| Project/Area Number |
14580692
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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 |
Cell biology
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| Research Institution | Yamaguchi University (2003)
The University of Tokyo (2002) |
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Principal Investigator |
MUROFUSHI Hiromu Yamaguchi University, Faculty of Science, Professor, 理学部, 教授 (70101128)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Xenopus egg / DNA replication / Cdc68p / Spt16p / SSRP1 / DNA repair / RPA / telomerase / DNA helicase / FACT / クロマチン / ヘリケース |
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| Research Abstract |
1. Alteration of chromatin structure by DUF
Chromatin was reconstituted in Xenopus egg extracts deprived of DUF with the aid of anti-DUF antibody and nuclease sensitivity of the chromatin was measured. It was revealed that chromatin made in the absence of DUF was less sensitive to nuclease than control chromatin made in the presence of DUF. It was also shown that DUF interacts not only with duplex DNA but also with core histones. These data suggest that DUF makes more relaxed chromatin accessible for replication factors
2. Search for factors interacting with DUF
Factors interacting with 140 kDa subunit of human DUF were screened by the yeast two-hybrid system. It was shown that CLKI, GU2, ribosomal proteins L15, and S26 interacted with DUF. Co-immunoprecipitation experiments revealed that Orc1 binds to DUF, suggesting that DUF in complex with ORC is involved in the initiation of DNA replication
3. Dynamics of RPA in DNA repair
Dynamics of RPA was investigated in Xenopus egg cell-free system. Double strand breaks in DNA induced RPA foci in the sites where DNA repair synthesis occurred. Inhibition of ATM/ATR showed little effects on the formation of RPA foci. These data suggest that DNA damage checkpoint mechanism is not involved in the formation of RPA foci (in collaboration with T.Enomoto, Tohoku University)
4. Study on DNA helicase with affinity for telomerase
In order to find new factors involved in DNA replication of mammalian telomeres, proteins co-purified with telomerase were investigated. cDNA cloning revealed that a protein with motifs of DNA helicase was co-purified with telomerase. (in collaboration with K.Murakami-Murofushi, Ochanomizu University). Expression of the protein was performed using the baculovirus system
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