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.