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Repair Mechanisms of DNA Double Strand Breaks

Research Project

Project/Area Number 10044206
Research Category

Grant-in-Aid for Scientific Research (A).

Allocation TypeSingle-year Grants
Section一般
Research Field Molecular biology
Research InstitutionOsaka University

Principal Investigator

SHINAGAWA Hideo  Research Institute for Microbial Diseases, Osaka Univ. Professor, 微生物病研究所, 教授 (40029799)

Co-Investigator(Kenkyū-buntansha) IWASAKI Hiroshi  Research Institute for Microbial Diseases, Research Assiate, 微生物病研究所, 助手 (60232659)
COX Michael  Univ.of Wisconsin, Dept.of Biochemistry
CLARK Alvin  Lawrence Berkeley National Laboratory, De, Professor
CARR Anthony  Univ.of Sussex, MRC Cell Mutation Unit, Lecturer
SHIRLEEN Rea  Yale University, Dept.of Biology, Professor
Project Period (FY) 1998 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥11,600,000 (Direct Cost: ¥11,600,000)
Fiscal Year 2000: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1999: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1998: ¥4,400,000 (Direct Cost: ¥4,400,000)
KeywordsDouble Stand breaks / DNA recombination / Hjc protein / Holliday junction / Escherichia coli / Thermophilic archaea / Thermophilic bacteria / Yeast / rhp57遺伝子 / 耐熱細菌 / RuvBタンパク質 / hjc遺伝子 / 古細菌 / 相同的組換え / DNA修復 / チェックポイント / RecAタンパク質 / RadAタンパク質
Research Abstract

DNA double strand breaks occur quite frequently during DNA replication not only by external genotoxic agents but also by genotoxic agents internally produced as a result of normal cellular metabolism. To elucidate the repair mechanisms of DNA double strand breaks, we employed Escherichia coli and a thermophilic bacterium as model prokaryotes, Saccharomyces cerevisiaea and Schizosaccharomyces pombe as model eukaryotes, and Pyrococcus furiosus as a model archaea.
Holliday junctions are formed as recombination intermediates and during DNA replication when replication fork is blocked. RuvABC proteins are involved in processing the Holliday junctions in prokaryotes. To gain insights into molecular mechanisms of Holliday junction resolution by RuvABC, we combined mutational analysis of these proteins with ctystallographic studies. A large number of RuvABC mutant proteins have been analyzed in vivo and in vitro. The three dimensional structure of RuvA-Holliday junction complex was revealed by … More crystallographic analysis and the three domains of RuvA were assigned to differerest functions. The crystal structure of monomeric RuvB protein from thermophilic bacteria was elucidated and the hexameric model structure of RuvB was proposed based on the similarity with the crystal structures of AAA^+ ATPases recently revealed. Based on mutational studies, two basic residues and a phenylalanine of RuvC critically important for the interactions with Holliday junction were revealed.
We identified 4 novel genes involved in repair of DNA double strand breaks in S.pombe. They are also involved in DNA replication and thus link DNA recombination with replication.
We studied the function of Mgs (Maintenance of Genome Stability) proteins which are homologous to RuvB motor protein and highly conserved from bacteria to humans. It has DNA-dependent ATPase and DNA annealing activities. We demonstrated that it is required to maintain genome stability by regulating DNA superhelicity during DNA replication together with RecQ helicases and topoisomerases. Less

Report

(4 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • 1998 Annual Research Report
  • Research Products

    (29 results)

All Other

All Publications (29 results)

  • [Publications] Yoshikawa M.,H.Iwasaki & H.Shinagawa: "Evidence that phenylalanine 69 in Escherichia coli RuvC resolvase forms a stacking interaction during binding and destabilization of a Holliday junction DNA substrate."J.Biol.Chem.. (in press). (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yamada,K.,N.Kunishima,K.Mayanagi,T.Ohnishi,T.Nishino,H.Iwasaki,H.Shinagawa & K.Morikawa: "Crystal structure of the Holliday junction migration motor protein RuvB from thermus Thermophilus HB8."Proc.Natl.Acad.Sci.USA. 98. 1442-1447 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Ohnishi T.,H.Iwasaki,Y.Ishino,S.Kuramitsu,A.Nakata and H.Shinagawa: "Identification and characterization of Thermus thermophilus HB8 RuvA protein, the subunit of the RuvAB protein complex that promotes branch migration of Holliday junctions."Genes & Genet.Syst.. 75. 233-243 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yoshikawa,M.,H.Iwasaki,K.Kinoshita & H.Shinagawa: "Two basic residues, Lys-107 and Lys-118, of RuvC resolvase are involved in critical contacts with the Holliday junction for its resolution."Genes to Cells. 5. 803-813 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Ariyoshi,M.,T.Nishino,H.Iwasaki,H.Shinagawa & K.Morikawa: "Crystal structure of the Holliday junction DNA in complex with a single RuvA tetramer."Proc.Natl.Acad.Sci.USA. 97. 8257-8262 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Iwasaki,H.,YW.Han,T.Okamoto,T.Ohnishi,M.Yoshikawa,K.Yamada,H.Toh,H.Daiyasu,T.Ogura & H.Shinagawa: "Mutational analysis of the functional motifs of RuvB, an AAA^+ class helicase and motor protein for Holliday junction branch migration."Mol.Microbiol.. 36. 528-538 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yoshikawa M., H.Iwasaki & H.Shinagawa.: "Evidence that phenylalanine 69 in Escherichia coli RuvC resolvase forms a stacking interaction during binding and destabilization of a Holliday junction DNA substrate."J.Biol.Chem.. (in press). (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yamada, K., N.Kunishima, K.Mayanagi, T.Ohnishi, T.Nishino, H.Iwasaki, H.Shinagawa & K.Morikawa: "Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8."Proc.Natl.Acad.Sci.USA. 98. 1442-1447 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Ohnishi T., H.Iwasa ki, Y.Ishino, S.Kuramitsu, A.Nakata and H.Shinagawa.: "Identification and characterization of Themus thermophilus HB8 RuvA protein, the subunit of the RuvAB protein complex that promotes branch migration of Holliday junctions."Genes & Genet.Syst.. 75. 233-243 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yoshikawa, M., H.Iwasaki, K.Kinoshita & H.Shinagawa.: "Two basic residues, Lys-107 and Lys-118, of RuvC resolvase are involved in critical contacts with the Holliday junction for its resolution."Genes to Cells. 5. 803-813 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Ariyoshi, M., T.Nishino, H.Iwasaki, H.Shinagawa & K.Morikawa.: "Crystal structure of the Holliday junction DNA in complex with a single RuvA tetramer."Proc.Natl.Acad.Sci.USA. 97. 8257-8262 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Iwasaki, H., YW.Han, T.Okamoto, T.Ohnishi, M.Yoshikawa, K.Yamada, H.Toh, H.Daiyasu, T.Ogura & H.Shinagawa.: "Mutational analysis of the functional motifs of RuvB, an AAA^+ class helicase and motor protein for Holliday junction branch migration."Mol.Microbiol.. 36. 528-538 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yoshikawa M.,H.Iwasaki & H.Shinagawa: "Evidence that phenylalanine 69 in Escherichia coli RuvC resolvasc forms a stacking interaction during binding and destabilization of a Holliday junction DNA substrate."J.Biol.Chem.. (in press). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Yamada,K.,N.Kunishima,K.Mayanagi,T.Ohnishi,T.Nishino,H.Iwasaki,H.Shinagawa & K.Morikawa: "Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8."Proc.Natl.Acad.Sci.USA. 98. 1442-1447 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Ohnishi T.,H.Iwasaki,Y.Ishino,S.Kuramitsu,A.Nakata and H.Shinagawa: "Identification and characterization of Thermus thermophilus HB8 RuvA protein, the subunit of the RuvAB protein complex that promotes branch migration of Holliday junctions."Genes & Genet.Syst.. 75. 233-243 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Yoshikawa,M.,H.Iwasaki,K.Kinoshita & H.Shinagawa: "Two basic residues, Lys-107 and Lys-118, of RuvC resolvase are involved in critical contacts with the Holliday junction for its resolution."Genes to Cells. 5. 831-841 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Ariyoshi,M.,T.Nishino,H.Iwasaki,H.Shinagawa & K.Morikawa: "Crystal structure of the Holliday junction DNA in complex with a single RuvA tetramer."Proc.Natl.Acad.Sci.USA. 97. 8257-8262 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Iwasaki,H.,YW.Han,T.Okamoto,T.Ohnishi,M.Yoshikawa,K.Yamada,H.Toh,H.Daiyasu,T.Ogura & H.Shinagawa: "Mutational analysis of the functional motifs of RuvB, and AAA^+ class helicase and motor protein for Holliday junction branch migration."Mol.Microbiol.. 36. 528-538 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Hishida, T., et al. and Shinaeawa, H.: "Role of Walker motif A of RuvB protein in promoting branch migration of Holliday junctions"J. Biol. Chem.. 274・36. 25335-25342 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Komori, K., Shinagawa, H., et al.: "A Holliday junction resolvase from Pyrococcus furiosus : Functional similarity to Escherichia coli RuvC provides evidence for conserved mechanism of homologous recombination in Bacteria, Eukarya, and Archaea"Proc. Natl. Acad. Sci. USA. 96. 8873-8878 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Komori, K., Shinagawa, H., et al.: "PI-PtuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification and identification of the homing-type endonuclease activities"Nucleic Acids Res.. 27・21. 4167-4174 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Yamada, K., et al. and Shinagawa, H.: "Novel properties of the Thermus thermophilus RuvB protein, which promotes branch migration of Holliday junctions"Mol. Gen. Genet.. 261. 1001-1011 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tsutsui, Y., et al., and Shinagawa, H.: "A Recombination repair gene of Schizosaccharomyses pombe, rhp57, is a functional homolog of the Saccharomyces cerevisiae RAD57 gene and is phylogenetically related to the Human XRCC3 gene"Genetics. 154(in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Ishioka,K.,et al.and Shinagawa,H.: "Abortive recombination in Escherichia coli ruv mutants blocks chromosome partitioning" Genes to Cells. 3・4. 209-220 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Ichiyanagi,K.,et al and Shinagawa,H.: "Mutational analysis on structure-fuction relationship of a Holliday junction specific endonuclease RuvC." Genes to Cells. 3・9. 575-586 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Ohsato,T.,Shinagawa,H.et al.: "R-loop in the replication origin of human mitochondrial DNA is resolved by RecG a Holliday junction specific helicase." Biochemical and Biophysical Research Communications. 255. 1-5 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Martinho,R.G.,Carr,A.M.,et al.: "Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses." EMBO Journal. 17・24. 7239-7249 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Sandler,S.J.et al.and Clark,A.J.: "Diversity of radA genes from cultured and uncltured archaea comparative analysis of putative RadA protein and thier use as a phylogenetic marler." Journal of Bacteriology. 181・3. 907-915 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Shan Q.and Cox M.M.: "On the mechanism of RecA-mediated repair of double-strand breaks: on role for four-strand DNA pairing intermediates." Molecular Cell. 1・2. 309-317 (1998)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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