2005 Fiscal Year Final Research Report Summary
Control of DNA replication mediated by the concentration of the initiator protein
| Project/Area Number |
16570143
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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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 | Nagoya University |
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Principal Investigator |
OGAWA Tohru Nagoya University, Graduate School of Science, Associate Professor, 大学院・理学研究科, 助教授 (80109256)
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| Project Period (FY) |
2004 – 2005
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| Keywords | DNA replication / initiation of replication / IHF / datA / oriC / DnaA |
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| Research Abstract |
Integration host factor (IHF) is a small basic protein that binds to specific DNA sequences and plays an architectural role in many cellular processes in prokaryotes. In this study, we found a novel function of IHF in the control of replication initiation in Escherichia coli. The initiation of replication is negatively controlled by a mechanism termed "initiator titration", a process where the initiator protein, DnaA, is titrated to newly replicated binding sequences on the chromosome. The initiator titration occurs predominantly at the datA locus that binds exceptionally large amounts of DnaA molecules. There is a consensus sequence for IHF binding in datA, which are essential for the high capacity DnaA binding. We found that IHF binds to this site in vitro. It is suggested that IHF supports assembly of highly ordered DnaA-DNA complex at datA during the E. coli cell cycle.
It was also found that the control of transcription of the dnaA gene is involved in the regulation of the initiation control. The dnaA gene of E coli is located close to oriC on the chromosome and is subjected to sequestration to the cell membrane for about one third of the cell cycle, beginning just after the initiation. Transcription of the gene is inhibited during the sequestration period. Movement of the gene locus on the chromosome, which alters the dnaA transcription in the cell cycle, disturbed initiation timing of replication.
In order to examine cellular localization and cell cycle dependent behavior of the DnaA protein we constructed strains that depend on DnaA-GFP fusion protein for growth. We are currently analyzing the behavior of the fusion protein.
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