2002 Fiscal Year Final Research Report Summary
In vitro reconstitution of the replication initiation complex
| Project/Area Number |
13680757
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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 |
Molecular biology
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
SHIRAKATA Masaki Medical Research Institute, Research Assistant, 難治疾患研究所, 助手 (70251551)
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| Project Period (FY) |
2001 – 2002
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| Keywords | Replication / EBV / Initiation complex |
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| Research Abstract |
The aim of this research is to reconstitute the replication initiation complex on the latent replication origin of Epstein Barr virus oriP from synthesized polypeptides of the replication initiation factors. These replication factors include the cellular replication licensing factors MCMs, cdc6, and the viral replication factor EBNA1, which is the only virus-encoded polypeptide required for the replication from oriP We expressed these polypeptides in insect cells using Baculo virus vector and prepared substantial amount of MCMs and cdc6 However, it was difficult to synthesize the full length EBNA1 protein. This problem was solved at last by synthesizing an EBNA1 mutant that lacked the Gly-Ala repeat in the amino terminal domain Moreover, a transient replication assay confirmed that the mutant held the replicator activity of the wild type. It has taken almost a year to solve this problem, and thereby the reconstitution experiment is still preliminary In addition to the reconstitution experiment, we investigated regulation of the oriP replication further in detail, and found that oriP is negatively regulated by the signal transduction mediated by TRAF5 and TRAF6 through p38 MAPK We also identified Thr534 of EBNA1, which is important for the negative regulation by p38 MAPK. The EBNA1 mutants of which Thr534 is substituted with Glu may provide a useful tool to examine the specificity of oriP replication in the reconstituted system.
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