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Study on Structure and Function of Mammalian DNA Replication Entyme

Research Project

Project/Area Number 04454598
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 物質生物化学
Research InstitutionThe Institute of Phisical and Chemical Research (RIKEN)

Principal Investigator

HANAOKA Fumio  RIKEN, Cell. Physiol. Lab., Chief Scientist, 細胞生理学研究室, 主任研究員 (50012670)

Co-Investigator(Kenkyū-buntansha) MASUTANI Chikahide  RIKEN, Cell. Physiol. Lab., Special Researcher, 細胞生理学研究室, 基礎特研 (40241252)
SUGASAWA Kaoru  RIKEN, Cell. Physiol. Lab., Research Scientist, 細胞生理学研究室, 研究員 (70202124)
Project Period (FY) 1992 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1993: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1992: ¥4,000,000 (Direct Cost: ¥4,000,000)
KeywordsDNA polumerase alpha / ts mutant / growth-revertants / primase / gene expression / overexpression / DNAポリメラーゼalpha / 細胞周期 / 温度感受性突然変異株 / PCR-SSCP法 / 遺伝子発現
Research Abstract

We isolated cDNA of the catalytic subunit of DNA polymerase alpha from tsFT20 cells, a temperature-sensitive mutant cell line derived from mouse FM3A cells. DNA sequence analysis revealed that the cDNA has a single mutation, a cytosine to thymine substitution that changes amino acid 1180 from serine to phenylalanine. We have also shown that tsFT20 cells could be rescued by transfection with the wild-type cDNA.In addition, we detected mutation sites in one spontaneous and six N-methyl-N'-nitro-N-nitrosoguanidine-induced growth revertants of tsFT20 cells. All revertant cell lines had a second point mutation adjacent to the first mutation site in tsFT20 cells.
During activation of quiescent Swiss mouse 3T3 cells to proliferate, the levels of mRNA of the four subunits of the DNA polymerase alpha-primase complex increased before DNA synthesis. In order to analyze how the expression of these genes are controlled, we have isolated upstream region of these genes and determined their nucleotide sequence. There are 1-2 E2F-binding sequence and 10-14 dp palindromic sequence, and one AP1-binding motif in each upstream region.
We overexpressed cDNAs of the DNA polymerase alpha-primase complex in E.coli or insect cells, purified the gene products and raised the specific antibodies against them. Co-precipitation experiments showed that 54K subunit has affinity to all other three subunits. We have also shown that 180K subunit has DNA polymerase activity and 46K subunit has primase activity.

Report

(3 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • Research Products

    (21 results)

All Other

All Publications (21 results)

  • [Publications] T.Sudo: "The c-myb protoencogene-product binds to but does not activate the promoter of the DNA polymorase α gene." Oncogene. 7. 1999-2006 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] F.Takeuchi: "Lobenzarit disodium(CCA)inhibits the proliferation of human endothelial cells and the activity of DNA polymerase-α" Agents Actions. 36. 312-316 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] H.Miyazawa: "Molecular cloning of the cDNAs for the four subunits of mouse DNA polymerase α-primase complex and their gene expression during cell proliferation and the cell cycle" J.Biol.Chem.268. 8111-8122 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] W.Pendergrass: "A murine temperature-sensitive DNA polymerase α mutant displays a diminished capacity to stimulate DNA synthesis in senescent human fibroblast nuclei in hetero|caryons at the nonpermissive condition" J.Cell.Physiol.158. 270-276 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] M.Izumi: "Identification of a point mutation in the cDNA of the catalytic subunit of DNA polymerase α from a temperature-sensitive mouse FM3A cell line" J.Biol.Chem.269. 7639-7644 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] 菅澤 薫: "無細胞系を用いたクロマチンDNAの複製と修復" 実験医学. 20. 2716-2721 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Sudo, T., Miyazawa, H., Hanaoka, F., & Ishii, S.: "The c-myb proto-oncogene product binds to but does not activate the promoter of the DNA polymerase alpha gene" Oncogene. 7. 1299-2006 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Takeuchi, F., Kosuge, E., Hashimoto, Y.Matsuta, K., Hanaoka, F., Tanimoto, K.& Ito, K.: "Lobenzarit disodium (CCA) inhibits the proliferation of human endotherial cells and the activity of DNA polymerase alpha" Agents Actions. 36. 312-316 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Miyazawa, H., Izumi, M., Tada, S., Takeda, R., Masutani, M., Ui, M., & Hanaoka, F.: "Molecular cloning of the cDNAs for the four subunits of mouse DNA polymerase alpha-primase complex and their gene expression during cell proliferation and the cell cycle" J.Biol.Chem.268. 8111-8122 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Pendergrass, W., Saulewicz, A.C., Hanaoka, F., & Norwood, T.H.: "Murine temperature-sensitive DNA polymerase alpha mutant displays a diminished capacity to stimulate DNA synthesis in senescent human fibroblast nuclei in heterokaryons at the nonpermissive condition" J.Cell.Physiol.158. 270-276 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Izumi, M., Miyazawa, H., Harakawa, S., Yatagai, F., & Hanaoka, F.: "Identification of a point mutation in the cDNAs of the catalytic subunit of DNA polymerase alpha from a temperature-sensitive mouse FM3A cell line" J.Biol.Chem.269. 7639-7644 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Sugawara, K., Masutani, C., & Hanaoka, F.: "Chromosome replication and repair in cell-free systems.(in Japanese)" Jikken Igaku. 11. 2716-2721 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] H.Miyazawa: "Molecular cloning of the cDNAsfor the few subunits of mouse DNA polymerize alpha-primso complex and their gene ezpression during cell preliferation abd the cell" J.Biol.Chem.268. 8111-8122 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] K.Matsumoto: "TAF-1,a novel host factor required to stimulate the adomires core DNA replication" J.Biol.Chem.268. 10582-10587 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] W.Pendergrass: "A murine temparature-sensitive DNA polymerase alpha mutant displays a diminished capacity to stimurate DNA synthesis in sonescent human fibroblast nucler in heterokaiyens at the nerpermissive condition" J.Cell.Physicl.158. 270-276 (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] M.Izumi: "Identification of a point mutation in the cDNA of the catalytre subunit of DNA polymerase alpha from a temperature-sensitive mouse FM3A cell line" J.Biol.Chem.269(印刷中). (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] 菅澤 薫: "無細胞系を用いたクロマチンDNAの複製と修復" 実験医学. 20. 2716-2721 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] T.Sudo: "The c-myb photo-oncogene product binds to but does not activate the promoter of the DNA polymerase α gene" Oncogene. 7. 1999-2006 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] F.Takeuchi: "Lobenzarit disodium (CCA) inhibits the proliferation of human endothelial cells and the activity of DNA polymerase α" Agents Actions. 36. 312-316 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] H.Miyazawa: "Molecular cloning of the cDNAs for the four subunits of mouse DNA polymerase α-primase complex and their gene expression during cell proliferation and the cell cycle" J.Biol.Chem. 268. (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] 泉 雅子: "DNA複製の開始機構" CRC(Cancer Research and Clinics). 2. 62-69 (1993)

    • Related Report
      1992 Annual Research Report

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Published: 1992-04-01   Modified: 2016-04-21  

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