| Project/Area Number |
62570108
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Okayama University |
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Principal Investigator |
SEKI Shuji Okayama University Medical School, 医学部, 助教授 (50032884)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1988: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1987: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | Repair DNA synthesis / Excision repair / Priming factor / Exonuclease / DNA polymerase / Bleomycin / ATP / 除去修復 / priming factor / primer activating enzyme |
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| Research Abstract |
To study the mechanism of DNA repair, a DNA repair system employing bleomycinpretreated, permeable mammalian cells was established and characterized. 1. The DNA repair system was shown to be useful to study removal of bleomycin-damaged ends by an exonuclease (priming), repair DNA synthesis by DNA polymerases and repair patch ligation by DNA ligase(s). 2. Aphidicolin-sensitive DNA polymerase (DNA polymerases and/or ) and DNA polymerase were shown to be involved in the repair DNA synthesis. Inhibitor studies suggested that aphidicolin-sensitive DNA polymerase plays a preferential role in repair label in the intranucleosomal regions of nuclear chromation and DNA polymerase in the completion of repair patches. 3. The DNA repair required ATP for at least two steps: repair DNA synthesis and repair patch ligation. ADP can apparently replaced ATP in both steps. The ADP effect on the repair patch ligation was attributed to ATP formed from ADP by adenylate kinase in permeable cells. 4. A protein factor having exonucleolytic activity on bleomycin-damaged DNA and providing priming sites for DNA polymerases existed in a DNA polymerase fraction pertially purified by ion exchange chromatography from an extract of permeable cells. The priming factor (exonuclease) was separated from DNA polymerase by single-stranded DNA-cellulose chromatography, and partially characterized. 5. A cell-free system for studying DNA repair was constructed from bleomycin-damaged DNA (or X-irradiated DNA), the priming factor, substrates for DNA synthesis, DNA polymerase , ATP and T4 DNA ligase. 6. The result obtained using the cell-free system suggested that the priming factor can initiate repair of not only bleomycin-damaged DNA but also single-strand breaks of X-irradiated DNA.
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