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Analysis of DNA Double-Strand Break Repair by Nonhomologous End Joining in Cell-Free Extracts From Mammalian Cells

  • Petra Pfeiffer
  • Elke Feldmann
  • Andrea Odersky
  • Steffi Kuhfittig-Kulle
  • Wolfgang Goedecke
Part of the Methods in Molecular Biology™ book series (MIMB, volume 291)

Abstract

Double-strand breaks (DSBs) in genomic DNA are induced by ionizing radiation or radiomimetic drugs, but they also occur spontaneously during the cell cycle at quite significant frequencies. In vertebrate cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of DSB repair. NHEJ is able to rejoin two broken DNA termini directly end-to-end irrespective of sequence and structure. Genetic studies in various radiosensitive and DSB repair-deficient hamster cell lines have yielded insights into the factors involved in NHEJ. Studies in cell-free systems derived from Xenopus eggs and mammalian cells have allowed the dissection of the underlying mechanisms. In the present chapter, we describe a protocol for the preparation of whole cell extracts from mammalian cells and a plasmid-based in vitro assay that permits the easy analysis of the efficiency and fidelity of DSB repair via NHEJ in different cell types.

Key Words

DSB repair NHEJ (nonhomologous DNA end joining) ligation illegitimate recombination cell-free extracts in vitro assays 

References

  1. 1.
    Haber, J. E. (1999) DNA recombination: the replication connection. Trends Biochem. Sci. 24, 271–275.PubMedCrossRefGoogle Scholar
  2. 2.
    Pfeiffer, P., Goedecke, W., and Obe, G. (2000) Mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations. Mutagenesis 15, 289–302.PubMedCrossRefGoogle Scholar
  3. 3.
    Haber, J. E. (1999) DNA repair. Gatekeepers of recombination. Nature 398, 665–667.PubMedCrossRefGoogle Scholar
  4. 4.
    Haber, J. E. (2000) Partners and pathways repairing a double-strand break. Trends Genet. 16, 259–264.PubMedCrossRefGoogle Scholar
  5. 5.
    Haber, J. E. (2000) Recombination: a frank view of exchanges and vice versa. Curr. Opin. Cell Biol. 12, 286–292.PubMedCrossRefGoogle Scholar
  6. 6.
    Pfeiffer, P. (1998) The mutagenic potential of DNA double-strand break repair. Toxicol. Lett. 96–97, 119–129.PubMedCrossRefGoogle Scholar
  7. 7.
    Critchlow, S. E. and Jackson, S. P. (1998) DNA end-joining: from yeast to man. Trends Biochem. Sci. 23, 394–398.PubMedCrossRefGoogle Scholar
  8. 8.
    Featherstone, C. and Jackson, S. P. (1999) DNA double-strand break repair. Curr. Biol. 9, R759–R761.PubMedCrossRefGoogle Scholar
  9. 9.
    Feldmann, E., Schmiemann, V., Goedecke, W., Reichenberger, S., and Pfeiffer, P. (2000) DNA double-strand break repair in cell-free extracts from Ku80-deficient cells: implications for Ku serving as an alignment factor in non-homologous DNA end joining. Nucleic Acids Res. 28, 2585–2596.PubMedCrossRefGoogle Scholar
  10. 10.
    Göttlich, B., Reichenberger, S., Feldmann, E., and Pfeiffer, P. (1998) Rejoining of DNA double-strand breaks in vitro by single-strand annealing. Eur. J. Biochem. 258, 387–395.PubMedCrossRefGoogle Scholar
  11. 11.
    Bryant, P. E. (1984) Enzymatic restriction of mammalian cell DNA using Pvu II and Bam H1: evidence for the double-strand break origin of chromosomal aberrations. Int. J. Radiat. Biol. 46, 57–65.CrossRefGoogle Scholar
  12. 12.
    Natarajan, A. T. and Obe, G. (1984) Molecular mechanisms involved in the production of chromosomal aberrations. III. Restriction endonucleases. Chromosoma 90, 120–127.PubMedCrossRefGoogle Scholar
  13. 13.
    Kabotyanski, E. B., Gomelsky, L., Han, J. O., Stamato, T. D., and Roth, D. B. (1998) Double-strand break repair in Ku86-and XRCC4-deficient cells. Nucleic Acids Res. 26, 5333–5342.PubMedCrossRefGoogle Scholar
  14. 14.
    King, J. S., Valcarcel, E. R., Rufer, J. T., Phillips, J. W., and Morgan, W. F. (1993) Noncomplementary DNA double-strand-break rejoining in bacterial and human cells. Nucleic Acids Res. 21, 1055–1059.PubMedCrossRefGoogle Scholar
  15. 15.
    Roth, D. B., Porter, T. N., and Wilson, J. H. (1985) Mechanisms of nonhomologous recombination in mammalian cells. Mol. Cell. Biol. 5, 2599–2607.PubMedGoogle Scholar
  16. 16.
    Roth, D. B. and Wilson, J. H. (1986) Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction. Mol. Cell. Biol. 6, 4295–4304.PubMedGoogle Scholar
  17. 17.
    Daza, P., Reichenberger, S., Göttlich, B., Hagmann, M., Feldmann, E., and Pfeiffer, P. (1996) Mechanisms of nonhomologous DNA end-joining in frogs, mice and men. Biol. Chem. 377, 775–786.PubMedGoogle Scholar
  18. 18.
    Pfeiffer, P. and Vielmetter, W. (1988) Joining of nonhomologous DNA double strand breaks in vitro. Nucleic Acids Res. 16, 907–924.PubMedCrossRefGoogle Scholar
  19. 19.
    Baumann, P. and West, S. C. (1998) DNA end-joining catalyzed by human cell-free extracts. Proc. Natl. Acad. Sci. USA 95, 14066–14070.PubMedCrossRefGoogle Scholar
  20. 20.
    Boe, S. O., Sodroski, J., Helland, D. E., and Farnet, C. M. (1995) DNA end-joining in extracts from human cells. Biochem. Biophys. Res. Commun. 215, 987–993.PubMedCrossRefGoogle Scholar
  21. 21.
    Cheong, N., Okayasu, R., Shah, S., Ganguly, T., Mammen, P., and Iliakis, G. (1996) In vitro rejoining of double-strand breaks in cellular DNA by factors present in extracts of HeLa cells. Int. J. Radiat. Biol. 69, 665–677.PubMedCrossRefGoogle Scholar
  22. 22.
    Cheong, N., Perrault, A. R., Wang, H., et al. (1999) DNA-PK-independent rejoining of DNA double-strand breaks in human cell extracts in vitro. Int. J. Radiat. Biol. 75, 67–81.PubMedCrossRefGoogle Scholar
  23. 23.
    Derbyshire, M. K., Epstein, L. H., Young, C. S., Munz, P. L., and Fishel, R. (1994) Nonhomologous recombination in human cells. Mol. Cell. Biol. 14, 156–169.PubMedGoogle Scholar
  24. 24.
    Labhart, P. (1999) Ku-dependent nonhomologous DNA end joining in Xenopus egg extracts. Mol. Cell. Biol. 19, 2585–2593.PubMedGoogle Scholar
  25. 25.
    Lakshmipathy, U. and Campbell, C. (1999) Double strand break rejoining by mammalian mitochondrial extracts. Nucleic Acids Res. 27, 1198–1204.PubMedCrossRefGoogle Scholar
  26. 26.
    Mason, R. M., Thacker, J., and Fairman, M. P. (1996) The joining of non-complementary DNA double-strand breaks by mammalian extracts. Nucleic Acids Res. 24, 4946–4953.PubMedCrossRefGoogle Scholar
  27. 27.
    Nicolas, A. L. and Young, C. S. (1994) Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific. Mol. Cell. Biol. 14, 170–180.PubMedGoogle Scholar
  28. 28.
    Nicolas, A. L., Munz, P. L., and Young, C. S. (1995) A modified single-strand annealing model best explains the joining of DNA double-strand breaks mammalian cells and cell extracts. Nucleic Acids Res. 23, 1036–1043.PubMedCrossRefGoogle Scholar
  29. 29.
    North, P., Ganesh, A., and Thacker, J. (1990) The rejoining of double-strand breaks in DNA by human cell extracts. Nucleic Acids Res. 18, 6205–6210.PubMedCrossRefGoogle Scholar
  30. 30.
    Sathees, C. R. and Raman, M. J. (1999) Mouse testicular extracts process DNA double-strand breaks efficiently by DNA end-to-end joining. Mutat. Res. 433, 1–13.PubMedGoogle Scholar
  31. 31.
    Labhart, P. (1999) Nonhomologous DNA end joining in cell-free systems. Eur. J. Biochem. 265, 849–861.PubMedCrossRefGoogle Scholar
  32. 32.
    Pfeiffer, P., Thode, S., Hancke, J., and Vielmetter, W. (1994) Mechanisms of overlap formation in nonhomologous DNA end joining. Mol. Cell. Biol. 14, 888–895.PubMedGoogle Scholar
  33. 33.
    Thode, S., Schäfer, A., Pfeiffer, P., and Vielmetter, W. (1990) A novel pathway of DNA end-to-end joining. Cell 60, 921–928.PubMedCrossRefGoogle Scholar
  34. 34.
    Sharma, R. C., and Schimke R. T. (1996) Preparation of electrocompetent E. coli using salt-free growth medium. BioTechniques 20, 42–44.PubMedGoogle Scholar
  35. 35.
    Zdzienicka, M. Z. (1999) Mammalian X-ray-sensitive mutants which are defective in nonhomologous (illegitimate) DNA double-strand break repair. Biochimie 81, 107–116.PubMedCrossRefGoogle Scholar
  36. 36.
    Pfeiffer, P., Thode, S., Hancke, J., Keohavong, P., and Thilly, W. G. (1994) Resolution and conservation of mismatches in DNA end joining. Mutagenesis 9, 527–535.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Petra Pfeiffer
    • 1
  • Elke Feldmann
    • 1
  • Andrea Odersky
    • 1
  • Steffi Kuhfittig-Kulle
    • 1
  • Wolfgang Goedecke
    • 1
  1. 1.Institute of GeneticsUniversity of EssenEssenGermany

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