Homologous Recombination Assay for Interstrand Cross-Link Repair

  • Koji Nakanishi
  • Francesca Cavallo
  • Erika Brunet
  • Maria JasinEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 745)


DNA interstrand cross-links (ICLs) covalently link both strands of the DNA duplex, impeding cellular processes like DNA replication. Homologous recombination (HR) is considered to be a major pathway for the repair of ICLs in mammalian cells as mutants for HR components are highly sensitive to DNA-damaging agents that cause ICLs. This chapter describes GFP assays to measure HR following site-specific ICL formation with psoralen through DNA triplex technology. This approach can be used to determine the genetic requirements for ICL-induced HR in relation to those involved in HR repair of other DNA lesions such as double-strand breaks.

Key words

Homologous recombination interstrand cross-link repair triplex-forming oligonucleotide GFP reporters 



This work was supported by the Byrne Fund and National Institutes for Health grants P01CA94060 (M.J.) and R01GM54668 (M.J.).


  1. 1.
    Guainazzi, A., and Schärer, O.D. (2010) Using synthetic DNA interstrand crosslinks to elucidate repair pathways and identify new therapeutic targets for cancer chemotherapy. Cell Mol Life Sci 67, 3683–3697.PubMedCrossRefGoogle Scholar
  2. 2.
    Hinz, J.M. (2010) Role of homologous recombination in DNA interstrand crosslink repair. Environ Mol Mutagen 51, 582–603.PubMedGoogle Scholar
  3. 3.
    Moynahan, M.E., Chiu, J.W., Koller, B.H., and Jasin, M. (1999) Brca1 controls homology-directed DNA repair. Mol Cell 4, 511–518.PubMedCrossRefGoogle Scholar
  4. 4.
    Moynahan, M.E., Cui, T.Y., and Jasin, M. (2001) Homology-directed DNA repair, mitomycin-c resistance, and chromosome stability is restored with correction of a Brca1 mutation. Cancer Res 61, 4842–4850.PubMedGoogle Scholar
  5. 5.
    Moynahan, M.E., Pierce, A.J., and Jasin, M. (2001) BRCA2 is required for homology-directed repair of chromosomal breaks. Mol Cell 7, 263–272.PubMedCrossRefGoogle Scholar
  6. 6.
    Kraakman-van der Zwet, M., et al. (2002) Brca2 (XRCC11) deficiency results in radioresistant DNA synthesis and a higher frequency of spontaneous deletions. Mol Cell Biol 22, 669–679.PubMedCrossRefGoogle Scholar
  7. 7.
    Wang, W. (2007) Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins. Nat Rev Genet 8, 735–748.PubMedCrossRefGoogle Scholar
  8. 8.
    Auerbach, A.D. (2009) Fanconi anemia and its diagnosis. Mutat Res 668, 4–10.PubMedGoogle Scholar
  9. 9.
    Nakanishi, K., et al. (2005) Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair. Proc Natl Acad Sci USA 102, 1110–1115.PubMedCrossRefGoogle Scholar
  10. 10.
    Nakanishi, K., Cavallo, F., Perrouault, L., Giovannangeli, C., Moynahan, M.E., Barchi, M., Brunet, E., and Jasin, M. (2011) Homology-directed Fanconi anemia pathway cross-link repair is dependent on DNA replication. Nat Struct Mol Biol doi:10.1038/nsmb.2029.Google Scholar
  11. 11.
    Pierce, A.J., Johnson, R.D., Thompson, L.H., and Jasin, M. (1999) XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev 13, 2633–2638.PubMedCrossRefGoogle Scholar
  12. 12.
    Pierce, A.J., and Jasin, M. (2005) Measuring recombination proficiency in mouse embryonic stem cells. Methods Mol Biol 291, 373–384.PubMedGoogle Scholar
  13. 13.
    Moynahan, M.E., and Jasin, M. (2010) Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat Rev Mol Cell Biol 11, 196–207.PubMedCrossRefGoogle Scholar
  14. 14.
    Slabicki, M., et al. (2010) A genome-scale DNA repair RNAi screen identifies SPG48 as a novel gene associated with hereditary spastic paraplegia. PLoS Biol 8, e1000408.PubMedCrossRefGoogle Scholar
  15. 15.
    Chin, J.Y., and Glazer, P.M. (2009) Repair of DNA lesions associated with triplex-forming oligonucleotides. Mol Carcinog 48, 389–399.PubMedCrossRefGoogle Scholar
  16. 16.
    Raha, M., Wang, G., Seidman, M.M., and Glazer, P.M. (1996) Mutagenesis by third-strand-directed psoralen adducts in repair-deficient human cells: high frequency and altered spectrum in a xeroderma pigmentosum variant. Proc Natl Acad Sci USA 93, 2941–2946.PubMedCrossRefGoogle Scholar
  17. 17.
    Reisman, D., Yates, J., and Sugden, B. (1985) A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components. Mol Cell Biol 5, 1822–1832.PubMedGoogle Scholar
  18. 18.
    Pierce, A.J., Hu, P., Han, M., Ellis, N., and Jasin, M. (2001) Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells. Genes Dev 15, 3237–3242.PubMedCrossRefGoogle Scholar
  19. 19.
    Richardson, C., Moynahan, M.E., and Jasin, M. (1998) Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations. Genes Dev 12, 3831–3842.PubMedCrossRefGoogle Scholar
  20. 20.
    Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199.PubMedCrossRefGoogle Scholar
  21. 21.
    Brunet, E., et al. (2005) Exploring cellular activity of locked nucleic acid-modified triplex-forming oligonucleotides and defining its molecular basis. J Biol Chem 280, 20076–20085.PubMedCrossRefGoogle Scholar
  22. 22.
    Brunet, E., Corgnali, M., Cannata, F., Perrouault, L., and Giovannangeli, C. (2006) Targeting chromosomal sites with locked nucleic acid-modified triplex-forming oligonucleotides: study of efficiency dependence on DNA nuclear environment. Nucleic Acids Res 34, 4546–4553.PubMedCrossRefGoogle Scholar
  23. 23.
    Raschle, M., et al. (2008) Mechanism of replication-coupled DNA interstrand crosslink repair. Cell 134, 969–980.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Koji Nakanishi
    • 1
  • Francesca Cavallo
    • 2
  • Erika Brunet
    • 3
  • Maria Jasin
    • 1
    Email author
  1. 1.Developmental Biology ProgramMemorial Sloan-Kettering Cancer CenterNew YorkUSA
  2. 2.Department of Public Health and Cell Biology, Section of AnatomyUniversity of Rome Tor VergataRomeItaly
  3. 3.Muséum National d’Histoire NaturelleParisFrance

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