| Project/Area Number |
14208079
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Tokyo Metropolitan Organization for Medical Research |
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Principal Investigator |
MASAI Hisao Tokyo Metropolitan Institute of Medical Science, Genome Dynamics Project, Project Leader, 東京都臨床医学総合研究所, 副参事研究員 (40229349)
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| Co-Investigator(Kenkyū-buntansha) |
SUGATA Naoko Tokyo Metropolitan Institute of Medical Science, Genome Dynamics Project, Senior Researcher, 東京都臨床医学総合研究所, 主任研究員 (30344071)
YOU Zhiying Tokyo Metropolitan Institute of Medical Science, Genome Dynamics Project, Senior Researcher, 東京都臨床医学総合研究所, 主任研究員 (90332270)
MORIYAMA Kenji Tokyo Metropolitan Institute of Medical Science, Genome Dynamics Project, Researcher, 東京都臨床医学総合研究所, 研究員 (00250217)
TSURIMOTO Toshiki Kyushu University, Faculty of Science, Professor, 理学部・生物学科, 教授 (30163885)
SIRAKATA Masaki Tokyo Medical and Dental University, Assistant Professor, 難治疾患研究所, 助手 (70251551)
TAMAI Katsuyuki Cyclex Co. Ltd., Chief Executive Queer, 代表取締役
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥54,080,000 (Direct Cost: ¥41,600,000、Indirect Cost: ¥12,480,000)
Fiscal Year 2004: ¥12,870,000 (Direct Cost: ¥9,900,000、Indirect Cost: ¥2,970,000)
Fiscal Year 2003: ¥11,830,000 (Direct Cost: ¥9,100,000、Indirect Cost: ¥2,730,000)
Fiscal Year 2002: ¥29,380,000 (Direct Cost: ¥22,600,000、Indirect Cost: ¥6,780,000)
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| Keywords | Epstein Barr Virus / in vitro DNA replication / MCM complex / ORC complex / preRC / oriP / EBNA1 protein / cell cycle / Epstein Brrr Virus / in viitro DNA複製 / in vitro DNA複製 |
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| Research Abstract |
Replication of Epstein Barr Viruses (EBV) occurs only once during S phase in coordination with the host cell cycle. The predominant replication origin of EBV, oriP, can support autonomous replication of a plasmid in the presence of virus-encoded EBNA-1 protein. oriP-dependent replication is cell cycle-regulated and depends on the host factors including ORC and most likely MCM, and supports a long-term maintenance of the plasmid in cells. Thus, EBV may serve as an excellent model replicon to probe the roles of cellular replication factors and to dissect the molecular mechanisms of the assembly and activation of eukaryotic replication complexes.
We first examined replication of the oriP plasmid on a cellular level. It required the passage through M phase and appears to occur during late S phase. The size of the episomal plasmid in a complex with chromatin proteins changes during cell cycle. We have also shown that Cdc7 function is required for oriP replication. We next attempted to assemb
… More
le the preRC (prereplicative complex) at the oriP. We speculated that proper chromatin structures would be required for assembly of a functional preRC and established a method for rapid isolation of the chromatin template form mammalian cells. The developed method has two features. 1) The oriP plasmid was linked to SV40 origin and the composite replicon plasmid was introduced into cells expressing the large T-antigen, which permitted highly efficient transient plasmid replication from the SV40 origin. This led to amplification of the plasmid copy number and allowed recovery of a sufficient amount of the template DNA for further analyses. 2) A multiple copies of the tetO sequence (the target of tetR protein) were added to the plasmid to facilitate the recovery of the template chromatin through tetR affinity beads. These modifications enabled us to rapidly isolate a sufficient quantity of the oriP plasmid chromatin.
We are now in the process of characterizing the isolated chromatin template to determine the replication proteins present in the complex. We also plan to reconstitute the preRC on the chromatin template using EBNA1 protein and the purified preRC components. We have already overexpressed and purified EBNA1, Cdt1 and MCM proteins, and are now purifying The Cdc6 and ORC complexes. We believe that the system we have established will be very useful for enzymatic characterization of the assembly and activation of the replication complexes at eukaryotic chromosomal replication origins. Less
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