| Project/Area Number |
02404088
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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 |
分子遺伝学・分子生理学
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| Research Institution | NATIONAL INSTITUTE OF GENETICS |
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Principal Investigator |
HORIUCHI Kensuke National Institute of Genetics, Dept.Cell Genetics, Professor, 細胞遺伝研究系, 教授 (70219210)
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| Co-Investigator(Kenkyū-buntansha) |
NISHIMURA Yukinobu Toho University, Faculty of Science, Professor, 理学部, 教授 (30029699)
NISHIMURA Akiko National Institute of Genetics, Genet.Stock Res.Center, Associate Professor, 微生物保存研究室, 助教授 (20142002)
HIGASHITANI Atsushi National Institute of Genetics, Dept.Cell Genetics, Assistant Professor, 細胞遺伝研究系, 助手 (40212162)
HARA Hiroshi National Institute of Genetics, Dept.Cell Genetics, Assistant Professor, 細胞遺伝研究系, 助手 (00173071)
YASUDA Seiichi National Institute of Genetics, Dept.Cell Genetics, Associate Professor, 細胞遺伝研究系, 助教授 (90037250)
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| Project Period (FY) |
1990 – 1993
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| Keywords | replication origin / filamentous phage / DNA bending / superhelix / rolling circle / Escherichia coli / SOS / cell division |
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| Research Abstract |
1) We studied protein-DNA interactions in initiation of DNA replication of filamentous bacteriophages of E.coli. In relation to replication of the leading (plus) strand, we found that (a) the initiation frequency is a function of negative superhelical density of template DNA, (b) the initiator protein (gpII) specifically binds and bends DNA in a stepwise fashion, (c) this leads to melting of duplex in the nicking region, (d) the melting is a prerequisite for the initiation, and (e) negative superhelicity is required for the melting to occur. The binding of gpII to DNA is co-operative and involves protein-protein interactions. These results indicate that the initiation of replication is preceded by an ordered series of protein-induced DNA conformational changes. The replication enhancer region contains a strong binding site for the host IHF protein, which bends DNA.Our results indicate that interaction between two regions flanking the IHF site is essential for the function of the enhancer. Replication of the lagging (minus) strand is initiated by a RNA primer that is synthesized by the host RNA polymerase. We studied the interaction between the enzyme and the origin DNA.The results suggested that there is certain similarity in recongnition mechanisms between initiation of transcription and primer synthesis. We also found that mutant phage that are defective in the origin for lagging strand synthesis induce the SOS response in the infected cells, and demonstrated that single-stranded DNA per se is the SOS signal in vivo. 2) We studied how a dnaK mutation in E.coli depresses initiation of chromosomal replication, and found that DnaK stabilizes the initiator protein DnaA in vitro. 3) We completed mapping of a large number of fts genes, which are involved in cell division. 4) We identified the natural promoter for penicillin-binding protein 3. This promoter appears to serve for transcription of a large operon that includes many genes involved in cell division.
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