Coupling of DNA Helicase Function to DNA Strand Exchange Activity

  • Frank G. Harmon
  • Stephen C. Kowalczykowski
Part of the Methods in Molecular Biology™ book series (MIMB, volume 152)


DNA repair can occur by a variety of mechanistically distinct pathways [for review, see (1)]. Recombinational DNA repair is one such pathway, and it requires the coordinated action of many different enzymes. In the best studied organism, Escherichia coli, more than 20 different proteins are involved [for review, see (2)]. The recombinational repair of a double-stranded DNA (dsDNA) break requires four general steps: (1) processing; (2) homologous pairing; (3) DNA heteroduplex extension; and (4) resolution. Here we describe assays to study aspects of the first two steps.


Vortex Mixer RecA Protein Homologous Pairing RecQ Helicase Linear dsDNA 
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  1. 1.
    Friedberg, E. C., Walker, G. C., and Siede, W. (1995) DNA Repair and Mutagenesis, ASM, Washington, DCGoogle Scholar
  2. 2.
    Kowalczykowski, S. C., Dixon, D. A., Eggleston, A. K., et al. (1994) Biochemistry of homologous recombination in Escherichia coli. Microbiol. Rev. 58, 401–465.PubMedGoogle Scholar
  3. 3.
    Kowalczykowski, S. C. and Eggleston, A. K. (1994) Homologous pairing and DNA strand-exchange proteins. Annu. Rev. Biochem. 63, 991–1043.PubMedCrossRefGoogle Scholar
  4. 4.
    Roca, A. I. and Cox, M. M. (1997) RecA protein: structure, function, and role in recombinational DNA repair. Prog. Nucleic Acid Res. Mol. Biol. 56, 129–223.PubMedCrossRefGoogle Scholar
  5. 5.
    Bianco, P. R., Tracy, R. B., and Kowalczykowski, S. C. (1998) DNA strand exchange proteins: a biochemical and physical comparison. Front Biosci. 3. D570–D603.PubMedGoogle Scholar
  6. 6.
    Ogawa, T., Shinohara, A., Nabetani, A., et al. (1993) RecA-like recombination proteins in eukaryotes: Function and structures of RAD51 genes. Cold Spring Harbor Symp. Quant. Biol. 58, 567–576.PubMedGoogle Scholar
  7. 7.
    Sargentini, N. J. and Smith, K. C. (1986) Characterization and quantitation of DNA strand breaks requiring recA-dependent repair in X-irradiated Escherichia coli. Radiat. Res. 105, 180–186.PubMedCrossRefGoogle Scholar
  8. 8.
    Clark, A. J. and Margulies, A. D. (1965) Isolation and characterization of recombination-deficient mutants of Escherichia coli K12. Proc. Natl. Acad. Sci. USA 53, 451–459.PubMedCrossRefGoogle Scholar
  9. 9.
    Cox, M. M. and Lehman, I. R. (1982) RecA protein-promoted DNA strand exchange: Stable complexes of recA protein and single-stranded DNA formed in the presence of ATP and single-stranded DNA binding protein. J. Biol. Chem. 257, 8523–8532.PubMedGoogle Scholar
  10. 10.
    Taylor, A. and Smith, G. R. (1980) Unwinding and rewinding of DNA by the recBC enzyme. Cell. 22, 447–457.PubMedCrossRefGoogle Scholar
  11. 11.
    Roman, L. J. and Kowalczykowski, S. C. (1989) Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay. Biochemistry 28, 2863–2873.PubMedCrossRefGoogle Scholar
  12. 12.
    Eggleston, A. K. and West, S. C. (1997) Recombination initiation: easy as A, B, C, D... chi? Curr. Biol. 7, R745–749.PubMedCrossRefGoogle Scholar
  13. 13.
    Arnold, D. A. and Kowalczykowski, S. C. (1999) RecBCD nuclease/helicase, in Encyclopedia of Life Sciences, Nature Publishing Group, London, Scholar
  14. 14.
    Nakayama, H., Nakayama, K., Nakayama, R., et al. (1984) Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichi coli K-12: Identification of a new mutation (recQ1) that blocks the recF recombination pathway. Mol. Gen. Genet. 195, 474–480.PubMedCrossRefGoogle Scholar
  15. 15.
    Lanzov, V., Stepanova, I., and Vinogradskaja, G. (1991) Genetic control of recombination exchange frequency in Escherichia coli K-12. Biochimie 73, 305–312.PubMedCrossRefGoogle Scholar
  16. 16.
    Harmon, F. G. and Kowalczykowski, S. C. (1998) RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination. Genes Dev. 12, 1134–1144.PubMedCrossRefGoogle Scholar
  17. 17.
    Roman, L. J., Dixon, D. A., and Kowalczykowski, S. C. (1991) RecBCD-dependent joint molecule formation promoted by the Escherichia coli RecA and SSB proteins. Proc. Natl. Acad. Sci. USA 88, 3367–3371.PubMedCrossRefGoogle Scholar
  18. 18.
    Dixon, D. A. and Kowalczykowski, S. C. (1991) Homologous pairing in vitro stimulated by the recombination hotspot, Chi. Cell 66, 361–371.PubMedCrossRefGoogle Scholar
  19. 19.
    West, S. C., Cassuto, E., and Howard-Flanders, P. (1981) RecA protein promotes homologous-pairing and strand-exchange reactions between duplex DNA molecules. Proc. Natl. Acad. Sci. USA 78, 2100–2104.PubMedCrossRefGoogle Scholar
  20. 20.
    Matson, S. W., Tabor, S., and Richardson, C. C. (1983) The gene 4 protein of bacteriophage T7 Characterization of helicase activity. J. Biol. Chem. 258, 14,017–14,024.PubMedGoogle Scholar
  21. 21.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds.) (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  22. 22.
    LeBowitz, J. (1985) Biochemical mechanism of strand initiation in bacteriophage lambda DNA replication. Ph.D. thesis, Johns Hopkins University, Baltimore, MD.Google Scholar
  23. 23.
    Cox, M. M., McEntee, K., and Lehman, I. R. (1981) A simple and rapid procedure for the large scale purification of the recA protein of Escherichia coli. J. Biol. Chem. 256, 4676–4678.PubMedGoogle Scholar
  24. 24.
    Griffith, J. and Shores, C. G. (1985) RecA protein rapidly crystallizes in the presence of spermidine: a valuable step in its purification and physical characterization. Biochemistry 24, 158–162.PubMedCrossRefGoogle Scholar
  25. 25.
    Matson, S. W. and George, J. W. (1987) DNA helicase II of Escherichia coli. Characterization of the single-stranded DNA-dependent NTPase and helicase activities. J. Biol. Chem. 262, 2066–2076.PubMedGoogle Scholar
  26. 26.
    Kowalczykowski, S. C. and Roman, L. J. (1990) Reconstitution of homologous pairing activity dependent upon the combined activities of purified E. coli RecA, RecBCD, and SSB proteins, in Molecular Mechanisms in DNA Replication and Recombination (Richardson, C. C. and Lehman, I. R., eds.), Wiley-Liss, New York,. 357–373.Google Scholar
  27. 27.
    Umezu, K. and Nakayama, H. (1993) RecQ DNA helicase of Escherichia coli. Characterization of the helix-unwinding activity with emphasis on the effect of single-stranded DNA-binding protein. J. Mol. Biol. 230, 1145–1150.PubMedCrossRefGoogle Scholar
  28. 28.
    Kowalczykowski, S. C. and Krupp, R. A. (1987) Effects of the Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein: evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA. J. Mol. Biol. 193, 97–113.PubMedCrossRefGoogle Scholar
  29. 29.
    Register, J. C., III and Griffith, J. (1985) The direction of RecA protein assembly onto single strand DNA is the same as the direction of strand assimilation during strand exchange. J. Biol. Chem. 260, 12,308–12,312.PubMedGoogle Scholar
  30. 30.
    Anderson, D. G. and Kowalczykowski, S. C. (1997) The recombination hot spot c is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme. Genes Dev. 11, 571–581.PubMedCrossRefGoogle Scholar
  31. 31.
    Bujalowski, W. and Lohman, T. M. (1986) Escherichia coli single-strand binding protein forms multiple, distinct complexes with single-stranded DNA. Biochemistry 25, 7799–7802.PubMedCrossRefGoogle Scholar
  32. 32.
    Lavery, P. E. and Kowalczykowski, S. C. (1990) Properties of recA441 proteincatalyzed DNA strand exchange can be attributed to an enhanced ability to compete with SSB protein. J. Biol. Chem. 265, 4004–4010.PubMedGoogle Scholar
  33. 33.
    Lavery, P. E. and Kowalczykowski, S. C. (1992) Enhancement of recA proteinpromoted DNA strand exchange activity by volume-occupying agents. J. Biol. Chem. 267, 9307–9314.PubMedGoogle Scholar
  34. 34.
    Cox, M. M. and Lehman, I. R. (1981) Directionality and polarity in recA proteinpromoted branch migration. Proc. Natl. Acad. Sci. USA 78, 6018–6022.PubMedCrossRefGoogle Scholar
  35. 35.
    Sung, P. (1994) Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science 265, 1241–1243.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Frank G. Harmon
    • 1
  • Stephen C. Kowalczykowski
    • 2
  1. 1.Division of Biological Sciences, Sections of Microbiology and Molecular and Cellular BiologyUniversity of CaliforniaDavis
  2. 2.Section of Microbiology, Division of Biological SciencesUniversity of CaliforniaDavis

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