Studies on the structure and functions of eukaryotic DNA-unwinding factor, DUF
| Project/Area Number |
12680686
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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 Graduate School of Science, The University of Tokyo, Associate professor, 大学院・理学系研究科, 助教授 (70101128)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2001: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | Xenopus egg / Hela cell / DNA replication / SSRP1 / Cdc68p / Spt16P / FACT / chromatin / HMG protein / Cde68p |
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| Research Abstract |
DUF was found in Xenopus egg extract as a factor necessary for DNA replication. Our previous study has revealed that DUF consists of SSRP1 and a homologue of Cdc68p/Sptl6p and that it binds to and introduces negative twist to duplex DNA.
Immunoprecipitation experiments using anti-DUF antibodies showed that p97 ATPase interacted with DUF in Xenopus egg extracts. Purified DUF and p97 made a complex in vitro. DUF was bound to isolated chromatin that had been reconstituted from demembranated sperm heads and from M13 DNA in egg extracts. It was revealed that chromatin constructed in egg extracts from which DUF had been immunodepleted was more resistant to micrococcal nuclease than that made in egg extracts containing DUF, showing that DUF made chromatin structure more accessible for the enzyme. DUF was shown to bind to affinity columns to which core histones, H2A, H2B, H3, and H4 were immobilized, respectively. These resultssuggest that DUF has a capability of binding to DNA and to histones to introduce change in chromatin structure to that suitable for DNA replication. Subunits of human DUF were co expressed in Sf9 cells and the expressed DUF was successfully purified as a stable complex. Expressed human DUF was firmly bound to histone-immobilized columns showing that recombinant human DUF had the same high affinity for histones as Xenopus DUF purified from egg extracts. When nucleosomes isolated from chicken erythrocytes were applied to DUF-immobilized columns, significant amount of nucleosomes were bound to the columns.Immunostaining of A6 and HeLa cells with antibodies against DUF revealed that DUF was mainly localized to the nucleus in the interphase cells. Nucleolar staining was stronger than other parts of the nucleus. In mitotic cells, DUF was distributed uniformly throughout the cytoplasm except for condensed chromatin, showing that DUF was released from chromatin in mitosis.
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Report
(3 results)
Research Products
(9 results)
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[Publications] Mizuno K, † Hasemi T, Ubukata T, Yamada T, Lehmann E, Kohli J, Watanabe Y, lino Y, Yamamoto M, Fox M E, Smith G R, Murofushi H, Shibata T, Ohta K: "Counteracting regulation of chromatin remodeling at a fission yeast cAMP responsive element-related recombination hotspot by stress-activated protein kinase, cAMP-dependent kinase and meiosis regulators"Genetics. 159. 1467-1478 (2001)
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[Publications] Kitazawa H, lida J, Uchida A, Haino-Fukushima K, Itoh T J, Hotani H, Ookata K, Murofushi H, Bulinski J C, Kishimoto T, Hisanaga S: "Ser787 in the proline-rich region of human MAP4 is a critical phosphorylatlon site that reduces its activity to promote tubulin polymerization"Cell Struct Funct. 25. 33-39 (2000)
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