaks is critical to maintain the genomic integrity of the cell. Break-induced replication (BIR) is a repair pathway that has been shown to repair DSBs where only one end of the break can locate homology, similar to ends seen at collapsed replication forks or eroded telomeres. BIR progresses by an unusual bubble-like intermediate. The asynchrony between the synthesis of leading and lagging strand synthesis during BIR leads to the accumulation of long single-stranded DNA (ssDNA) behind the bubble. This mechanism leads to the conservative inheritance of newly synthesized DNA. BIR repair can lead to increased mutations, loss of heterozygosity and gross chromosomal rearrangements. In this thesis I investigated the deleterious effects of the ssDNA formed during BIR. Using yeast, Saccharomyces cerevisiae, I showed that the regulation of Rad51 that binds ssDNA during BIR is important to prevent the accumulation of toxic joint intermediates. Here, I demonstrate that a known Rad51-interacting protein, Srs2, plays two key roles in counteracting the accumulation of lethal recombination intermediates. First, Srs2 dislodges Rad51 from long ssDNA formed during DSB repair and therefore prevents promiscuous strand invasions that generate lethal joint molecules. Second, Srs2 helicase dismantles toxic intermediates that have already formed. We also demonstrate that the structure-specific endonucleases, Mus81 and Yen1, can resolve toxic joint molecules formed in the absence of Srs2, thus promoting cell survival. The other goal of this thesis was to study the effects of ssDNA accumulated during BIR in the formation of base-substitution mutagenesis. I test whether this ssDNA is mutagenic by analyzing BIR with and without the presence of DNA damaging agents, including methyl methanesulfonate (MMS) and APOBEC3A. I observed a hypermutagenic effect of BIR with respect to base- substitutions in both cases. Importantly, BIR synergizes with ssDNA damaging agents to produce mutation clusters similar to those previously observed in cancer. I also report the critical role translesion polymerase Polζ plays in the formation of base-substitutions resulting from BIR. Finally, I have discovered a completely novel, UNG1-dependent mechanism of supposed error-free bypasses of APOBEC-induced DNA lesions during BIR that promotes chromosomal rearrangements.

Yes / We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilises Rad51-DNA filaments and is thought to regulate strand invasion and prevent hyper-recombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although in such cells sister centromeres often separate. Undivided nuclei contain aggregates of Rad51 colocalised with the ssDNA-binding protein RPA, suggesting the presence of persistent single-strand DNA. Rad51 aggregate formation requires Spo11-induced DSBs, Rad51 strand-invasion activity and progression past the pachytene stage of meiosis, but not the DSB end-resection or the bias towards interhomologue strand invasion characteristic of normal meiosis. srs2 mutants also display altered meiotic recombination intermediate metabolism, revealed by defects in the formation of stable joint molecules. We suggest that Srs2, by limiting Rad51 accumulation on DNA, prevents the formation of aberrant recombination intermediates that otherwise would persist and interfere with normal chromosome segregation and nuclear division. / Biotechnology and Biological Sciences Research Council (BB/K009346/1)

<p>The genetic information necessary for the survival and propagation of a species is contained within a physical structure, DNA. However, this molecule is sensitive to damage arising from both exogenous and endogenous sources. DNA damage can prevent metabolic processes such as replication and transcription; thus, systems to bypass or repair DNA lesions are essential. One type of lesion in particular - the double strand break (DSB) - is extremely dangerous as inappropriate repair of DSBs can lead to deletions, mutations and rearrangements. Homologous recombination (HR) uses a template with sequence homology to the region near the DSB to restore the damaged molecule. However, this high-fidelity pathway can contribute to genome instability when recombination occurs between diverged substrates. To further our understanding of the regulation of HR during vegetative growth, we have used the budding yeast Saccharomyces cerevisiae as a model system and a plasmid-based assay to model repair of a DSB. In the first part of this work, the molecular structures of noncrossover (NCO) and crossover (CO) products of recombination were examined. While the majority of NCOs had regions of heteroduplex DNA (hDNA) on one side of the gap in the repaired allele and no change to the donor allele, most COs had two tracts of hDNA. They were present on opposite sides of the gap, one in each allele. Our results suggest that the majority of NCOs are generated through synthesis-dependent strand annealing (SDSA), and COs are the result of constrained cleavage of a Holliday junction (HJ) intermediate. To clarify the mechanisms regulating NCO production, the effects of three DNA helicases - Mph1, Sgs1 and Srs2 - on the structures of NCO events were examined. All three helicases promote NCO formation by SDSA, but Sgs1 and Srs2 also assist in NCO formation arising from an HJ-containing intermediate, consistent with HJ-dissolution. To study how CO products are generated, we have investigated the contribution of the following candidate HJ resolvases to the structures of CO events: Mus81, Yen1 and Rad1. The results suggest that Rad1 is important to normal CO formation in this assay, but Mus81 and Yen1 are largely dispensable. Together, this work advances our knowledge of how the NCO versus CO outcome is determined during HR, expanding our understanding of how mitotic recombination is regulated.</p> / Dissertation

L’usage de composé chimique causant des dommages à l’ADN en phase S est une stratégie couramment utilisée en chimiothérapie du cancer. Ainsi, l’étude de la réponse cellulaire aux dommages subit en phase S s’avère indispensable afin de mieux comprendre les mécanismes cellulaires sous jacents à la réparation de ces dommages et pour permettre le développement ou l’amélioration de nouvelles stratégies antitumorales. Lors de chaque phase S, les nouvelles histones sont acétylées par des histones acétyltransférases (HAT) et déacétylées en fin de phase S et en début de phase G2, par des histones déacétylases (HDAC). Ce cycle d’acétylation des histones est conservé chez tous les eucaryotes. Chez la levure Saccharomyces cerevisiae, l’acétylation de de la lysine 56 de l’histone H3 (H3K56ac) est une marque des nouvelles histones qui est ajoutée par la HAT Rtt109 et retirée par les sirtuines Hst3 et Hst4, des HDAC de classe III. Lors de l’induction de dommages à l’ADN au cours de la phase S par des agents génotoxiques, une persistance de l’acétylation de H3K56 est observée, ce qui suggère un rôle de l’acétylation de H3K56 dans la réponse aux stress réplicatifs subits en phase S. Notre objectif est de comprendre la base moléculaire des défauts de réparation observés dans les mutants de la voie de l’acétylation de H3K56. Précédemment, nous avons réalisé des cribles chémogénétique au nicotinamide (NAM), un inhibiteur des sirtuines, afin d’identifier des gènes influençant la croissance cellulaire en absence de l’activité des sirtuines. SRS2 a été identifié parmi les gènes importants pour le maintien de la viabilité en absence des sirtuines. Srs2 est une hélicase dont l’une de ses principales fonctions est de retirer les nucléofilaments de Rad51, l’une des principales protéines de la recombinaison homologue, de l’ADN simple brin. À l’inverse, RIF1 fut trouvé parmi les gènes dont la délétion confère une meilleure résistance au NAM. Rif1 est impliqué dans le maintien de la taille des télomères, mais également dans l’inhibition des origines de réplication. Dans cette thèse, je présenterai les résultats d’un crible avec des mutants hétérozygotes diploïdes pour évaluer l’importance des gènes essentiels à la croissance cellulaire en absence des sirtuines. Plusieurs gènes impliqués dans l’initiation de la phase S sont ressortis des deux cribles, ce qui suggère que l’acétylation de H3K56 a une fonction dans le processus de réplication de l’ADN qui a lieu en phase S du cycle cellulaire. Par des méthodes de génétique classique, nous avons validé que l’inactivation de membres du complexe DDK, DBF4 et CDC7, dont la fonction est requise par l’initiation des origines de réplication, sensibilise les cellules à la présence d’acétylation constitutive de H3K56. Nous avons confirmé que l’activité toxique de Rif1 pour la viabilité cellulaire en absence des sirtuines Hst3 et Hst4 est sa fonction répressive des origines de réplication. Nous avons observé que l’activation du point de contrôle intra-S n’expliquait pas la perte de viabilité d’un mutant H3K56 constitutivement acétylé alors que l’activité des origines est compromise. Finalement, nous avons identifié un rôle de l’acétylation de H3K56 dans l’initiation des origines de réplication. La progression dans le cycle cellulaire d’une souche constitutivement acétylée sur H3K56 n’est pas ralentie lorsque le complexe DDK est fonctionnel. Toutefois, des dommages spontanés à l’ADN sont observés au cours de la phase S dans les souches dépourvues des protéines Hst3 et Hst4. Ceci suggère que le stress réplicatif observé dans les mutants de la voie de l’acétylation de H3K56 ne peut être entièrement expliqués par un ralentissement de l’initiation des origines de réplication. Nous avons utilisé un mutant srs2Δ qui présente des dommages spontanés à l’ADN et une très forte sensibilité au NAM afin d’exacerber les problèmes réplicatifs observés dans des mutant constitutivement acétylés sur H3K56. Par des méthodes de génétique classique, nous avons observé que la léthalité synthétique entre l’acétylation constitutive de H3K56 et la perte de SRS2 ne peut pas être renversé par la délétion des membres de la voie canonique de l’acétylation de H3K56 suggérant un rôle important de cette modification dans la réparaiton des dommages à l’ADN. De plus, lors d’une persistance de l’acétylation de H3K56, nous avons constaté que la présence de Rad51 s’avère toxique pour des cellules srs2∆. Ensemble, nos résultats suggèrent un rôle de l’acétylation de H3K56 complémentaire au point de contrôle intra-S pour réguler l’initiation des origines de réplication lors de stress réplicatif. Nos données révèlent des fonctions encore méconnues de l’acétylation de H3K56 ainsi que de nouveaux liens entre la structure de la chromatine et la dynamique de réplication. / The use of chemical compounds causing S-phase damage is a common strategy used in cancer chemotherapy. Thus, the study of the cellular response to S-phase DNA damage is essential to better understand the cellular mechanisms underlying the repair of this damage and to allow the development or improvement of antitumor strategies. During each S-phase, new histones are acetylated by histone acetyltransferases (HATs) and deacetylated at the end of S-phase and at the beginning of G2 phase by histone deacetylases (HDACs). This histone acetylation cycle is conserved in all eukaryotes. In the yeast Saccharomyces cerevisiae, acetylation of lysine 56 of histone H3 (H3K56ac) is a hallmark of new histones that is added by the HAT Rtt109 and removed by sirtuins Hst3 and Hst4, class III HDACs. Upon induction of DNA damage during S-phase by genotoxic agents, persistence of H3K56 acetylation is observed suggesting a role for H3K56 acetylation in the response to replicative stresses. Our goal was to understand the molecular basis of the DNA damage defects observed in H3K56 acetylation pathway mutants. Previously, we performed chemogenetic screens with nicotinamide (NAM), a sirtuin inhibitor, to identify genes that influence cell growth in the absence of sirtuin activity. SRS2 emerged as one of the important genes for maintaining viability in the absence of sirtuins. Srs2 is a helicase whose main function is to remove the nucleofilaments of Rad51, one of the major homologous recombination proteins, from single-stranded DNA. Conversely, RIF1 has emerged as one of the genes whose deletion enhances resistance to NAM. Rif1 is involved in the maintenance of telomere size, but also in the inhibition of replication origins. In this thesis, I will present the results of a screen with diploid heterozygous mutants to assess the importance of genes essential for cell growth in the absence of sirtuins. Several genes involved in S-phase initiation emerged from both screens, suggesting that H3K56 acetylation has a function in the DNA replication process that occurs in the S-phase of the cell cycle. By classical genetic methods, we validated that defective activity of the DDK complex members, DBF4 and CDC7, whose function is required by the initiation of replication origins, sensitize cells in the presence of constitutive H3K56 acetylation. We confirmed that the toxic activity of Rif1 8 for cell viability in the absence of Hst3 and Hst4 sirtuins is its repressive function of the origins of replication. We observed that activation of the intra-S checkpoint did not explain the loss of viability of a constitutively acetylated H3K56 mutant while the activity of the origins is compromised. Finally, we identified a role for H3K56 acetylation in the initiation of replication origins. By classical genetic methods, we also observed that the synthetic lethality between constitutive acetylation of H3K56 and loss of SRS2 cannot be reversed by deletion of members of the canonical H3K56 acetylation pathway. Furthermore, upon persistence of H3K56 acetylation, we found that the presence of Rad51 proves toxic to srs2Δ cells. Taken together, our results reveal previously unknown functions of H3K56 acetylation as well as novel links between chromatin structure and DNA replication dynamics.