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Nonhomologous end-joining in a cell-free extract from the cultured silkworm cell line BmN4

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Abstract

Nonhomologous end-joining (NHEJ) is one of the repair pathways for double-strand breaks (DSBs) in eukaryotic cells. By using linearized plasmid substrates, we have detected intramolecular NHEJ activity in a cell-free extract from the cultured silkworm cell line BmN4. The efficiency of NHEJ differed according to the structure of DNA ends; approximately 1% of input DNA was repaired when the substrate had cohesive ends. The reaction required the hydrolysis of nucleotide triphosphate; interestingly, all of four rNTPs or four dNTPs could support the reaction. A substrate with non-complementary DNA ends was mainly repaired by the DNA polymerase-mediated pathway. These results indicate that the present cell-free system will be useful to analyze the molecular mechanisms of DSB repair and NHEJ in insect cells.

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References

  • M Takata MS Sasaki E Sonoda C Morrison M Hashimoto H Utsumi Y Yamaguchi-Iwai A Shinohara S Takeda (1998) EMBO J 17 5497–5508 Occurrence Handle1:CAS:528:DyaK1cXmsFOgsLg%3D Occurrence Handle9736627

    CAS  PubMed  Google Scholar 

  • C Featherstone SP Jackson (1999) Curr. Biol 9 R759–761 Occurrence Handle1:CAS:528:DyaK1MXntVSlsbc%3D Occurrence Handle10531043

    CAS  PubMed  Google Scholar 

  • KP Hopfner CD Putnam JA Tainer (2002) Curr. Opin. Struct. Biol 12 115–122 Occurrence Handle1:CAS:528:DC%2BD38XhtF2itLk%3D Occurrence Handle11839498

    CAS  PubMed  Google Scholar 

  • F Pâques JE Haber (1999) Microbiol. Mol. Biol. Rev 63 349–404 Occurrence Handle10357855

    PubMed  Google Scholar 

  • RD Johnson M Jasin (2001) Biochem. Soc. Trans 29 196–201 Occurrence Handle1:CAS:528:DC%2BD3MXkslentbk%3D Occurrence Handle11356153

    CAS  PubMed  Google Scholar 

  • F Liang M Han PJ Romanienko M Jasin (1988) Proc. Natl. Acad. Sci. USA 95 5172–5177

    Google Scholar 

  • PA Jeggo (1998) Radiat Res 150 IssueID(5 Suppl S80–91 Occurrence Handle1:CAS:528:DyaK1cXnt1Cqsrk%3D Occurrence Handle9806611

    CAS  PubMed  Google Scholar 

  • SE Critchlow SP Jackson (1998) Trends Biochem. Sci 23 394–398 Occurrence Handle1:CAS:528:DyaK1cXntlansL8%3D Occurrence Handle9810228

    CAS  PubMed  Google Scholar 

  • P Baumann SC West (1998) Proc. Natl. Acad. Sci. USA 95 14066–14070 Occurrence Handle1:CAS:528:DyaK1cXns1GmtLo%3D Occurrence Handle9826654

    CAS  PubMed  Google Scholar 

  • L Chen K Trujillo P Sung AE Tomkinson (2000) J. Biol. Chem 275 26196–26206 Occurrence Handle1:CAS:528:DC%2BD3cXmt1Oksb8%3D Occurrence Handle10854421

    CAS  PubMed  Google Scholar 

  • KJ Lee J Huang Y Takeda WS Dynan (2000) J. Biol. Chem 275 34787–34796 Occurrence Handle1:CAS:528:DC%2BD3cXotFWlur4%3D Occurrence Handle10945980

    CAS  PubMed  Google Scholar 

  • N Adachi T Ishino Y Isii S Takeda H Koyama (2001) Proc. Natl. Acad. Sci. USA 98 12109–12113 Occurrence Handle1:CAS:528:DC%2BD3MXns1CjsLc%3D Occurrence Handle11593023

    CAS  PubMed  Google Scholar 

  • S Mårtensson O Hammarsten (2002) J. Biol. Chem 277 3020–3029 Occurrence Handle11700303

    PubMed  Google Scholar 

  • H Ouyang A Nussenzweig A Kurimasa VC Soares X Li C Cardon-Cardo W Li N Cheong M Nussenzweig G Iliakis DJ Chen GC Li (1997) J. Exp. Med 186 921–929 Occurrence Handle1:CAS:528:DyaK2sXmtV2itbw%3D Occurrence Handle9294146

    CAS  PubMed  Google Scholar 

  • S Jin DT Weaver (1997) EMBO. J 16 6874–6885 Occurrence Handle1:CAS:528:DyaK1cXisVWksw%3D%3D Occurrence Handle9362500

    CAS  PubMed  Google Scholar 

  • BK Singleton A Priestley H Steingrimsdottir D Gell T Blunt SP Jackson AR Lehmann PA Jeggo (1997) Mol. Cell. Biol 17 1264–1273 Occurrence Handle1:CAS:528:DyaK2sXhtlGhu74%3D Occurrence Handle9032253

    CAS  PubMed  Google Scholar 

  • PA Jeggo GE Taccioli SP Jackson (1995) Bioessays 11 949–957

    Google Scholar 

  • M Lieber (1996) Curr Biol 6 134–136 Occurrence Handle1:CAS:528:DyaK28XhtFOjtLw%3D Occurrence Handle8673457

    CAS  PubMed  Google Scholar 

  • M Bogue DB Roth (1996) Curr. Opin. Immunol 8 175–180 Occurrence Handle1:CAS:528:DyaK28Xjtl2iurk%3D Occurrence Handle8725940

    CAS  PubMed  Google Scholar 

  • GC Smith SP Jacson (1999) Genes Dev 13 916–934 Occurrence Handle1:CAS:528:DyaK1MXivFGkur4%3D Occurrence Handle10215620

    CAS  PubMed  Google Scholar 

  • C Chappell LA Hanakahi F Karimi-Busheri M Weinfeld SC West (2002) EMBO J 21 2827–2832 Occurrence Handle1:CAS:528:DC%2BD38XkvV2isLY%3D Occurrence Handle12032095

    CAS  PubMed  Google Scholar 

  • B-BS Zhou SJ Elledge (2000) Nature 408 433–439 Occurrence Handle1:CAS:528:DC%2BD3cXos1Srsb0%3D Occurrence Handle11100718

    CAS  PubMed  Google Scholar 

  • RT Abraham (2001) Genes Dev 15 2177–2196 Occurrence Handle1:CAS:528:DC%2BD3MXmvFSruro%3D Occurrence Handle11544175

    CAS  PubMed  Google Scholar 

  • J Yang Y Yu HE Hamrick PJ Duerksen-Hughes (2003) Carcinogenesis 24 1571–80 Occurrence Handle1:CAS:528:DC%2BD3sXotVClsb4%3D Occurrence Handle12919958

    CAS  PubMed  Google Scholar 

  • G Iliakis Y Wang J Guan H Wang (2003) Oncogene 22 5834–47 Occurrence Handle1:CAS:528:DC%2BD3sXmslemt7c%3D Occurrence Handle12947390

    CAS  PubMed  Google Scholar 

  • BA Edgar TL Orr-Weaver (2001) Cell 105 297–306 Occurrence Handle1:CAS:528:DC%2BD3MXjs1aisLg%3D Occurrence Handle11348589

    CAS  PubMed  Google Scholar 

  • JJ Blow B Hodgson (2002) Trends. Cell. Biol 12 72–78 Occurrence Handle1:CAS:528:DC%2BD38XhtFyrtbc%3D Occurrence Handle11849970

    CAS  PubMed  Google Scholar 

  • M Ikeda M Kobayashi (1999) Virology 258 176–188 Occurrence Handle1:CAS:528:DyaK1MXivFOqt7w%3D Occurrence Handle10329579

    CAS  PubMed  Google Scholar 

  • P Pfeiffer W Vielmetter (1988) Nucleic Acids Res 16 907–924 Occurrence Handle1:CAS:528:DyaL1cXhsVGmtLo%3D Occurrence Handle3344222

    CAS  PubMed  Google Scholar 

  • P Daza S Reichenberger B Gottlich M Hagmann E Feldmann P Pfeiffer (1996) Biol Chem 377 775–86 Occurrence Handle1:CAS:528:DyaK2sXoslCq Occurrence Handle8997488

    CAS  PubMed  Google Scholar 

  • P Labhart (1999a) Mol. Cell. Biol 19 2585–2593 Occurrence Handle1:CAS:528:DyaK1MXit1amsbk%3D

    CAS  Google Scholar 

  • G Gilliland S Perrin K Blanchard F Bunn (1990) Proc. Natl. Acad. Sci. USA 87 2725–2729 Occurrence Handle1:CAS:528:DyaK3cXhvFKhurs%3D Occurrence Handle2181447

    CAS  PubMed  Google Scholar 

  • U Grawunder M Wilm X Wu P Kulesza TE Wilson M Mann MR Lieber (1997) Nature 388 492–495 Occurrence Handle1:CAS:528:DyaK2sXltFKjs74%3D Occurrence Handle9242410

    CAS  PubMed  Google Scholar 

  • E Feldmann V Schmiemann W Goedecke S Reichenberger P Pfeiffer (2000) Nucleic Acids Res 28 2585–2596 Occurrence Handle1:CAS:528:DC%2BD3cXlvFWqsLY%3D Occurrence Handle10871410

    CAS  PubMed  Google Scholar 

  • P Robins T Lindahl (1996) J. Biol. Chem 271 24257–24261 Occurrence Handle1:CAS:528:DyaK28XlvFWju7o%3D Occurrence Handle8798671

    CAS  PubMed  Google Scholar 

  • KJ Lee J Huang Y Takeda WS Dynan (2000) J. Biol. Chem 275 34787–34796 Occurrence Handle1:CAS:528:DC%2BD3cXotFWlur4%3D Occurrence Handle10945980

    CAS  PubMed  Google Scholar 

  • E Riballo AJ Doherty Y Dai T Stiff MA Oettinger PA Jeggo B Kysela (2001) J. Biol. Chem 276 31124–31132 Occurrence Handle1:CAS:528:DC%2BD3MXmsVejsro%3D Occurrence Handle11349135

    CAS  PubMed  Google Scholar 

  • S Thode A Schafer P Pfeiffer W Vielmetter (1990) Cell 60 921–8 Occurrence Handle1:CAS:528:DyaK3cXitFamuro%3D Occurrence Handle2317864

    CAS  PubMed  Google Scholar 

  • JS King CF Fairley WF Morgan (1994) J. Biol. Chem 269 13061–13064 Occurrence Handle1:CAS:528:DyaK2cXjtFOhs7Y%3D Occurrence Handle8175727

    CAS  PubMed  Google Scholar 

  • TE Wilson MR Lieber (1999) J. Biol. Chem 275 23599–23609

    Google Scholar 

  • KN Mahajan SA Nick McElhinny BS Mitchell DA Ramsden (2002) Mol. Cell. Biol 22 5194–5202 Occurrence Handle1:CAS:528:DC%2BD38XltVOqt7w%3D Occurrence Handle12077346

    CAS  PubMed  Google Scholar 

  • X Wu TE Wilson MR Lieber (1999) Proc. Natl. Acad. Sci. USA 96 1303–1308 Occurrence Handle1:CAS:528:DyaK1MXhsFSrtLs%3D Occurrence Handle9990019

    CAS  PubMed  Google Scholar 

  • D Moshous I Callebaut R Chasseval B Corneo M Cavazzana-Calvo F Deist I Tezcan O Sanal Y Bertrand N Philippe A Fischer J Villartay (2001) Cell 105 177–186 Occurrence Handle1:CAS:528:DC%2BD3MXjtFOkt7s%3D Occurrence Handle11336668

    CAS  PubMed  Google Scholar 

  • Y May U Pannicke K Schwarz MR Lieber (2002) Cell 108 781–794 Occurrence Handle11955432

    PubMed  Google Scholar 

  • Hanakahi LA & West SC (2002) EMBO J. 2038–2044.

  • P Labhart (1999b) Eur. J. Biochem 265 849–861 Occurrence Handle1:CAS:528:DyaK1MXntFKku78%3D

    CAS  Google Scholar 

  • A Odersky IV Panyutin IG Panyutin C Schunck E Feldmann W Goedecke RD Neumann G Obe Pfeiffer (2002) J Biol Chem 277 11756–11764 Occurrence Handle1:CAS:528:DC%2BD38XivVeks7o%3D Occurrence Handle11821407

    CAS  PubMed  Google Scholar 

  • Q Zhong TG Boyer PL Chen WH Lee (2002) Cancer Res 62 3966–3970 Occurrence Handle1:CAS:528:DC%2BD38XlsV2ls7c%3D Occurrence Handle12124328

    CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan

    Arisa Ohsaki, Yoshitaka Miyagawa, Yutaka Kawaguchi, Katsumi Koga & Takahiro Kusakabe

  2. Institute of Biological Control, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan

    Kazuhiro Iiyama

Authors
  1. Arisa Ohsaki
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  2. Kazuhiro Iiyama
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  3. Yoshitaka Miyagawa
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  4. Yutaka Kawaguchi
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  5. Katsumi Koga
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  6. Takahiro Kusakabe
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Correspondence to Takahiro Kusakabe.

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Ohsaki, A., Iiyama, K., Miyagawa, Y. et al. Nonhomologous end-joining in a cell-free extract from the cultured silkworm cell line BmN4. Mol Biol Rep 32, 25–34 (2005). https://doi.org/10.1007/s11033-004-2474-y

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  • DOI: https://doi.org/10.1007/s11033-004-2474-y

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