The Dbf4 protein is involved in the initiation of DNA replication, in complex with Cdc7 kinase, and also plays a role in the intra-S-phase checkpoint response via an interaction with Rad53 in Saccharomyces cerevisiae. The Dbf4 protein has three highly conserved motifs, called the N, M and C motifs. In view of the fact that a comprehensive analysis of the roles of the three motifs in the initiation of DNA replication and checkpoint response was not previously available, this study was, therefore, conducted. The objectives of the study were: (1) to assess the function of the three conserved motifs, with respect to their essentiality for cell viability, (2) to determine their roles in mediating interactions with other proteins (i.e. Cdc7, Orc2, Mcm2) involved in the initiation of DNA replication and with Rad53 in the intra-S-phase checkpoint response, and (3) to obtain the three-dimensional structure of the Dbf4 N-motif by X-ray crystallography. The Dbf4 N-motif was found to be nonessential for cell viability, mediates the interaction between Dbf4 and Rad53, and as well as the interaction with Orc2. A mutant lacking the N-motif (dbf4N), was found to have a growth defect and was hypersensitive to the genotoxic agents: hydroxyurea (HU) and methyl methane sulfonate (MMS), suggesting that a disruption in the intra-S-phase checkpoint occurred because of an abrogated Dbf4-Rad53 interaction. Double point mutation of two threonine residues of the N-motif (threonines 171 and 175) to alanines also caused an abrogated Dbf4-Rad53 FHA1 domain interaction. The Dbf4 M-motif was found to be essential for cell viability and mediates the interaction between Dbf4 and Mcm2. A single proline to leucine point mutation at amino acid residue 277 conferred resistance to HU and MMS and caused disrupted Dbf4-Mcm2 and Dbf4-Orc2 interaction, while Dbf4-Rad53 interaction was maintained. Thus, the alteration of the M-motif may facilitate the role of Dbf4 as a checkpoint target. The Dbf4-C motif was also found to be essential for cell viability. Deletion and point mutations to the C-motif affected the interactions between Dbf4 and Rad53, Orc2, Mcm2 and also with Mcm4. Attempts were also made to obtain the three-dimensional structure of Dbf4, using X-ray crystallography methods. The work presented here represents a thorough functional analysis of the three conserved domains of Dbf4 in Saccharomyces cerevisiae. These results can be used as a baseline for further research involving higher eukaryotic organisims, including humans. This is particularly of relevance in light of recent evidence demonstrating an overexpression of the human Dbf4 orthologue overexpression as a cancer phenotype in human cancer cells.

The Dbf4 protein is involved in the initiation of DNA replication, in complex with Cdc7 kinase, and also plays a role in the intra-S-phase checkpoint response via an interaction with Rad53 in Saccharomyces cerevisiae. The Dbf4 protein has three highly conserved motifs, called the N, M and C motifs. In view of the fact that a comprehensive analysis of the roles of the three motifs in the initiation of DNA replication and checkpoint response was not previously available, this study was, therefore, conducted. The objectives of the study were: (1) to assess the function of the three conserved motifs, with respect to their essentiality for cell viability, (2) to determine their roles in mediating interactions with other proteins (i.e. Cdc7, Orc2, Mcm2) involved in the initiation of DNA replication and with Rad53 in the intra-S-phase checkpoint response, and (3) to obtain the three-dimensional structure of the Dbf4 N-motif by X-ray crystallography. The Dbf4 N-motif was found to be nonessential for cell viability, mediates the interaction between Dbf4 and Rad53, and as well as the interaction with Orc2. A mutant lacking the N-motif (dbf4N), was found to have a growth defect and was hypersensitive to the genotoxic agents: hydroxyurea (HU) and methyl methane sulfonate (MMS), suggesting that a disruption in the intra-S-phase checkpoint occurred because of an abrogated Dbf4-Rad53 interaction. Double point mutation of two threonine residues of the N-motif (threonines 171 and 175) to alanines also caused an abrogated Dbf4-Rad53 FHA1 domain interaction. The Dbf4 M-motif was found to be essential for cell viability and mediates the interaction between Dbf4 and Mcm2. A single proline to leucine point mutation at amino acid residue 277 conferred resistance to HU and MMS and caused disrupted Dbf4-Mcm2 and Dbf4-Orc2 interaction, while Dbf4-Rad53 interaction was maintained. Thus, the alteration of the M-motif may facilitate the role of Dbf4 as a checkpoint target. The Dbf4-C motif was also found to be essential for cell viability. Deletion and point mutations to the C-motif affected the interactions between Dbf4 and Rad53, Orc2, Mcm2 and also with Mcm4. Attempts were also made to obtain the three-dimensional structure of Dbf4, using X-ray crystallography methods. The work presented here represents a thorough functional analysis of the three conserved domains of Dbf4 in Saccharomyces cerevisiae. These results can be used as a baseline for further research involving higher eukaryotic organisims, including humans. This is particularly of relevance in light of recent evidence demonstrating an overexpression of the human Dbf4 orthologue overexpression as a cancer phenotype in human cancer cells.

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.

<p>All living things must replicate their DNA. Despite being essential for life, this process is also inherently dangerous. Replication stress, which induces replication fork stalling, is an unavoidable risk that can trigger potentially harmful changes to the genome. Eukaryotes have a replication checkpoint pathway that stabilizes stalled replication forks to prevent damage. One of the critical protein interactions in this pathway, between Dbf4 and Rad53, pauses the cell cycle in budding yeast. This is important to give the cell time to recover from stress. The molecular details of this interaction were investigated to shed light on how this association is regulated by the cell. The structure of an N-terminal domain from Dbf4 was solved through X-ray crystallography and discovered to have a modified <em>BR</em>CA-1 <em>C</em>-<em>t</em>erminal (BRCT) fold, which included an additional N-terminal helix. This domain could interact with the <em>F</em>ork<em>H</em>ead <em>A</em>ssociated 1 (FHA1) domain from Rad53 <em>in vitro</em>, and the additional helix was necessary for complex formation. Although the FHA1 domain has a well-characterized binding site for phospho-epitopes, a combination of chemical cross-linking and NMR spectroscopy experiments demonstrated that the N-terminal domain from Dbf4 is contacting an alternative surface. However, the full-length Dbf4 protein <em>in vivo</em> may be contacting both this distal site and the phospho-epitope binding pocket. This bipartite interaction between Dbf4 and Rad53 would lend specificity to the complex and also suggests a kinase may be regulating the association. As FHA and BRCT domains are prevalent in eukaryotic nuclear proteins, these findings are instructive for how these domains mediate interactions in other signaling pathways.</p> / Doctor of Philosophy (PhD)