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EXPRESSION OF EUKARYOTIC DNA REPLICATION PROTEIN COMPLEXS AND THEIR SIMPLE PURIFICATION METHOD

Research Project

Project/Area Number 07558101
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section試験
Research Field Molecular biology
Research InstitutionOsaka University

Principal Investigator

SUGINO Akio  OSAKA UNIV,RES.INSTIT.FOR MICROBIAL DISEASES,PROFESSOR, 微生物病研究所, 教授 (90231737)

Co-Investigator(Kenkyū-buntansha) MASAI Hisao  UNIV.TOKYO,INSTIT.MED.SCI., ASSOCIATE PROFESSOR, 医科学研究所, 助教授 (40229349)
TSURIMOTO Toshiki  NARA INSTIT.SCI.TECH., ASSOCIATE PROFESSOR, バイオサイエンス科, 助教授 (30163885)
ITOH Tateo  SHINSHU UNIV., FACULTY OF SCIENCE,PROFESSOR, 理学部, 教授 (40051817)
MAKI Hisaji  NARA INSTIT.SCI.TECH,PROFESSOR, バイオサイエンス科, 教授 (20199649)
MAKI Satoko  OSAKA UNIV.RES.INSTIT.FOR MICROBIAL DISEASES,RESEARCH ASSOCIATE, 微生物病研究所, 助手 (60212205)
Project Period (FY) 1995 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥21,300,000 (Direct Cost: ¥21,300,000)
Fiscal Year 1996: ¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1995: ¥14,300,000 (Direct Cost: ¥14,300,000)
KeywordsDNA Replication protein complex / Expression vector / Baculovirus expression vector / Insect cells / Budding yeast / Cdc7 / Dbf4 protein kinase complex / RF-C complex / DNA polymerase complex / YACベクター / Cdc7-Dbf4複合体 / RFC複合体
Research Abstract

In last year, we have succeeded in expressing yeast Saccharomyces cerevisae Cdc7/Dbf4 protein kinase complex which is required for the initiation of yeast chromosomal DNA replication using Vaculovirus vector in Sf9 insect cells. In this year, we tried to develop a new and simple purification method of Cdc7/Dbf4 protein complex from Sf9 cells infected with the recombinant virus containing CDC7 and DBF4 genes using Mcm2 protein as a kinase substrate. Using MonoQ,hydroxylapatite, and Heparin-Sepharose columns, we were able to purify the protein complex. Using the purified Cdc7/Dbf4 protein kinase complex, we found that the kinase phosphorylates not only S.cerevisiae Mcm2 protein, but also Mcm3, Mcm4, Mcm6, and Dbf4 protein. However, Mcm proteins expressed in and purified from E.coli were not good substrates for the kinase, suggesting that the kinase prefers partially phosphorylated proteins.
Using yeast expression vector containing Gal 1 and Gal10 promotors, we were able to express yeast R … More F-C complex consisting of five different subunits (Rfc1p (Cdc44p), Rfc2p, Rfc3p, Rfc4p and Rfc5p) in yeast. In the presence of galactose, yeast cells expressed at least 50 time more RF-C activity than in the absence of galactose. Since PCNA stimulates the ATPase activity of RF-C complex, RF-C complex should tightly interact with PCNA.Based un this, we made PCNA-conjugated Sepharose column. Using this column, we were able to obtain more than mg of highly purified RF-C complex from 1 L culture of yeast cells. This amount of pure RF-C complex makes more detailed study on protein-protein interaction between RF-C complex, DNA polymerases and PCNA.
Finally, we tried to express yeast DNA polymerases II and III complex using either yeast expression vectors or vaculovirus vector. However, so far we were not able to obtain any system expressing DNA polymerase complex. The main reason why we could not obtain any system that expresses yeast DNA polymerase complex was due to unstable recombinant vector DNA containing the gene encoding the DNA polymerase catalytic subunit in E.coli. Less

Report

(3 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report
  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] Sugimoto, K.: "Rfc5, a small subunit of replicaton factor C complex, couples DNA replication and mitosis in budding yeast." Proc. Natl. Acad. Sci., USA. 93. 7048-7052 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Noskov, V.: "The RFC2 gene encoding the third largest subunit of the RF-C complex is required for chromosomal DNA replication and cell cycle checkpoint in the yeast Saccharomyces cerevisiae." Mol. Cell Biol.17 (in press). (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Sugino, A.: "Overproduction and affinity purification of Saccharomyces cerevisiae replication factor C." J. Biol. Chem.272 (in press). (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Hashimoto, K.: "The second subunit of DNA polymerse III (δ)is encoded by the HYS2 gene in Saccharomyces cerevisiae." J. Biol. Chem.272 (in press). (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Sugimoto, K,Shimomura, T., Hashimoto, K., Araki, H., Sugino, A., and Matsumoto, K.: "Rfc5, a smalll subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast" Proc.Natl.Acad.Sci., USA,93. 7048-7052 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Noskov, V., Araki, H., and Sugino, A.: "The RFC2 gene encoding the third subunit of the RF-C complex is required for chromosomal DNA replication and cell cycle checkpoint in the yeast Saccharomyces cerevisiae." Mol.Cell.Biol.(in press). (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Sugino, A., Hashimoto, K., and Maki, S.: "Overproduction and affinity purification of Saccharomyces cerevisiae replication factor C." J.Biol.Chem.(in press). (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Hashimoto, K., Ohara, T., Maki, S., and Sugino, A.: "The second subunit of DNA polymerase III (delta) is encoded by the HYS2 gene in Saccharomyces cerevisiae." J.Biol.Chem.(in press). (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Sugimoto,K.: "Rfc5,a small subunit of replication factor C complex,couples DNA replication and mitosis in budding yeast" Proc.Natl.Acad.Sci.,USA. 93. 7048-7052 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Noskov,V.: "The RFC2 gene encoding the third largest subunit of the RF-C complex is required for chromosomal DNA replication and cell cycle checkpoint" Mol.Cell Biol.17(in press). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Lei,M.: "Mcm proteins are phosphorylated by Dbf4/Cdc7 protein kinase at the G1/S transition in Saccharomyces cerevisiae" Cell. (in press). (1997)

    • Related Report
      1996 Annual Research Report
  • [Publications] Sugimoto,K.,et al.: "RFC 5,a small subunit of replication factor C complex,couples DNA replication and mitosis in budding yeast." Proc.Natl.Acad.Sci.,U.S.A.92(in press). (1996)

    • Related Report
      1995 Annual Research Report

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Published: 1995-04-01   Modified: 2016-04-21  

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