| Project/Area Number |
11480212
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cell biology
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| Research Institution | NARA INSTITUTE OF SCIENCE AND TECHNOLOGY |
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Principal Investigator |
OGASAWARA Naotake Nara Institute of Science and Technology, Graduate School of Information Science Professor, 情報科学研究科, 教授 (10110553)
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| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Kazuo Nara Institute of Science and Technology, Graduate School of Information Science Assistant Professor, 情報科学研究科, 助手 (70324978)
KASAHARA Yasuhiro Nara Institute of Science and Technology, Graduate School of Information Science Assistant Professor, 情報科学研究科, 助手 (20273849)
MORIYA Shigeki Nara Institute of Science and Technology, Graduate School of Information Science Associate Professor, 情報科学研究科, 助教授 (40191051)
朝井 計 奈良先端科学技術大学院大学, バイオサイエンス研究科, 助手 (70283934)
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| Project Period (FY) |
1999 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥12,700,000 (Direct Cost: ¥12,700,000)
Fiscal Year 2002: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2001: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥3,500,000 (Direct Cost: ¥3,500,000)
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| Keywords | Bacillus subtilis / Chromosome replication / Chromosome segregation / DnaA protein / Initiation complex / Replisome / SpoOJ protein / SMC protein / 染色体構造 / Spo0J蛋白質 / Smc蛋白質 / 複製開始 / DnaA / 細胞周期 / フローサイトメトリー / 染色体分離 / シス配列 / FISH / 蛋白質相互作用 / 酵母2ハイブリッド系 / DNAヘリカーゼ / 細胞内局在 / GFP / SpoOJ |
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| Research Abstract |
(1) Initiation of chromosome replication starts with formation of a complex of initiator protein DnaA and the oriC sequence. Then, DNA helicase, DnaC, is loaded on the complex by the action of DnaB, DnaD and DnaI proteins. We showed that DnaI acts as a helicase loader and DnaD interact with DnaA.
(2) DnaA was distributed throughout the cytoplasm, but both DnaB and DnaI seemed to be localized near the outer or inner edges of the nucleoids at initiation of replication, together with oriC. Furthermore, DnaX (a component of DNA polymerase) foci were detected near either of the edges of the nucleoids at the onset of replication, suggesting that the replisome is recruited into oriC near either edge of the nucleoids to initiate chromosome replication in B.subtilis.
(3) We obtained results indicating that, if the DnaA level increases with progression of the replication cycle, it could act as a rate-limiting factor of replication initiation in B.subtilis. We found that SpoOJ contributes to the po
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sitioning of the chromosomal oriC region. In addition, the spoOJ mutant produced a significant proportion of cells with increased chromosome content Furthermore we demonstrated that Soj interferes with tight control of replication initiation and causes early and asynchronous initiation and SpoOJ counteracts this Soj function in wild-type cells. Additionally we found that essential GTP-binding proteins, Bex and YqeH, appeared to participate in the regulation of initiation of chromosome replication.
(4) We demonstrated that the two DNA sequences in and downstream of the dnaA gene participate in correct positioning of the replication origin region within the cell and that this function is associated with accurate chromosome segregation in B.subtilis.
(5) When visualized simultaneously, SMC and the replisome were often in similar regions of the cell. Persistence of SMC foci did not depend on ongoing replication, but did depend on ScpA and ScpB, two proteins interacting with SMC. Our results indicate that SMC is bound to many sites on the chromosome and a concentration of SMC is localized near replication forks, perhaps there to bind and organize newly replicated DNA. Less
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