Molecular biological and genetical study on the mechanism of chromosomal DNA replicaiton in eukaryotes -Yeast as a model-

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1. Identification and purification of protein factor (s) interacting with either DNA polymerase II (epsilon) or III (delta) of Saccharromyces cerevisiae We have shown that DNA polymerase II (epsilon) is required for chromosomal DNA replication as well as two other DNA polymerase, DNA polymerase I (alpha) and III (delta) and have been proposing "three DNA polymerase model for eukaryotic chromosomal DNA replication." To prove this model, we have been reconstituting both leading and lagging strand replication complexes. In this study, we have identified and cloned the DPB11 gene whose product (Dpb11) interacts with DNA polymerase II (epsilon) catalytic and the second largest subunits.S.cerevisiae DNA polymerase III (delta) complex has been extensively purified and found to consist of at least three subunits, the 125-kDa catalytic subunit encoded by CDC2 (or POL3), the 55-kDa second largest subunit, and the 50-kDa subunit. We have determined partial amino acid sequence of both 55-kDa and 5 … More 0-kDa subunit polypeptides. The results showed that the 55-kDa subunit is the product of HUS2, which Matsumoto's group recently isolated and sequenced, and the 50-kDa subunit is the product of newly identified gene, YHR065C,which encodes a polypeptide having RNA helicase domain. These results suggest that DNA polymerase III (delta) complex participates in process of RNA primar removal from Okazaki-fragments (lagging strand synthesis product) during chromosome DNA replication.2. Identification of protein factor (s) which regulates the initiation of chromosomal DNA replication in S.cerevisiae. In the budding yeast S.cerevisiae, it has been known that the initiation of chromosomal DNA replication is regulated by Cdc7-Dbf4 cell-cycle protein kinase. In order to further understand its regulation, we have tried to isolate multicopy suppressors of the temperature-sensitive dbf4-1 mutation. From this experiments, we have identified, cloned, and sequenced two new genes, MSD3 and GLU8. MSD3 is an essential gene for cell growth and its temperature sensitive mutant cells (msd3-1) exhibited temperature-sensitive initiation of chromosomal DNA replication. GLU8 is known to be a reglator of the GLU7 gene product, a type I phosphatase. Therefore, the control of the initiation of chromosomal DNA replication is regulated by dephosphorylation as well as phosphorylation. Less