| Project/Area Number |
05404083
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
Molecular biology
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| Research Institution | Osaka University |
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Principal Investigator |
SUGINO Akio Osaka Univ., Res.Inst.Microbial Diseases, Professor, 微生物病研究所, 教授 (90231737)
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| Co-Investigator(Kenkyū-buntansha) |
LEEM Sun-hee Osaka Univ., Res.Inst.Microbial Diseases, Research Associate, 微生物病研究所, 助手 (40263313)
MAKI Satoko Osaka Univ., Res.Inst.Microbial Diseases, Research Associate, 微生物病研究所, 助手 (60212205)
川崎 泰生 大阪大学, 微生物病研究所, 助手 (30243257)
荒木 弘之 大阪大学, 微生物病研究所, 助教授 (20151160)
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| Project Period (FY) |
1993 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥32,000,000 (Direct Cost: ¥32,000,000)
Fiscal Year 1995: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1994: ¥14,100,000 (Direct Cost: ¥14,100,000)
Fiscal Year 1993: ¥14,200,000 (Direct Cost: ¥14,200,000)
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| Keywords | Eukaryotic chromosomal DNA replication / Saccharomyces cerevisiae / DNA polymerase II (epsilon) / DNA polymerase III (delta) / DNA polymerase subunits / three DNA polymerase model / Cdc 7-Dbf4 complex / phosphatase / DNAポリメラーゼ / 細胞周期チェックポイント制御 / ORC蛋白 / 複製開始部位 / DNAヘリカーゼ / DNAポリメラーゼ補助因子 / In vitro DNA複製 / 蛋白精製 / クローニング |
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| Research Abstract |
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
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