Initiation of DNA replication requires the action of the Dbf4/Cdc7 kinase complex (DDK) which is also a phosphorylation target of Rad53 kinase in the S-phase checkpoint. DDK is thought to trigger DNA replication by phosphorylating members of the Mcm2-7 complex present at origins of replication. While DDK phosphorylation sites have been identified on Mcm2-7, the contributions made by Dbf4 and Cdc7 to the targeting of the complex have not been established. DDK has also been implicated in the S-phase checkpoint response since it is removed from chromatin in a Rad53-dependent manner. The interaction of Dbf4 and Cdc7 with each of the Mcm2-7 subunits was assessed and showed an interaction between Dbf4 and Mcm2 and Mcm6, while interactions between Cdc7 and Mcm4 and Mcm5 were observed. Mutations in Mcm2 and Mcm4 that disrupt the interactions with Dbf4 or Cdc7 showed modest growth impairment and compromised DNA replication, while simultaneous abrogation of both interactions resulted in lethality. Strains overexpressing Mcm2 or Mcm4 were sensitive to genotoxic agents, while overexpression of Mcm2 in a Mcm4Δ175-333 strain background resulted in a severe growth impairment as well as sensitivity to genotoxic stress. ChIP analysis revealed the possibility of Dbf4/Cdc7 localization to origin flanking regions through most of S-phase, which may redistribute to origins at the time of firing. Fluorescence microscopy of Mcm2 and Dbf4 in S-phase seem to show a punctate pattern of staining, consistent with these factors’ localization to ‘replication factories.’ By using a Dbf4ΔN mutant, the N-motif was shown to be required for the Rad53-mediated removal of Dbf4 from chromatin under checkpoint conditions. Initial optimization of a DNA combing protocol was also performed, which along with Dbf4ΔN mutant and the fluorescently-epitope tagged strains, will be useful tools for evaluating a role for DDK in the S-phase checkpoint response. Altered levels of DNA replication factors have been implicated in many human cancers. The data presented in this study provide novel insight into the normal process of the initiation of DNA replication which can be applied to research involving higher eukaryotes, including humans, and can serve as a benchmark for comparison with the cancer phenotype.

A crucial step in eukaryotic cell proliferation is the initiation of DNA replication, a tightly regulated process mediated by a multitude of protein factors. In Saccharomyces cerevisiae, this occurs as a result of the concerted action of an assembly of proteins acting at origins of replication, known as the pre-replicative complex (pre-RC). While many of the mechanisms pertaining to the functions of these proteins and the associations amongst them have been explored experimentally, mathematical models are needed to effectively explore the network’s dynamic behaviour. An ordinary differential equation (ODE)-based model of the protein-protein interaction network describing DNA replication initiation was constructed. The model was validated against quantified levels of protein factors determined in vivo and from the literature over a range of cell cycle timepoints. The model behaviour conforms to perturbation trials previously reported in the literature and accurately predicts the results of knockdown experiments performed herein. Furthermore, the DNA replication model was successfully incorporated into an established model of the entire yeast cell cycle, thus providing a comprehensive description of these processes. A screen for novel DNA damage response proteins was investigated using a unique proteomics approach that uses chromatin fractionation samples to enrich for factors bound to the DNA. This form of sub-cellular fractionation was combined with differential-in-gel-electrophoresis (DIGE) to detect and quantify low abundance chromatin proteins in the budding yeast proteome. The method was applied to analyze the effect of the DNA damaging agent methyl methanesulfonate (MMS) on levels of chromatin-associated proteins. Up-regulation of several previously characterized DNA damage checkpoint-regulated proteins, such as Rnr4, Rpa1 and Rpa2, was observed. In addition, several novel DNA damage responsive proteins were identified and assessed for genotoxic sensitivity. A strain in which the expression of the Ran-GTPase binding protein Yrb1 was reduced was found to be hypersensitive to genotoxic stress, pointing to a role for this nuclear import-associated protein in DNA damage response. The model presented in this thesis provides a tool for exploring the biochemical network of DNA replication. This is germane to the exploration of new cancer therapeutics considering the link between this disease (and others) and errors in proper cell cycle regulation. The high functional conservation between cell cycle mechanisms in humans and yeast allows predictive analyses of the model to be extrapolated towards understanding aberrant human cell proliferation. Importantly, the model is useful in identifying potential targets for cancer treatment and provides insights into developing highly specific anti-cancer drugs. Finally, the characterization of factors in the proteomic screen opens the door to further investigation of the roles of potential DNA damage response proteins.

Errata pasted on front fly-leaf. / Bibliography: leaves 139-173. / iv, 173, [88] leaves, [1] leaf of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis identifies DNA structures and genes involved in the process of homologous chromosome pairing in allohexaploid bread wheat (Triticum aestivum). In addition to studying late replicating DNA, a speculative model on the action of the pairing genes in allohexaploid wheat and the putative function of the AWWM5 gene is discussed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Errata pasted on front fly-leaf. / Bibliography: leaves 139-173. / iv, 173, [88] leaves, [1] leaf of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis identifies DNA structures and genes involved in the process of homologous chromosome pairing in allohexaploid bread wheat (Triticum aestivum). In addition to studying late replicating DNA, a speculative model on the action of the pairing genes in allohexaploid wheat and the putative function of the AWWM5 gene is discussed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Mitochondrial DNA encodes thirteen protein subunits in the oxidative phosphorylation system (OXPHOS) that is responsible for cellular energy production. Mitochondrial disorders have been identified to be associated with mtDNA mutations. However, the molecular mechanisms of specific mtDNA mutations are still being explored in order to establish causative links. This study tries to elucidate the mutational effects of mtDNA on OXPHOS complex activities and cell growths. Using mouse 3T3 fibroblasts as a cell model, single-cell clones with different growth rates were isolated. The entire mtDNA genome was sequenced for mutations. The enzymatic activities of OXPHOS complex I to V were analysed. Three growth patterns represented by five clones were identified. Three clones (clone #2, #3, and #6) had the shortest doubling times (11.5 - 14.9 hours). Clone #1 had a medium growth rate (19.2 hous); and clone #5 had a significantly slow growth rate (22 hours). MtDNA sequencing results revealed that clone #5 had several heteroplasmic mutations (one in 16S rRNA, two in tRNAser (UCN), three in tRNAasp, one in tRNAlys, one in COI, five in COII, and one in ATPase8) while the other four clones showed sequence homology. Enzymatic analyses showed that on average clone #5 had significantly low complex III, IV, and V activities (p < 0.05). Changes in biochemical properties and protein structure were analyzed to deduct possible mechanisms for reduced respiration. In conclusion, the slow growth rate is associated with reduced OXPHOS enzyme functions. It is most likely that the combination of COI and COII mutations resulted in the reduction of complex IV function. It is still unclear whether the ATPase8 mutation (T7869A) in the non-conserved region alone can have such a pronounced phenotypic effect. A reduction in complex III also cannot be explained since there were no mutations in the only mtDNA-encoded complex III gene, but it is possible that there are mutations in the nDNA-encoded complex III genes. Mutations in tRNA and rRNA genes may also be responsible for reduced protein syntheses and consequently reduced OXPHOS activities. It is unclear why complex I activity was not affected. Although the mutational effect of individual mtDNA mutation observed cannot be clearly identified, this study establishes a correlation between mtDNA mutation and cell energy production and growth.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 67-74). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 6 uppsatser.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 78-82). Also available in electronic version.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 1999. / Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.