| Project/Area Number |
11480202
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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 |
Molecular biology
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| Research Institution | The University of Tokyo |
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Principal Investigator |
SEKIMIZU Kazuhisa Graduate School of Pharmaceutical Sciences, The University of Tokyo, Professor, 大学院・薬学系研究科, 教授 (90126095)
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| Co-Investigator(Kenkyū-buntansha) |
KUBO Takeo Graduate School of Pharmaceutical Sciences, The University of Tokyo, Associate Professor, 大学院・薬学系研究科, 助教授 (10201469)
小林 綾子 東京大学, 大学院・薬学系研究科, 助手 (90272484)
本間 光一 東京大学, 大学院・薬学系研究科, 助手 (90251438)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥15,300,000 (Direct Cost: ¥15,300,000)
Fiscal Year 2000: ¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1999: ¥8,300,000 (Direct Cost: ¥8,300,000)
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| Keywords | Escherichia coli / initiation of DNA replication / DnaA / ATP / ATPase / RIDA / replication fork / phospholipid / DnaA蛋白質 / DNA複製 / 複製開始制御 / DNAポリメラーゼ / 抗菌剤 |
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| Research Abstract |
The purpose of this study is to understand the regulatory mechanism of DnaA protein, the initiator of DNA replication in Escherichia coli. The head investigator et al. previously reported that (i) DnaA protein has a high affinity for ATP, (ii) ATP bound to DnaA protein is hydrolyzed to form the ADP-binding form of DnaA protein, which is inactive for DNA replication, and, (iii) DNA polymerase III holoenzyme, the replicase in Escherichia coli, stimulates the hydrolysis of ATP bound to DnaA protein. In this study, we established a new method to determine adenine nucleotide bound to DnaA protein in vivo. Radio labeled cells with [^<32>P] orthophosphate were homogenized, and DnaA protein was recovered by immuno-precipitation, followed by analysis by thin layer chromatography. By using various temperature-sensitive mutants of DNA replication, we demonstrated that replication proteins responsible for the formation of the replication fork participate in the reaction of the hydrolysis ATP bound to the initiator protein. We also established a method to determine phospholipid bound to DnaA protein. The radiolabeled immuno-precipitates were extracted with organic solvents, and analyzed by thin layer chromatography. We detected cardiolipin and phosphatidylglycerol, which are previously shown to bind to DnaA protein in vitro, in the immuno-precipitates. The result demonstrates that DnaA protein binds to acidic phospholipids in cells.
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