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Analysis of DNA strand break repair using human knockout cells

Research Project

Project/Area Number 18590063
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Biological pharmacy
Research InstitutionYokohama City University

Principal Investigator

ADACHI Noritaka  Yokohama City University, International Graduate School of Arts and Sciences, Associate Professor (30264675)

Project Period (FY) 2006 – 2007
Project Status Completed (Fiscal Year 2007)
Budget Amount *help
¥3,890,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
KeywordsDNA strand break / DNA repair / end-ioining / random integration / gene targeting / nonhomologous recombination / 非相同組換之
Research Abstract

We recently developed a system using the human cell line Nalm-6 that enables rapid production of human gene knockout cells. In this study, we used this system to genetically analyze the function of human genes that have been implicated in DNA strand break repair. Specifically, we disrupted a series of genes involving those for Rad54, FancB, Mus81, Tdp1, Ku70/80, and Lig3, which all have some roles in DNA single and/or double-strand break repair in human cells. In addition, we tried to produce as many double-knockout mutant cells as possible, particularly those with a Lig4 mutation. Using these mutants, we have successfully analyzed the cellular sensitivity to anticancer agents involving cisplatin and topoisomerase inhibitors as well as the genetic interaction between p53 and BLM and between Mus81 and FancB, We also performed gene knockdown experiments with siRNAs for DNA ligases that are potentially involved in random integration and/or gene targeting. The results suggested that suppressing nonhomologous end-joining reactions could enhance the efficiency of gene targeting. Our findings also indicated that there must be another mechanism for random integration that does not rely on the nonhomologous end-joining pathway.

Report

(3 results)
  • 2007 Annual Research Report   Final Research Report Summary
  • 2006 Annual Research Report
  • Research Products

    (51 results)

All 2008 2007 2006

All Journal Article (23 results) (of which Peer Reviewed: 10 results) Presentation (28 results)

  • [Journal Article] The requirement of Artemis in doublestrand break repair depends on the type of DNA damage2008

    • Author(s)
      Kurosawa, A., et. al.
    • Journal Title

      DNA Cell Biol. 27

      Pages: 55-61

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] The requirement of Artemis in double-strand break repair depends on the type of DNA damage.2008

    • Author(s)
      Kurosawa, A., et. al.
    • Journal Title

      DNA Cell Biol 27

      Pages: 55-61

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] The requirement of Artemis in double-strand break repair depends on the type of DNA damage.2008

    • Author(s)
      Kurosawa, A., et. al.
    • Journal Title

      DNA Cell Biol. 27

      Pages: 55-61

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Absence of p53 enhances growth defects and etoposide sensitivity of human cells lacking the Bloom syndrome helicase BLM2007

    • Author(s)
      So, S., et. al.
    • Journal Title

      DNA Cell Biol. 26

      Pages: 517-525

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Human Mus81 and FANCB independently contribute to repair of DNA damage during rephcation2007

    • Author(s)
      Nomura, Y., et. al.
    • Journal Title

      Genes Cells 12

      Pages: 1111-1122

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Absence of p53 enhances growth defects and etoposide sensitivity of human cells lacking the Bloom syndrome helicase BLM.2007

    • Author(s)
      So, S., et. al.
    • Journal Title

      DNA Cell Biol 26

      Pages: 517-525

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Human Mus81 and FANCB independently contribute to repair of DNA damage during replication.2007

    • Author(s)
      Nomura, Y., et. al.
    • Journal Title

      Genes Cells 12

      Pages: 1111-1122

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Annual Research Report 2007 Final Research Report Summary
  • [Journal Article] Absence of p53 enhances growth defects and etoposide sensitivity of human cells lacking the Bloom syndrome helicase BLM.2007

    • Author(s)
      So, S., et. al.
    • Journal Title

      DNA Cell Biol. 26

      Pages: 517-525

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Absence of p53 enhances growth defects and etoposide sensitivity of human cells lacking the Bloom syndrome helicase BLM.2007

    • Author(s)
      So, S.et al.
    • Journal Title

      DNA Cell Biol. (In press)

    • Related Report
      2006 Annual Research Report
  • [Journal Article] The human pre-B cell line Nalm-6 is highly proficient in gene targeting by homologous recombination2006

    • Author(s)
      Adachi, N., et. al.
    • Journal Title

      DNA Cell Biol. 25

      Pages: 19-24

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Simple one-week nethod to construct gene-targeting vectors application to production of human knockout cell lines2006

    • Author(s)
      Iiizum, S., et. al.
    • Journal Title

      BioTechniques 41

      Pages: 311-316

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] 53BP1 contributes to survival of cells irradiated with X-ray durig G1 without Ku70 or Artemis2006

    • Author(s)
      Iwabuchi, K., et. al.
    • Journal Title

      Genes Cells 11

      Pages: 935-948

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells2006

    • Author(s)
      Hochegger, H., et. al.
    • Journal Title

      EMBO J. 25

      Pages: 1305-1314

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Enhanced gene targeting efficiency by siRNA that silences the expression of the Bloom syndrome gene in human cells2006

    • Author(s)
      So, S., et. al.
    • Journal Title

      Genes Cells 11

      Pages: 363-371

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] The human pre-B cell line Nalm-6 is highly proficient in gene targeting by homologous recombination.2006

    • Author(s)
      Adachi, N., et. al.
    • Journal Title

      DNA Cell Biol 25

      Pages: 19-24

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines.2006

    • Author(s)
      Iiizumi, S., et. al.
    • Journal Title

      Bio Techniques 41

      Pages: 311-316

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] 53BP1 contributes to survival of cells irradiated with X-ray during G1 without Ku70 or Artemis.2006

    • Author(s)
      Iwabuchi, K., et. al.
    • Journal Title

      Genes Cells 11

      Pages: 935-948

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells.2006

    • Author(s)
      Hochegger, H., et. al.
    • Journal Title

      EMBO J 25

      Pages: 1305-1314

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Enhanced gene targeting efficiency by siRNA that silences the expression of the Bloom syndrome gene in human cells.2006

    • Author(s)
      So, S., et. al.
    • Journal Title

      Genes Cells 11

      Pages: 363-371

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Enhanced gene targeting efficiency by siRNA that silences the expression of the Bloom syndrone gene in human cells.2006

    • Author(s)
      So, S et al.
    • Journal Title

      Genes Cells 11(4)

      Pages: 363-371

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Parp-1 protects homologous recombination from interference by Ku and Ligase VI in vertebtate cells.2006

    • Author(s)
      Hochegger, H. et al.
    • Journal Title

      EMBO J. 25(6)

      Pages: 1305-1314

    • Related Report
      2006 Annual Research Report
  • [Journal Article] 53BP1 contributes to survival of cells irradiated with X-ray during G1 without Ku70 or Artemis.2006

    • Author(s)
      Iwabuchi, K. et al.
    • Journal Title

      Genes Cells 11(8)

      Pages: 935-948

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Simple one-week method to construct gene-targeting vectors : application to production of human knockout cell lines.2006

    • Author(s)
      Iiizumi, S. et al.
    • Journal Title

      BioTechniques 41(1)

      Pages: 311-316

    • Related Report
      2006 Annual Research Report
  • [Presentation] Human DNA ligase IV and Artemis independently contribute to cytotoxicity in the presence of replication-associated DNA damage:Implications for double-strand break repair2008

    • Author(s)
      Kurosawa, A., et. al.
    • Organizer
      日本薬学会第128年会
    • Place of Presentation
      横浜
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Human DNA ligase IV and Artemis independently contribute to cytotoxicity in the presence of replication-associated DNA damage: Implications for double-strand break repair.2008

    • Author(s)
      Kurosawa, A., et. al.
    • Organizer
      128th Annual Meeting of the Pharmaceutical Society of Japan
    • Place of Presentation
      Yokohama
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Genetic studies on the repair of topoisomerase-mediated DNA damage using human gene knock out cell lines2007

    • Author(s)
      Kurosawa, A., et. al.
    • Organizer
      EMBO Workshop 2007 DNA Supercoiling & Topoisomerases
    • Place of Presentation
      Frejus,France
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] NK314 is a topoisomerase IIα specific inhibitor inducing stable cleavage complex and rapid DNA double-strand breaks2007

    • Author(s)
      Toyoda, E., et. al.
    • Organizer
      2007 AACR Annual Meeting
    • Place of Presentation
      Los Angeles,CA,USA
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Impact of DSB repair deficiency in human cells:NHEJ negatively affects cell survival in the presence of replication-associated DNA damage2007

    • Author(s)
      Kurosawa, A., et. al.
    • Organizer
      BMB2007
    • Place of Presentation
      Yokohama
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Human Mus81 and FANCB independently contribute to repair of DNA damage during replication2007

    • Author(s)
      Nomura, Y., et. al.
    • Organizer
      BMB2007
    • Place of Presentation
      Yokohama
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Generation and characterization of human Aprataxin knockout cells2007

    • Author(s)
      Iiizumi, S., et. al.
    • Organizer
      BMB2007
    • Place of Presentation
      Yokohama
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] NK314 is a topoisomerase IIalpha specific inhibitor forming stable DNA cleavage complex2007

    • Author(s)
      Toyoda, E., et. al.
    • Organizer
      The 66th Annual Meeting of the Japanese Cancer Association(JCA2007)
    • Place of Presentation
      Yokohama
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Genetic studies on the repair of topoisomerase-mediated DNA damage using human gene knockout cell lines2007

    • Author(s)
      Kurosawa, A., et. al.
    • Organizer
      The 66th Annual Meeting of the Japanese Cancer Association(JCA2007)
    • Place of Presentation
      Yokohama
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Gene knockout studies on DNA strand break repair in human cells2007

    • Author(s)
      Adachi, N., et. al.
    • Organizer
      日本薬学会第127年会
    • Place of Presentation
      富山
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Genetic studies on the repair of topoisomerase-mediated DNA damage using human gene knockout cell lines.2007

    • Author(s)
      Kurosawa, A., et. al.
    • Organizer
      EMBO Workshop 2007 DNA Supercoiling & Topoisomerases
    • Place of Presentation
      Frejus (France)
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] NK314 is a topoisomerase IIa specific inhibitor inducing stable cleavage complex and rapid DNA double-strand breaks.2007

    • Author(s)
      Toyoda, E., et. al.
    • Organizer
      2007 AACR Annual Meeting
    • Place of Presentation
      Los Angeles (USA)
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Impact of DSB repair deficiency in human cells: NHEJ negatively affects cell survival in the presence of replication-associated DNA damage.2007

    • Author(s)
      Kurosawa, A., et. al.
    • Organizer
      BMB2007
    • Place of Presentation
      Yokohama
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Human Mus81 and FANCB independently contribute to repair of DNA damage during replication.2007

    • Author(s)
      Nomura, Y., et. al.
    • Organizer
      BMB2007
    • Place of Presentation
      Yokohama.
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Generation and characterization of human Aprataxin knockout cells.2007

    • Author(s)
      Iiizumi, S., et. al.
    • Organizer
      BMB2007
    • Place of Presentation
      Yokohama
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] NK314 is a topoisomerase IIalpha specific inhibitor forming stable DNA cleavage complex.2007

    • Author(s)
      Toyoda, E., et. al.
    • Organizer
      JCA2007
    • Place of Presentation
      Yokohama
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Genetic studies on the repair of topoisomerase-mediated DNA damage using human gene knockout cell lines.2007

    • Author(s)
      Kurosawa, A., et. al.
    • Organizer
      JCA2007
    • Place of Presentation
      Yokohama
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Gene knockout studies on DNA strand break repair in human cells2007

    • Author(s)
      Adachi, N., et. al.
    • Organizer
      127th Annual Meeting of the Pharmaceutical Society of Japan
    • Place of Presentation
      Toyama
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Impact of DSB repair deficiency on damage sensitivity:NHEJ negatively affects cell survival in the presence of DNA lesions that rely on homologous DNA repair2006

    • Author(s)
      Adachi, N., et. al.
    • Organizer
      The Salk/Caltech conference on DNA Replication and Genome Integrity
    • Place of Presentation
      La Jolla,CA,USA
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Gene knockout studies on DNA double-strand break epair in human cells2006

    • Author(s)
      Adachi, N., et. al.
    • Organizer
      第65回日本癌学会学術総会
    • Place of Presentation
      横浜
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Nonhomologous end-joining negatively affects cell survival in the presence of DNA lesions that rely on homologous DNA repair2006

    • Author(s)
      Adachi, N., et. al.
    • Organizer
      20th IUBMB Int'l Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress
    • Place of Presentation
      Kyoto
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Impact of DNA ligase IV deficiency on random and targeted integration2006

    • Author(s)
      Iiizumi, S., et. al.
    • Organizer
      20th IUBMB Int'l Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress
    • Place of Presentation
      Kyoto
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Genetic analysis of human Artemis2006

    • Author(s)
      Kurosawa, A., et. al.
    • Organizer
      2006年分子生物学フォーラム
    • Place of Presentation
      名古屋
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Impact of DSB repair deficiency on damage sensitivity: NHEJ negatively affects cell survival in the presence of DNA lesions that rely on homologous DNA repair.The Salk/Caltech conference on DNA.2006

    • Author(s)
      Adachi, N., et. al.
    • Organizer
      Replication and Genome Integrity
    • Place of Presentation
      La Jolla (USA)
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Gene knockout studies on DNA double-strand break repair in human cells.2006

    • Author(s)
      Adachi, N., et. al.
    • Organizer
      JCA2006
    • Place of Presentation
      Yokohama
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Nonhomologous end-joining negatively affects cell survival in the presence of DNA lesions that rely on homologous DNA repair.2006

    • Author(s)
      Adachi, N., et. al.
    • Organizer
      20th IUBMB Int'l and 11th FAOBMB Congress
    • Place of Presentation
      Kyoto
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Impact of DNA ligase IV deficiency on random and targeted integration.2006

    • Author(s)
      Iiizumi, S., et. al.
    • Organizer
      20th IUBMB Int'l and 11th FAOBMB Congress
    • Place of Presentation
      Kyoto
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Genetic analysis of human Artemis.2006

    • Author(s)
      Kurosawa, A., et. al.
    • Organizer
      2006 Mol. Biol. Forum
    • Place of Presentation
      Nagoya.
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary

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Published: 2006-04-01   Modified: 2016-04-21  

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