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

1.与酿酒酵母DNA聚合酶II(Epsilon)和DNA聚合酶III(Delta)相互作用的蛋白因子(S)的鉴定和纯化我们已经证明，染色体DNA复制需要DNA聚合酶II(Epsilon)，以及另外两个DNA聚合酶I(α)和DNA聚合酶III(Delta)，并提出了真核染色体DNA复制的三种DNA聚合酶模型。为了证明这一模型，我们一直在重组领先和滞后的链复制复合体。在本研究中，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。酿酒酵母DNA聚合酶III(Delta)复合体被广泛纯化，发现至少由三个亚基组成，即由CDC2(或POL3)编码的125 kDa催化亚基、55 kDa第二大亚基和50 kDa亚基。我们测定了55-kDa和5-…的部分氨基酸序列更多的0-kDa亚基多肽。结果表明，55 kDa亚基是松本团队最近分离和测序的HUS2的产物，50 kDa亚基是新发现的基因YHR065C的产物，YHR065C编码一种具有RNA解旋酶结构域的多肽。这些结果表明，DNA聚合酶III(Delta)复合体参与了染色体DNA复制过程中从冈崎片段(滞后链合成产物)中去除RNA引物的过程。调控酿酒酵母染色体复制起始的蛋白因子(S)的鉴定在萌芽酵母中，已知染色体DNA复制的启动是由细胞周期蛋白激酶CDC7-Dbf4调控的。为了进一步了解它的调控，我们试图分离温度敏感型DBF4-1突变的多拷贝抑制子。通过这次实验，我们已经鉴定、克隆和测序了两个新的基因，MSD3和GLU8。Msd3是细胞生长所必需的基因，其温度敏感型突变细胞(msd3-1)对温度敏感，可启动染色体DNA复制。已知GLU8是GLU7基因产物的调节子，GLU7是一种I型磷酸酶。因此，染色体DNA复制起始的控制既受去磷酸化的调控，也受磷酸化的调控。较少

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