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In vitro reconstitution of the replication initiation complex

Research Project

Project/Area Number 13680757
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Molecular biology
Research InstitutionTokyo Medical and Dental University

Principal Investigator

SHIRAKATA Masaki  Medical Research Institute, Research Assistant, 難治疾患研究所, 助手 (70251551)

Project Period (FY) 2001 – 2002
Project Status Completed (Fiscal Year 2002)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
KeywordsReplication / EBV / Initiation complex / ORC
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.

Report

(3 results)
  • 2002 Annual Research Report   Final Research Report Summary
  • 2001 Annual Research Report
  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] 白形正樹: "Activation of TRAF5 and TRAF6 signal cascades negatively regulates the latent replication origin of Epstein-Barr virus through p38 MAPK"Journal of Virology. 75. 5059-5068 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 白形正樹: "Novel immediate-early protein IE19 of human cytomegalovirus activates origin recognition complex I promoter in a coopeative manner with IE72"Journal of Virology. 76. 3158-3167 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 白形正樹: "Replication licensing of the EBV oriP minichromosome"Current Topics in Microbiology and Immunology. 258. 13-33 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] K Hirai, and M. Shirakata.: "Replication licensing of the EBV oriP minichromosome"Current Topics in Microbiology and Immunology. 258. 13-33 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] M Shirakata, K Imadome, K Okazaki, and K Hirai.: "Activation of TRAF5 and TRAF6 signal cascades negatively regulates the latent replication origin of Epstein-Barr virus through p38 mitogen-activated protein kinase"J. Virol.. 75. 5059-5068 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] M. Shirakata, M. Terauchi, M Ablikim, K Imadome, K Hirai, T. Aso, and Y. Yamanashi.: "Novel immediate-early protein IE19 of human cytomegalovirus activates the origin recognition complex I promoter in a cooperative manner with IE72"J. Virol.. 76. 3158-3167 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 白形正樹: "Activation of TRAF5 and TRAF6 signal cascades negatively regulates the latent replication origin of Epstein-Barr virus through p38 MAPK"Journal of Virology. 75. 5059-5068 (2001)

    • Related Report
      2002 Annual Research Report
  • [Publications] 白形正樹: "Novel immediate-early protein IE19 of human cytomegalovirus activates origin recognition complex I promoter in a coopeative manner with IE72"Journal of Virology. 76. 3158-3167 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 白形正樹: "Replication licensing of the EBV oriP minichromosome"Current Topics in Microbiology and Immunology. 258. 13-33 (2001)

    • Related Report
      2002 Annual Research Report
  • [Publications] 白形正樹: "Novel immediate-early protein IE19 of humancytomegalovirus activates originarecognition complex I promoter ina cooperative manner with IE72"Journal of Virology. (印刷中). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] 白形正樹: "Activation of TRAF5 and TRAF6 signal cascades negatively regulates the latent replication origin of Epsterin-Barr virus through p38 MAPK"Journal of Virology. 75. 5059-5068 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] 白形正樹: "Replication licensing of the EBV oriP minichromosome"Current Topics in Microbiology and lmmunology. 258. 13-33 (2001)

    • Related Report
      2001 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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