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1993 Fiscal Year Final Research Report Summary

Biochemical studies of yeast replication or gin binding protein sand initiate complexes

Research Project

Project/Area Number 04680182
Research Category

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

Allocation TypeSingle-year Grants
Research Field 代謝生物化学
Research InstitutionThe University of Tokyo

Principal Investigator

MASAI Hisao  The University of Tokyo Associate Professor, 医科学研究所, 助教授 (40229349)

Project Period (FY) 1992 – 1993
Keywordsreplication origin / in vitro DNA replication / DNA binding proteins / initiation of DNA replication / single-stranded DNA / RNA recognition motif / GST-fusion protein / primosome
Research Abstract

South-western screening of a glutathione-S-transferase fusion protein library constructed from the yeast Saccharomyces cerevisiae genomic DNA lead to isolation of core T-rich strand binding protein (CTBP) clones that bound to single-stranded DNA containing the T-rich strand of the 11 base-pair core sequence of autonomously replicating sequences. One of these clones, CTBP1, contains a portion of previously described RBP1 which is an RNA- and single-stranded DNA binding protein of Saccharomyces cerevisiae. GST-CTBP1 fusion protein binds exclusively to the T-rich strand of the core sequence with apparent dissociation constant of 5*10^<-9> M but not to the A-rich strand or double-strand of the same sequence. Mutations within the core which reduce the number of T or C residues decrease the affinity to this protein. In keeping with this, binding to the core sequence is efficiently competed by poly [dT] or ply [dT-dC] as well as by poly [dC] albeit to a lesser extent, but not by poly [dA] or … More poly [dG] to any significant extent. Among polyribonucleic acids, it binds to poly [U] and poly [T] with affinity similar to but not greater than that of poly [dT] and poly [dC], respectively. Our results indicate that CTBP1 is a polydeoxypyrimidine binding protein of Saccharomyces cerevisiae. CTBP1 contains two sets of an RNA recognition motif (RRM) and a glutamine-stretch. The binding affinity of the N-terminal or C-terminal set containing one RRM and one glutamine stretch is nearly two orders of magnitude lower than that of the "wild-type" CTBP1 containing both sets. The isolated N-terminal or C-terminal RRM alone (RRM1 and RRM2, respectively) is sufficient for binding nucleic acids with the binding specificity similar to that of the "wild-type", although binding affinity of the isolated RRM2 is nearly two orders of magnitude lower than that of RRM1. Our results indicate that two RRMs present in CTBP1/RBP1 have differential binding affinity and that its high affinity to polydeoxypyrimidine results from synergy between two lower affinity RRMs. Less

  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] Ikeda,M., Arai,K and Masai,H.: ""A fusion protein library : an improved method for rapid screening and characterization of DNA binding or interacting proteins"" Gene. 181. 167-171 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ikeda,M., Arai,K. and Masai,H.: ""CTBP-1/RBP-1 of Saccharomyces cerevisiae protein which binds to T-rich single-stranded DNA containing the 11 base-pair core sequence of autonomously replicating sequence,is a polydeoxypyrimidine binding protein"" Eur.J.Biochem.238. 38-47 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Masai,H., Arai,K.: ""DanA- and PriA-dependent primosomes : two distinct replicaation complexes for replication of Escherichia coli chromosome"" Frontiers in Bioscience. 1. 49-59 (1996)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Masai,H., and Arai,K.: ""DanA-dependent assembly of the ABC primosome at the A site,a single-stranded DNA containing a dnaA box"" Eur.J.Biochem.230. 384-395 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Masai,H., Miyake,T.and Arai,K.: ""hsk1+,a Schizosaccharomyces Pombe gene related to Saccharomyces cerevisiae CDC7 kinase,is required for chromosomal replication"" EMBO J.14. 3094-3104 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Masai,H., Asai,T., Kubota,Y., Arai,K.,and Kogoma,T.: ""Escherichia coli PriA" protein is essential for inducible and constitutive stable DNA replications"" EMBO J.13. 5338-5345 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 正井 久雄: "バイオマニュアルシリーズ4 遺伝子導入と発現・解析法" 羊土社, 14 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 正井 久雄: "遺伝子工学実験法" 中外医学社, 8 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ikeda, M., Arai, K and Masai, H.: ""A fusion protein liberary : an improved method for rapid screening and characterization of DNA binding or interacting proteins"" Gene. 181. 167-171 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ikeda, M., Arai, K.and Masai, H.: ""CTBP-1/RBP-1 of Saccharomyces cerevisiae protein which binds to T-rich single-stranded DNA containing the 11 base-pair core sequence of autonomously replicationg sequence, is a polydeoxypyrimidine binding protein"" Eur.J.Biochem.238. 38-47 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Masai, H., Arai, K.: ""DnaA- and PriA-dependent primosomes : two distinct replication complexes for replication of Escherichia coli chromosome"" Frontiers in Bioscience. 1. 49-59 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Masai, H., and Arai, K.: ""DnaA-dependent assembly of the ABC primosome at the A site, a single-stranded DNA containing a dnaA box"" Eur.J.Biochem. 230. 384-395 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Masai, H., Miyake, T.and Arai, K.: ""hsk1+, a Schizosaccharomyces Pombe gene related to Saccharomyces cerevisiae CDC7 kinase, is required for chromosomal replication"" EMBO J.14. 3094-3104 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Masai, H., Asai, T., Kubota, Y., Arai K., and Kogoma, T.: ""Escherichia coli PriA protein is essential for inducible and constitutive stable DNA replications"" EMBOJ.13. 5338-5345 (1994)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-03-16  

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