The Dbf4/Cdc7 kinase complex is required for the initiation of DNA replication and promotes this by acting upon members of the Mcm2-7 helicase. In addition to its role in replication, Dbf4/Cdc7 is a target of the S-phase checkpoint response through the Rad53 checkpoint kinase. In the budding yeast Saccharomyces cerevisiae, the regulatory subunit of this complex, Dbf4, is essential for kinase activity. Dbf4 is conserved throughout eukaryotes and contains three regions of discrete homology, termed the N, M, and C motifs, based on their location in the polypeptide chain. Motif C shows the highest conservation of all the motifs of Dbf4 and contains a CCHH type zinc finger. Mutation of the conserved cysteine and histidine residues of this zinc finger impair interactions with origin DNA and the Mcm2-7 helicase subunit Mcm2, but do not disrupt associations with Cdc7, Orc2, or Rad53. Cells where the endogenous Dbf4 CCHH zinc finger has been mutated exhibit slowed growth, and are delayed in their entry to, and progression through S-phase. These cells also display sensitivity upon long-term exposure to the ribonucleotide reductase inhibitor hydroxyurea (HU) and the DNA alkylating agent methyl methanesulfonate (MMS). The crystal structure of an amino-terminal region of Dbf4 containing motif N folds as a BRCA1-carboxy-terminal (BRCT) domain. This domain is required for the interaction with Rad53, but is not sufficient. A fragment of Dbf4 containing the BRCT domain and its fifteen preceding amino acids is sufficient to interact with Rad53 and folds as a modified BRCT domain containing an integral amino-terminal helical projection. Denoted the Helix-BRCT (HBRCT) domain, mutations that destabilize it abrogate the interaction with Rad53, and result in sensitivity to genotoxic agents. Dbf4 is recognized by the forkhead-associated FHA1 domain of Rad53, and the HBRCT domain of Dbf4 interacts directly with FHA1 in vitro. This interaction is phosphorylation independent and relies on a conserved lateral surface of FHA1, distinct from the phosphoepitope binding surface, which when mutated abrogates the interaction between Dbf4 and Rad53 and results in sensitivity to HU and MMS. The in vitro interaction between FHA1 and HBRCT does not require the ability of FHA1 to bind a phosphoepitope, while the in vivo interaction between full-length Rad53 and Dbf4 does. The FHA1 domain of Rad53 can simultaneously bind to a phosphopeptide and HBRCT, indicating that Rad53 recognition of Dbf4 may occur through a bipartite interaction using two surfaces of FHA1.

The cell cycle is a highly orchestrated developmental process that eventually leads to the reproduction of a cell. In metazoans, it is driven by the successive activation of cyclin-dependent kinases (Cdk) and proper coordination of cell cycle transitions and processes ensure genomic stability. DNA replication takes place during S phase to faithfully duplicate a cell’s genetic material. In eukaryotes, S phase onset involves the initiation of numerous licensed replication origins across the genome and requires the activities of two protein kinases, S phase-Cdk and Cdc7. In this thesis, I present work relating to the role of the S phase-promoting kinases in DNA replication and S phase regulation. Using the cell-free system of Xenopus egg extracts, a small molecule inhibitor of Cdc7, PHA-767491, was characterised. PHA-767491 was then used to demonstrate that Cdc7 executes its activity early in S phase before the Cdk-dependent step. Cdc7 is not rate limiting for the progression of the replication timing programme once its essential function has been executed, unlike S-Cdk whose activity is required throughout S phase. Protein Phosphatase 1 (PP1) was identified as a modulator of Cdc7 activity in egg extracts, which rapidly reverses Cdc7-dependent phosphorylation of chromatin-bound Mcm4 and likely functionally lowers Cdc7 activity during an etoposide-induced checkpoint response. This provides a novel mechanism for regulating Cdc7 by counteracting its activity on essential replication substrates in the event of replicative stress. In the second part of the thesis, the design strategy for generating a Cdc7-conditional knockout mouse (cko) is outlined and results from the screen for a transgenic founder are presented. A Cdc7-cko mouse will be a valuable tool to further dissect Cdc7 function and regulation in mammalian cells. In the final section, S phase entry and progression in mouse embryonic fibroblasts lacking both Cdk1 and Cdk2 was examined. Contrary to expectations, Cdk1/Cdk2 double knockout cells can enter S phase in the absence of detectable S phase-Cdk activity. S phase progression, however, was inefficient. Cdc6 and cyclin E1 proteins were found to accumulate in high levels in these cells. The exact function(s) and mechanism(s) for these observations remain to be discovered. With this work, I hope to provide additional insight into the roles and regulation of S phase kinases in eukaryotic DNA replication.

Obesity increases the risk of colon cancer as well as the expression of many cancer markers, ostensibly due to the interaction between insulin resistance and adipocyte production of hormones, mitogens and cytokines which collaborate to enhance proliferation signaling and impair the DNA damage response. Cdc7 and PCNA are both proteins involved in the DNA damage response as well as DNA replication. Both have also been shown to be upregulated in human tumours. To assess Cdc7 and PCNA roles during the DNA damage response in obese and lean animals, we administered azoxymethane (AOM), a colon-specific carcinogen, to obese and lean rats. Cdc7 and PCNA levels in colonic mucosal protein extracts from obese Zucker rats were compared with those from their lean counterparts. Significant differences were seen between lean and obese animals 3 hours post-AOM (lean Cdc7 levels > obese Cdc7 levels) and 24 hours post-AOM (lean PCNA levels > obese PCNA levels). This result suggests an impaired checkpoint response in obese animals relative to lean animals and supports a previously reported early role for Cdc7 in the checkpoint signaling cascade relative to a later role of PCNA in DNA damage repair. At the time tumours appeared (32 weeks post-AOM), colonic mucosal Cdc7 levels of obese rats exceeded that of their lean counterparts, suggesting that the obese metabolic environment causes upregulation of Cdc7 in obese rat epithelia. Cdc7 and PCNA levels were then compared between tumours and mucosa in obese and Sprague Dawley rats. Tumour Cdc7 levels were upregulated relative to mucosal levels in more samples than tumour PCNA levels, suggesting Cdc7 may be a more sensitive tumour marker. No significant differences in Cdc7 levels were seen between obese tumours and mucosa, likely due to elevation of obese mucosal Cdc7 levels. However, Sprague Dawley (non-obese) rats showed significantly higher Cdc7 and PCNA levels in tumours than mucosa, consistent with previous studies in human tissues. These results suggest that Cdc7 may be a more sensitive tumour marker than PCNA, but that its utility as a biomarker of colon cancer is dependent on the metabolic state (leanness) of the individual.

Obesity increases the risk of colon cancer as well as the expression of many cancer markers, ostensibly due to the interaction between insulin resistance and adipocyte production of hormones, mitogens and cytokines which collaborate to enhance proliferation signaling and impair the DNA damage response. Cdc7 and PCNA are both proteins involved in the DNA damage response as well as DNA replication. Both have also been shown to be upregulated in human tumours. To assess Cdc7 and PCNA roles during the DNA damage response in obese and lean animals, we administered azoxymethane (AOM), a colon-specific carcinogen, to obese and lean rats. Cdc7 and PCNA levels in colonic mucosal protein extracts from obese Zucker rats were compared with those from their lean counterparts. Significant differences were seen between lean and obese animals 3 hours post-AOM (lean Cdc7 levels > obese Cdc7 levels) and 24 hours post-AOM (lean PCNA levels > obese PCNA levels). This result suggests an impaired checkpoint response in obese animals relative to lean animals and supports a previously reported early role for Cdc7 in the checkpoint signaling cascade relative to a later role of PCNA in DNA damage repair. At the time tumours appeared (32 weeks post-AOM), colonic mucosal Cdc7 levels of obese rats exceeded that of their lean counterparts, suggesting that the obese metabolic environment causes upregulation of Cdc7 in obese rat epithelia. Cdc7 and PCNA levels were then compared between tumours and mucosa in obese and Sprague Dawley rats. Tumour Cdc7 levels were upregulated relative to mucosal levels in more samples than tumour PCNA levels, suggesting Cdc7 may be a more sensitive tumour marker. No significant differences in Cdc7 levels were seen between obese tumours and mucosa, likely due to elevation of obese mucosal Cdc7 levels. However, Sprague Dawley (non-obese) rats showed significantly higher Cdc7 and PCNA levels in tumours than mucosa, consistent with previous studies in human tissues. These results suggest that Cdc7 may be a more sensitive tumour marker than PCNA, but that its utility as a biomarker of colon cancer is dependent on the metabolic state (leanness) of the individual.

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.