| Project/Area Number |
07457446
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
病態科学系歯学(含放射線系歯学)
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
SASAKI Takehito Tokyo Medical and Dental Univ., Faculty of Dent., Prof., 歯学部, 教授 (90013896)
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| Co-Investigator(Kenkyū-buntansha) |
MIURA Masahiko Tokyo Medical and Dental Univ., Faculty of Dent., Instructor, 歯学部, 助手 (10272600)
DOMON Masaharu Tokyo Medical and Dental Univ., Faculty of Dent., Ass.Prof., 歯学部, 助教授 (60014198)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1997: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1996: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1995: ¥3,400,000 (Direct Cost: ¥3,400,000)
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| Keywords | PCNA / DNA Repair / DNA Polymerase / Excision Repair / Radiation / UV / 紫外線 / DNA修復 / 除去修復 / 放射線 / 突然変異 / ヌクレオチド |
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| Research Abstract |
The study supported by this grant has aleady been published as three papers in international journals as described elsewhere. The results are summarized as follows :
1. The ionizing-radiation induced PCNA-DNA complex formation was characterized in human diploid fibroblasts using an immunofluorescence method. It was revealed that complex formation is dependent on ATP and temperature, but not on protein synthesis or or activities of DNA polymerases delta/epsilon. The complex was lost 12-15 h after irradiation, which implies completion of the PCNA-dependent DNA synthesis, while it persisted when protein synthesis or activities of DNA polymerases delta epsilon were inhibited. Cells derived from a xeroderma pigmentosum group A patient, characteristic of complete deficiency in nucleotide excision repair (NER) , showed exactly the same time course as that in normal cells. These findings suggest that the repair of ionizing-radiation induced DNA damge requires newly synthesized proteins and DNA polymerases delta/epsilon activities to carry out DNA synthesis, and the initiation step is different from that in NER.
2. We pinpointed at which steps the PCNA complex is formed in NER employing XP-F and XP-G cells deficient in DNA endonucleases. Neither of cells showed PCNA complex formation, implicating that the complex is formed after coordinately regulated nicks at 3'and 5'sides of the DNA lesions in the NER process.
3. Mutational analysis of the XPA function on PCNA complex formation was performed using various mutations of XP-A cells. Mutations in the C4 type zinc finger domain completely lost the UV-induced complex formation, but those in the C2H2-like zinc finger domain did not affect the formation. These results were well correlated to the capacities of NER as reported previously. It was thus demonstrated that PCNA is closely linked the function of the XPA protein.
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