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The role of Utp7p in DNA replication initiation in yeast saccharomyces cerevisiae

Authors Cheng, Xue
Issue Date 2010
Summary In Saccharomyces cerevisiae, initiation of DNA replication (IDR) requires the replicators ARS (autonomously replicating sequence) elements and replication-initiation proteins. Pre-replication Complex (pre-RC) assembly on ARS elements strictly control initiation of DNA replication, while it is still unknown if there are specific pre-RC assembly foci in nucleus and what are other components involving in this process. Here we identified a novel protein Utp7p (U3-associated protein 7) by yeast genetic screen which suggests that Utp7p may play a role in DNA replication initiation. Synthetic lethality data showed that Utp7p had genetic interactions with ORC, Cdc6 and MCM proteins which suggested that Utp7p may play a role in G1/S transition. Multicopy UTP7 suppressed orc5-1 temperature-sensitive phenotype. Western Blot using anti-Orc5 antibody detected Orc5p and its degradation bands showed that Utp7p inhibited Orc5-1 protein degradation by physical interaction. Co-IP assay showed that Utp7p had physical interactions with ORC and MCM proteins. ChIP assay showed that Utp7p bound to ARS elements. Utp7p depletion in vivo caused a G1/S transition defect by DNA content analysis. In vivo loading chromatin binding assay showed that Utp7p was required for MCM proteins loading onto chromatin. By using a high salt buffer extraction method, we found that ORC and Utp7p associated with nuclear matrix in G1 phase and disassociated with nuclear matrix in G2/M phase. Further, from M phase to G1 phase, ORC and Utp7p attached to nuclear matrix and then pre-RC assembled on nuclear matrix. At last, we found that ORC attachment to nuclear matrix depended on Utp7p.
Note Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2010
Language English
Format Thesis
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