| Project/Area Number |
13116201
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Biological Sciences
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| Research Institution | Hiroshima University |
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Principal Investigator |
MATSUURA Shinya Hiroshima University, RIRBM, Professor, 原爆放射線医科学研究所, 教授 (90274133)
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| Co-Investigator(Kenkyū-buntansha) |
KOMATSU Kenshi Kyoto University, Radiation Biology Center, Professor, 放射線生物研究センター, 教授 (80124577)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥12,300,000 (Direct Cost: ¥12,300,000)
Fiscal Year 2002: ¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 2001: ¥6,200,000 (Direct Cost: ¥6,200,000)
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| Keywords | Nijmegen breakage syndrome / Nbs1 / Knockout mouse / Radiation scnsitivity / DT40 cells / Homologous recombination / Histone H2AX / Nuclear foci / NBS / AT / NBS1 / 電離放射線 / DN二重鎖切断 / ATM / フォーカス / dsb / テロメア |
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| Research Abstract |
Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder, resulting from the defects in DSB repair. We have isolated the NBS1 gene, mutated in NBS patients. To analyze the NBS1 function in DSB repair, we established mouse Nbs1 knockout mutant. The Nbs1 mutants exhibited embryonic lethality. The embryos died at 8.5-9.5 dpc. On the other hands, the Nbs1-/-primary fibroblast cells were virtually viable. Chromosome analysis of the transformed MEF showed high frequencies of breaks and gaps after irradiation. Clonogenic analysis revealed cellular hypersensitivity to irradiation. These results clearly demonstrated that mouse Nbs1 is also responsible for genomic integrity.
Next, we carried out targeted disruption of the NBS1 gene using chicken DT4O cells. Scneo reporter assays revealed that NBS1-disrupted cells showed marked reduction of HR events ollowing generation of DSB. On the other hand, NHEJ assays using linearized plasmid DNA showed that NBS1 mutant DT40 cells were proficient in end-joining activity. These results demonstrated that NBS1 is essential for HR-mediated repair, but not for NHEJ repair. NBS1 might be required to process recombinant intermediates and to suppress inter-chromosomal translocation.
Histone H2AX is the first protein, which is phosphorylated by ATM and forms discrete foci immediately after irradiation. NBS1 complex also forms foci on sites of DSBs after irradiation. We reported that NBS1 directly binds to gamma-H2AX, via the FHA/BRCT domain, and form foci to co-localize with that of gamma-H2AX. Such foci formation could facilitate DNA repair and cell cycle monitoring. We proposed the two-step binding model of NBS1 complex for DNA repair and checkpoint control.
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