| Project/Area Number |
09680688
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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 | The University of Tokyo |
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Principal Investigator |
MUROFUSHI Hiromu The University of Tokyo, Graduate School of Sciences, Associate Professor, 大学院・理学系研究科, 助教授 (70101128)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1998: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1997: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Xenopus egg / DNA replication / SSRP1 / Cdc68 / HMG protein / P97 ATPase / chromatin / p97ATPアーゼ / DNA untwisting因子 / P97ATPアーゼ |
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| Research Abstract |
The modulation of DNA structure is a key step in the initiation of various DNA metabolic events such as transcription and replication. We purified from Xenopus eggs a novel heterodimeric factor that binds to and unwinds closed circular duplex DNA in the presence of eukaryotic topoisomerase I.The unwinding activity of the factor was observed equally when various plasmid or bacteriophage DNAs were incubated with the factor, indicating that a specific DNA sequence is not required for the unwinding reaction. This factor, termed DUF, consists of 87- and 140-kDa polypeptides. cDNA cloning and sequencing of the two polypeptides revealed the 87-kDa polypeptide to show homology to a mammalian HMG protein, T160/structure-specific recognition protein (SSRP1), and the 140-kDa polypeptide to show homology to Cdc68, a protein that controls the expression of several genes during G1 phase. Immunodepletion of DUF from Xenopus egg extracts drastically reduced the ability of the extract to replicate exogenously added sperm chromatin and plasmid DNA.These findings suggest that DUF is a novel factor involved in DNA replication in Xenopus egg extracts. We also revealed that DUF interacted with histones and loosened the chromatin reconstituted in vitro. These data strongly suggest that DUF functions in the alteration of the chromatin structure to that favorable for DNA replication. Immunoprecipitation experiments using anti-DUF revealed that DUF interacted with p97 ATPase in the egg extract. Both proteins were shown to be incorporated into nuclei assembled from sperm chromatin in egg extracts. These results suggest that p97 ATPase, that is involved in membrane fusion in the cytoplasm, may also function in DNA replication in the nucleus with DUF.
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