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Development of antibiotics by monitoring the inhibition of the ATP-binding to DnaA, the initiator protein of chromosomal DNA replication in bacteria

Research Project

Project/Area Number 12557210
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field Biological pharmacy
Research InstitutionThe University of Tokyo

Principal Investigator

SEKIMIZU Kazuhisa  The University of Tokyo, Graduate School of Pharmaceutical Sciences, Developmental Biochemistry, Professor, 大学院・薬学系研究科, 教授 (90126095)

Co-Investigator(Kenkyū-buntansha) 久保 健雄  東京大学, 大学院・薬学系研究科, 助教授 (10201469)
Project Period (FY) 2000 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥12,000,000 (Direct Cost: ¥12,000,000)
Fiscal Year 2001: ¥5,700,000 (Direct Cost: ¥5,700,000)
Fiscal Year 2000: ¥6,300,000 (Direct Cost: ¥6,300,000)
KeywordsDnaA protein / Staphylococcus aureus / ATP-binding / basic phospholipid / acidic phospholipid / DNA replication / DNA polymerase III / silkworm / 大腸菌 / DnaA蛋白質 / 阻害剤 / 抗菌物質 / 抗菌剤 / ATP / 蛋白質の精製
Research Abstract

Staphylococcus aureus causes opportunistic diseases for humans. In this study, we examined biochemical nature of Staphylococcus aureus DnaA protein, the replication initiator. Previous studies with Escherichia coli DnaA protein demonstrated that the ATP-binding form of DnaA protein is active, whereas the ADP-binding form is inactive. Purified Staphylococcus aureus DnaA proteins also showed high affinity for ATP and ADP. We showed that phosphatidylglycerol, an acidic phospholipids, stimulated the release of ADP from the ADP-DnaA complex, resulting in the activation of DnaA protein. Lysylphosphatidylglycerol, a basic phospholipid, was shown to inhibit the action of phosphatidylglycerol. Thus, phosphatidylglycerol may negatively regulate re-activation of DnaA protein. We propose here a new model of the regulation of initiation of DNA replication by increase in the content of basic phospholipids in cytoplasmic membranes.
We isolated mutants of Staphylococcus aureus whose DNA replication were temperature-sensitive. By complementation analysis, we identified polC, dnaE, and dnaC genes which are essential for DNA replication in the bacteria. We also established an animal model of infectious diseases by using silkworms. The system will provide a useful screening system for medicines against infectious diseases.

Report

(3 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • Research Products

    (26 results)

All Other

All Publications (26 results)

  • [Publications] Nishida S. et al.: "A nucleotide switch in the E. coil DnaA protein initiates chromosomal replication : Evidence from a mutant DnaA protein defective in regulatory ATP hydrolysis in vitro and in vivo"J Biol Chem. (in press). (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kaito C. et al.: "Silkworm larvae as an animal model of bacterial infection pathogenic to humans"Microbial Pathogenesis. (in press). (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kuroda M. et al.: "Whole genome sequencing of meticillin-resistant Staphylococcus aureus"Lancet. 357・9264. 1225-1240 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Inoue R. et al.: "Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus"Mol Genet Genomics. 266・4. 564-571 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kubota T. et al.: "DnaA protein Lys-415 is close to the ATP-binding site : ATP-pvridoxal affinity labeling"Biochem Biophys Res Commun. 288・5. 1141-1148 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kawakami H. et al.: "Arrest of cell division and nucleoid partition by genetic alterations in the sliding clamp of the replicase and in DnaA"Mol Genet Genomics. 266・2. 167-179 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kondo T. et al.: "Suppression of temperature-sensitivity of a dnaA46 mutant by excessive DNA supercoiling"Biochem J.. 348・2. 375-379 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Nishida S. et al.: "A nucleotide switch in the E. coli DnaA protein initiates chromosomal replication : Evidence from a mutant DnaA protein defective in regulatory ATP hydrolysis in vitro and in vivo"J Biol Chem. (in press). (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kaito C. et al.: "Silkworm larvae as an animal model of bacterial infection pathogenic to humans"Microbial Pathogenesis. (in press). (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kuroda M. et al.: "Whole genome sequencing of meticillin-resistant Staphylococcus aureus"Lancet. 357. 1225-1240 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Inoue R. et al.: "Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus"Mol Genet Genomics. 266. 564-571 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kubota T. et al.: "DnaA protein Lys-415 is close to the ATP-binding site : ATP-pyridoxal affinity labeling"Biochem Biophys Res Commun. 288. 1141-1148 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kawakami H. et al.: "Arrest of cell division and nucleoid partition by genetic alterations in the sliding clamp of the replicase and in DnaA"Mol Genet Genomics. 266. 167-179 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kondo T. et al.: "Suppression of temperature-sensitivity of a dnaA46 mutant by excessive DNA supercoiling"Biochem J.. 348. 375-379 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Nishida S. et al.: "A nucleotide switch in the E.coli DnaA protein initiates chromosomal replication : Evidence from a mutant DnaA protein defective in regulatory ATP hydrolysis in vitro and in vivo"J Biol Chem. (in press). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Kaito C. et al.: "Silkworm larvae as an animal model of bacterial infection pathogenic to humans"Microbial Pathogenesis. (in press). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Kuroda M. et al.: "Whole genome sequencing of meticillin-resistant Staphylococcus aureus"Lancet. 357・9264. 1225-1240 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Inoue R. et al.: "Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus"Mol Genet Genomics. 266・4. 564-571 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Kubota T. et al.: "DnaA protein Lys-415 is close to the ATP-binding site : ATP-pyridoxal affinity labeling"Biochem Biophys Res Commun.. 288・5. 1141-1148 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Kawakami H. et al.: "Arrest of cell division and nucleoid partition by genetic alterations in the sliding clamp of the replicase and in DnaA"Mol Genet Genomics. 266・2. 167-179 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Kondo T.et al.: "Suppression of temperature-sensitivity of a dnaA46 mutant by excessive DNA supercoiling"Biochem J.. 348・2. 375-379 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] Kenji Kurokawa: "Replication cycle-coordinated change of the adenine nucleotide-bound forms of DnaA protein in Escherichia coli"The EMBO Journal. 18・23. 6642-6652 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Tsutomu Katayama: "Inactivation of Escherichia coli DnaA protein by DNA polymerase III and negative regulations for initiation of chromosomal replication"Biochimie. 81. 835-840 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Nobuyoshi Akimitsu: "Increase in Resistance of Methicillin-Resistant Staphylococcus aureus to β-Lactams Caused by Mutations Conferring Resistance to Benzalkonlum Chloride, a Disinfectant Widely Used in liospitals"ANTIMICROBIAL AGENTS AND CHEMOTHERAPY. 43・12. 3042-3043 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Makoto Takata: "Mutant DnaA proteins defective in duplex opening of oriC, the orlgin of chromosomal DNA replication in Escherichia coli"Molecular Microbiology. 35・2. 454-462 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Taemi KONDO: "Suppression of temperature-sensitivity of a dnaA46 mutant by excessive DNA supercoiling"Biochem.J.. 348. 375-379 (2000)

    • Related Report
      2000 Annual Research Report

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Published: 2000-04-01   Modified: 2016-04-21  

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