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

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