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DNA Repair Protocols pp 75–89Cite as

Coupling of DNA Helicase Function to DNA Strand Exchange Activity

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 152))

Abstract

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.

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References

  1. Friedberg, E. C., Walker, G. C., and Siede, W. (1995) DNA Repair and Mutagenesis, ASM, Washington, DC

    Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  3. Kowalczykowski, S. C. and Eggleston, A. K. (1994) Homologous pairing and DNA strand-exchange proteins. Annu. Rev. Biochem. 63, 991–1043.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  10. Taylor, A. and Smith, G. R. (1980) Unwinding and rewinding of DNA by the recBC enzyme. Cell. 22, 447–457.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  12. Eggleston, A. K. and West, S. C. (1997) Recombination initiation: easy as A, B, C, D... chi? Curr. Biol. 7, R745–749.

    Article  PubMed  CAS  Google Scholar 

  13. Arnold, D. A. and Kowalczykowski, S. C. (1999) RecBCD nuclease/helicase, in Encyclopedia of Life Sciences, Nature Publishing Group, London, http://www.els.net.

    Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  15. Lanzov, V., Stepanova, I., and Vinogradskaja, G. (1991) Genetic control of recombination exchange frequency in Escherichia coli K-12. Biochimie 73, 305–312.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  18. Dixon, D. A. and Kowalczykowski, S. C. (1991) Homologous pairing in vitro stimulated by the recombination hotspot, Chi. Cell 66, 361–371.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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. LeBowitz, J. (1985) Biochemical mechanism of strand initiation in bacteriophage lambda DNA replication. Ph.D. thesis, Johns Hopkins University, Baltimore, MD.

    Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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. 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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  35. Sung, P. (1994) Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science 265, 1241–1243.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Division of Biological Sciences, Sections of Microbiology and Molecular and Cellular Biology, University of California, Davis, CA

    Frank G. Harmon

  2. Section of Microbiology, Division of Biological Sciences, University of California, Davis, CA

    Stephen C. Kowalczykowski

Authors
  1. Frank G. Harmon
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  2. Stephen C. Kowalczykowski
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Editor information

Editors and Affiliations

  1. HiberGen Ltd. and University College, Cork, Ireland

    Pat Vaughan

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© 2000 Humana Press Inc.

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Harmon, F.G., Kowalczykowski, S.C. (2000). Coupling of DNA Helicase Function to DNA Strand Exchange Activity. In: Vaughan, P. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 152. Humana Press. https://doi.org/10.1385/1-59259-068-3:75

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  • DOI: https://doi.org/10.1385/1-59259-068-3:75

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-643-7

  • Online ISBN: 978-1-59259-068-1

  • eBook Packages: Springer Protocols

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