| Project/Area Number |
15570146
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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 | Yokohama City University, Kihara Institute for Biological Research |
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Principal Investigator |
KOYAMA Hideki Yokohama City University, Kihara Institute for Biological Research, Dev. Mol. Cell Genetics, Professor, 木原生物学研究所, 教授 (40085626)
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| Co-Investigator(Kenkyū-buntansha) |
ADACHI Noritaka Yokohama City University, Kihara Institute for Biological Research, Dev. Mol. Cell Genetics, Assistant Professor, 助手 (30264675)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2003: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | Base excision repair / DNA polymerase beta / FEN1 / chicken DT40 cell / Gene targeting / knockout cell / Short-patch BER / Long-patch BER / FEN1 / シーンターゲティング / 非相同組換え / 相同組換え / BER活性 |
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| Research Abstract |
Base excision repair (BER) is the major pathway in repair of DNA base lesions such as apurinic/apyrimidinic (AP) sites, or deaminated, alkylated or oxidative bases. The BER pathway is divided into DNA polymerase β(Polβ)-dependent short-patch BER and PCNA/flap endonuclease-1 (FEN-1)-dependent long-patch BER. We generated knockout cell lines deficient in either FEN-1 or Polβ, or both from the chicken DT40 cell line and studied differential roles of the proteins in the two subpathways. Surprisingly, double mutant cells could survive nevertheless of deficiency in the subpathways, implying the redundancy of both proteins or the existence of alternative pathways to backup the defects. Three mutant cell lines were all hypersensitive to methyl methanesulfonate, but FEN1-null and double mutants were hypersensitive to hydrogen peroxide compared with wild-type cells. In vitro BER assay, using cell-free extracts and a double-stranded DNA substrate containing one uracil, revealed that while wild-type and Polβ-null cells had an activity to repair the defect, FEN1-null and double mutant cells showed almost no activity. Importantly, this result indicates that FEN1, but not Polβ, is essential for repairing one base defect. Therefore, we are further studying the reason why cells lacking FEN1 but not Polβ are unable to repair the defect.
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