| Project/Area Number |
61065007
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| Research Category |
Grant-in-Aid for Specially Promoted Research
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| Allocation Type | Single-year Grants |
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| Research Institution | Kyushu University |
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Principal Investigator |
SEKIGUCHI Mutsuo Kyushu University, Faculty of Medicine Professor, 医学部, 教授 (00037342)
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| Co-Investigator(Kenkyū-buntansha) |
SAKUMI Kunihiko Kyushu University, Medical Institute of Bioregulation Research Associate, 生体防御医学研究所, 助手 (50211933)
NAKATSU Yoshimichi Kyushu University, Faculty of Medicine Research Associate, 医学部, 助手 (00207820)
HAYAKAWA Hiroshi Kyushu University, Faculty of Medicine Research Associate, 医学部, 助手 (70150422)
MAKI Hisaji Kyushu University, Faculty of Medicine Research Associate, 医学部, 助手 (20199649)
NAKABEPPU Yusaku Kyushu University, Faculty of Medicine Research Associate, 医学部, 助手 (30180350)
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| Project Period (FY) |
1986 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥242,000,000 (Direct Cost: ¥242,000,000)
Fiscal Year 1990: ¥33,000,000 (Direct Cost: ¥33,000,000)
Fiscal Year 1989: ¥34,000,000 (Direct Cost: ¥34,000,000)
Fiscal Year 1988: ¥60,000,000 (Direct Cost: ¥60,000,000)
Fiscal Year 1987: ¥60,000,000 (Direct Cost: ¥60,000,000)
Fiscal Year 1986: ¥55,000,000 (Direct Cost: ¥55,000,000)
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| Keywords | DNA repair / replication fidelity / DNA polymerase / alkylating agent / gene cloning / spontaneous mutation / induced mutation / methyltransferase / 突然変異 / DNA修復酵素 / DNA複製 / 転写調節 / ミューテーター / メチルトランスフェラーゼ / アルキル科剤 / 遺伝情報 / 遺伝子のクローニング / 発がんの抑制 / DNAポリメラーゼ |
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| Research Abstract |
It is of utmost importance for organisms to conserve their genetic information through generations, and this is achieved by accurate DNA replication and repair of damaged DNA. To resolve molecular mechanisms of these processes, we have studied the following three subjects.
1. Molecular mechanism of adaptive response of Escherichia coli cells to alkylating agents : Methylating agents trigger the adaptive response in E. coli generating an intracellular signal for its induction. This signal has been identified as one of the minor DNA methylation product, a methyl phosphortiester. The regulatory Ada protein, coded by the ada gene, transfers this particular methyl group to one of its own cysteine residues, and this post translational modification event converts the protein from a weak to a strong transcriptional activator. The methylated protein binds tightly to a specific DNA sequence in the promoter regions of the own gene, ada, and other genes induced in the response, thereby facilitating
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the initiation of transcription. Detailed molecular mechanisms have been analyzed by using various mutants having mutations in the structural gene and its regulatory region.
2. Roles of DNA polymerase in control of mutation frequency of cell : To achieve DNA replication with an exceedingly high accuracy, correction must be made of errors occurring during DNA synthesis. Most potent mutators heretofore defected in E. coli are associated with defects in epsilon subunit of DNA polymerase lll, encoded by the dnaQ gene. We have determined the nucleotide sequences of the wild type and the mutator dnaQ mutants and clarified structural and functional relationships. To elucidate the role of the alpha subunit. The catalytic subunit of the polymerase, in maintaining the high fidelity of DNA replication, we isolated mutator mutants having mutations in the dnaE gene, encoding the alpha subunit. The DNA polymerase lll purified from the mutant had a decreased proofreading capacity though it carried the normal level of editing exonuclease activity. A specific role of MutT protein to prevent dG・dA mispairing in DNA replication was also elucidated.
3. Mammalian genes for DNA repair and control of mutagenesis : O^6-methylguanine-DNA mthyltransferase plays a major role in preventing mutations as well as cell death after exposure to alkylating agents. By using an expression vector, we were able to clone cDNA for human methyltransferase enzyme and determine its nucleotide sequence. Taking advantage of overexpression of the cDNA, the enzyme was purified to apparent physical homogeneity and the physical and biochemical properties investigated. The human methyltransferase exhibits an extensive homology with those of the bacterial counter parts, and they can be replaced each other to replace genetic defects of the mutants. Less
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