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

1. 我们已经证明DNA聚合酶II （epsilon）和其他两种DNA聚合酶，DNA聚合酶I （α）和DNA聚合酶III （δ）是染色体DNA复制所必需的，并提出了“真核生物染色体DNA复制的三种DNA聚合酶模型”。为了证明这个模型，我们已经重构了前导链和后链复制复合体。在这项研究中，我们鉴定并克隆了DPB11基因，其产物（DPB11）与DNA聚合酶II （epsilon）催化和第二大亚基相互作用。s.c reevisiae DNA聚合酶III （delta）复合物已被广泛纯化，并发现至少由三个亚基组成，由CDC2（或POL3）编码的125-kDa催化亚基，55-kDa第二大亚基和50-kDa亚基。我们已经确定了55-kDa和5…多个0-kDa亚基多肽的部分氨基酸序列。结果表明，55-kDa亚基是最近分离并测序的HUS2的产物，50-kDa亚基是新发现的基因YHR065C的产物，该基因编码具有RNA解旋酶结构域的多肽。这些结果表明，在染色体DNA复制过程中，DNA聚合酶III （delta）复合体参与了冈崎片段（滞后链合成产物）的RNA原代去除过程。调控酿酒酵母染色体DNA复制起始的蛋白因子的鉴定。在出芽酵母中，已经知道染色体DNA复制的起始是由Cdc7-Dbf4细胞周期蛋白激酶调控的。为了进一步了解其调控，我们试图分离温度敏感的dbf4-1突变的多拷贝抑制子。从这个实验中，我们已经鉴定、克隆和测序了两个新的基因，MSD3和GLU8。MSD3是细胞生长的重要基因，其温度敏感突变细胞（MSD3 -1）表现出温度敏感的染色体DNA复制起始。已知GLU8是GLU7基因产物（一种I型磷酸酶）的调节因子。因此，染色体DNA复制起始的控制既受去磷酸化调控，也受磷酸化调控。少

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