Enzymatic analyses of eukaryotic chromosomal DNA replication using the Epstein Barr Virus-derived oriP replicon

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 14208079
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Functional biochemistry
• Research Institution: Tokyo Metropolitan Organization for Medical Research
• 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)
• 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
• Project Period (FY): 2002 – 2004
• Keywords: Epstein Barr Virus / in vitro DNA replication / MCM complex / ORC complex / preRC / oriP / EBNA1 protein / cell cycle

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 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.

Research Products

• [Journal Article] Cell cycle and developmental regulations of replication factors in mouse embryonic stem cells. (2005)
  • Author(s): Fujii-Yamamoto, H, Kim, J-M., Arai, K, Masai,H.
  • Journal Title: J. Biol. Chem. 280 Pages: 12976-12987
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A second human Dbf4/ASK-related protein, Drf1/ASKL1 is required for efficient completion of S and M phases. (2005)
  • Author(s): Yoshizawa, N., Ishii, A., Taniyama, C., Matsui, E., Arai, K., Masai,H.
  • Journal Title: J. Biol. Chem. 280 Pages: 13062-13070
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Functional analyses of mouse ASK, an activator subunit for C dc7 kinase, using conditional ASK knockout ES cells. (2005)
  • Author(s): Yamashita, N., Kim, J-M, Koiwai, 0, Arai, K., Masai, H.
  • Journal Title: Genes to Cells (In press)
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Cell cycle and developmental regulations of replication factors in mouse embryonic stem cells. (2005)
  • Author(s): Fujii-Yamamoto, H., Kim, J-M., Arai, K., Masai, H.
  • Journal Title: J.Biol.Chem. 280 Pages: 12976-12987
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] A second human Dbf4/ASK-related protein, Drf1/ASKL1 is required for efficient completion of S and M phases. (2005)
  • Author(s): Yoshizawa, N., Ishii, A., Taniyama, C., Matsui, E., Arai, K., Masai, H.
  • Journal Title: J.Biol.Chem. 280 Pages: 11362-113070
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Functional analyses of mouse ASK, an activator subunit for Cdc7 kinase, using conditional ASK knockout ES cells. (2005)
  • Author(s): Yamashita, N., Kim, J-M., Koiwai, O., Arai, K., Masai, H.
  • Journal Title: Genes to Cells (In press)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Genetic dissection of mammalian Cdc7 kinase : Cell cycle and developmental roles. (2004)
  • Author(s): Kim, J-M, Masai, H.
  • Journal Title: Cell Cycle 3 Pages: 300-304
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Functional interaction between tumor suppressor menin and activator of S-phase kinase. (2004)
  • Author(s): Schnepp, R.W., Hou, Z., Wang, H., Petersen, C., Silva, A, Masai, H, Hua, X.
  • Journal Title: Cancer Res. 64 Pages: 6791-6796
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Overexpression of CR/periphilin downregulates Cdc7 expression and induces S-phase arrest. (2004)
  • Author(s): Kurita, M., Suzuki, H., Masai, H., Mizumoto, K., 0gala, E., Nishimoto, I., Aiso, S., Matsuoka,M.
  • Journal Title: Biochem. Biophys. Res. Commun. 32 Pages: 554-561
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Genetic dissection of mammalian Cdc7 kinase : Cell cycle and developmental roles. (2004)
  • Author(s): Kim, J-M., Masai, H.
  • Journal Title: Cell Cycle 3 Pages: 300-304
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Functional interaction between tumor suppressor menin and activator of S-phase kinase. (2004)
  • Author(s): Schnepp, R.W., Hou, Z., Wang, H., Petersen, C., Silva, A., Masai, H., Hua, X.
  • Journal Title: Cancer Res. 64 Pages: 6791-6796
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Overexpression of CR/periphilin downregulates Cdc7 expression and induces S-phase arrest. (2004)
  • Author(s): Kurita, M., Suzuki, H., Masai, H., Mizumoto, K., Ogata, E., Nishimoto, I., Aiso, S., Matsuoka, M.
  • Journal Title: Biochem.Biophys.Res.Commun. 32 Pages: 554-561
  • Description: 「研究成果報告書概要(欧文)」より