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Functional studies of the interaction between human MCM6 and Cdt1 & Characterization of Nog1p for its novel function in DNA replication initiation in budding yeast

Authors Yu, Lan
Issue Date 2008
Summary DNA replication is one of the most fundamental cellular processes. To ensure proper execution of this process, eukaryotic cells exert strict controls over the initiation of DNA replication by stepwise assembly and disassembly of a series of replication-initiation proteins. In this thesis, I investigated the functional significance of the interaction between two replication-initiation proteins MCM6 and Cdt1 in human cells and the mechanism of a novel replication-initiation protein in budding yeast. One crucial step of initiation of DNA replication is the loading of the MCM complex onto chromatin. Cdt1 is required for this process in budding yeast and has been reported to physically interact with MCM6 in human cells. However, whether this interaction is responsible for the loading of the MCM complex has not been verified. Previously in our laboratory, the interacting domains have been identified in both proteins. In this project, our BrdU incorporation and chromatin binding results show that over-expression of the interacting fragments will lead to defects in DNA replication and chromatin association of MCM complex. These data suggest that the interaction is the key for Cdt1 to facilitate the MCM loading onto the chromatin. Since cells must reach a critical cell size before it can divide, DNA replication and ribosome biogenesis are thought to be coordinated via mechanisms that have not been fully understood. In the other project, I investigated Nog1p, a ribosome biogenesis protein, on its novel role in DNA replication. Previously in our laboratory, Nog1p was found to give an IDR (Initiation of DNA Replication) phenotype. Further studies indicate that it binds to ARS and is required for the G1/S phase transition. In this project, chromatin binding assay, co-IP and multicopy suppression assay have been carried out. The results show that Nog1p constantly binds to chromatin throughout the cell cycle and interacts with ORC both physically and genetically. The physical interaction between Nog1p and ORC takes place in G1 phase only. Our results suggest that Nog1p may play a regulatory role in DNA replication besides ribosome biogenesis. Further characterization of this protein may give more insight into the coordination between DNA replication and ribosomal biogenesis.
Note Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2008
Language English
Format Thesis
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