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Characterization of the association of Dbf4 and Cdc7 with Mcm2-7 and chromatin in Saccharomyces cerevisiae.Ramer, Matthew January 2011 (has links)
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.
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Characterization of the association of Dbf4 and Cdc7 with Mcm2-7 and chromatin in Saccharomyces cerevisiae.Ramer, Matthew January 2011 (has links)
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.
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Characterizing the Associations and Roles of DDK and Mcm2-7 DNA Replication Proteins in Saccharomyces CerevisiaeSuman, Evelyin 20 May 2014 (has links)
The essential cell cycle kinase Dbf4/Cdc7 (DDK) triggers DNA replication through phosphorylation of the Mcm2-7 helicase at replication origins. Prior work has implicated various Mcm2-7 subunits as targets of DDK, however it is not well understood which specific subunits mediate the docking of the DDK complex. Through yeast two-hybrid and co-immunoprecipitation analyses, we found that Dbf4 and Cdc7 interact with distinct subunits of the Mcm2-7 helicase complex. Dbf4 showed the strongest interaction with Mcm2 while Cdc7 associated with Mcm4 and Mcm5. Dissection of the N-terminal region of Mcm2 revealed two regions that mediate the interaction with Dbf4, whereas in Mcm4, a region near the N-terminus has been previously identified by another group as the DDK docking domain. Mutant forms of Mcm2 (Mcm2ΔDDD) or Mcm4 (Mcm4ΔDDD) lacking the DDK docking domain were expressed in cells and resulted in modest growth and replication defects. Combining the two mutations resulted in synthetic lethality, suggesting a redundant mechanism of Mcm2 and Mcm4 in targeting the DDK complex to Mcm rings. Furthermore, growth inhibition could be induced in a Mcm4ΔDDD background by overexpressing Mcm2 to titrate Dbf4 from Mcm rings. These growth defects were exacerbated in the presence of genotoxic agents such as hydroxyurea and methyl methanesulfonate, suggesting that DDK-Mcm interactions may play a role in stabilizing replication forks under S-phase checkpoint conditions. Regions of Cdc7 were examined for their interaction with Mcm4 and Dbf4. Results have shown that the N-terminal amino acid region 55-124 and the C-terminal region 453-507 of Cdc7 are likely target regions for Dbf4-binding. Several conserved residues were identified within the N-terminal 55-124 Cdc7 region that interface with conserved residues within motif-C of Dbf4. Conserved residues were identified within the DDD domain of Mcm2 and mutating these residues resulted in a decreased interaction with Dbf4. Lastly, bioinformatics analysis has revealed potential conserved residues within the Mcm4DDD region, which may play a role in binding to Cdc7. This research is significant because these factors, which are conserved in all eukaryotes studied to date, should give further insight as to how DNA replication is triggered and how it is affected when cells are exposed to DNA damaging or replication compromising agents. This research also has implications in cancer genetics, as prior studies have shown elevated DDK and Mcm protein levels in tumour cell lines and melanomas, with Cdc7 showing great promise as a cancer therapeutic target. Such knowledge will further enhance our understanding of the DNA replication process and the roles of cell cycle proteins involved, under both normal and checkpoint conditions.
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Interaction of DUE-B and Treslin during the initiation of DNA replicationPoudel, Sumeet January 2016 (has links)
No description available.
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Genetic studies of genes involved in the initiation of DNA replication in the fission yeast Schizosaccharomyces pombeWang, Zhuo 28 October 2010 (has links)
No description available.
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