Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Feb 16;17(4):1161–1168. doi: 10.1093/emboj/17.4.1161

The function of the secondary DNA-binding site of RecA protein during DNA strand exchange.

A V Mazin 1, S C Kowalczykowski 1
PMCID: PMC1170464  PMID: 9463393

Abstract

RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product. Although the high affinity of the secondary site for ssDNA is essential for DNA strand exchange, it renders DNA strand exchange sensitive to an excess of ssDNA which competes with dsDNA for binding. We further demonstrate that single-stranded DNA-binding protein can sequester ssDNA, preventing its binding to the secondary site and thereby assisting at two levels: it averts the inhibition caused by an excess of ssDNA and prevents the reversal of DNA strand exchange by removing the displaced strand from the secondary site.

Full Text

The Full Text of this article is available as a PDF (369.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bryant F. R., Taylor A. R., Lehman I. R. Interaction of the recA protein of Escherichia coli with single-stranded DNA. J Biol Chem. 1985 Jan 25;260(2):1196–1202. [PubMed] [Google Scholar]
  2. Camerini-Otero R. D., Hsieh P. Homologous recombination proteins in prokaryotes and eukaryotes. Annu Rev Genet. 1995;29:509–552. doi: 10.1146/annurev.ge.29.120195.002453. [DOI] [PubMed] [Google Scholar]
  3. Chow S. A., Honigberg S. M., Bainton R. J., Radding C. M. Patterns of nuclease protection during strand exchange. recA protein forms heteroduplex DNA by binding to strands of the same polarity. J Biol Chem. 1986 May 25;261(15):6961–6971. [PubMed] [Google Scholar]
  4. Chow S. A., Radding C. M. Ionic inhibition of formation of RecA nucleoprotein networks blocks homologous pairing. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5646–5650. doi: 10.1073/pnas.82.17.5646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Conley E. C., West S. C. Underwinding of DNA associated with duplex-duplex pairing by RecA protein. J Biol Chem. 1990 Jun 15;265(17):10156–10163. [PubMed] [Google Scholar]
  6. Cox M. M., Lehman I. R. 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. 1982 Jul 25;257(14):8523–8532. [PubMed] [Google Scholar]
  7. Dunderdale H. J., West S. C. Recombination genes and proteins. Curr Opin Genet Dev. 1994 Apr;4(2):221–228. doi: 10.1016/s0959-437x(05)80048-6. [DOI] [PubMed] [Google Scholar]
  8. Howard-Flanders P., West S. C., Stasiak A. Role of RecA protein spiral filaments in genetic recombination. Nature. 1984 May 17;309(5965):215–219. doi: 10.1038/309215a0. [DOI] [PubMed] [Google Scholar]
  9. Kahn R., Radding C. M. Separation of the presynaptic and synaptic phases of homologous pairing promoted by recA protein. J Biol Chem. 1984 Jun 25;259(12):7495–7503. [PubMed] [Google Scholar]
  10. Kowalczykowski S. C., Eggleston A. K. Homologous pairing and DNA strand-exchange proteins. Annu Rev Biochem. 1994;63:991–1043. doi: 10.1146/annurev.bi.63.070194.005015. [DOI] [PubMed] [Google Scholar]
  11. Kowalczykowski S. C., Krupp R. A. Effects of 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. 1987 Jan 5;193(1):97–113. doi: 10.1016/0022-2836(87)90630-9. [DOI] [PubMed] [Google Scholar]
  12. Lavery P. E., Kowalczykowski S. C. A postsynaptic role for single-stranded DNA-binding protein in recA protein-promoted DNA strand exchange. J Biol Chem. 1992 May 5;267(13):9315–9320. [PubMed] [Google Scholar]
  13. Mazin A. V., Kowalczykowski S. C. The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10673–10678. doi: 10.1073/pnas.93.20.10673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. McEntee K., Weinstock G. M., Lehman I. R. Binding of the recA protein of Escherichia coli to single- and double-stranded DNA. J Biol Chem. 1981 Aug 25;256(16):8835–8844. [PubMed] [Google Scholar]
  15. McEntee K., Weinstock G. M., Lehman I. R. recA protein-catalyzed strand assimilation: stimulation by Escherichia coli single-stranded DNA-binding protein. Proc Natl Acad Sci U S A. 1980 Feb;77(2):857–861. doi: 10.1073/pnas.77.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Menetski J. P., Kowalczykowski S. C. Transfer of recA protein from one polynucleotide to another. Kinetic evidence for a ternary intermediate during the transfer reaction. J Biol Chem. 1987 Feb 15;262(5):2085–2092. [PubMed] [Google Scholar]
  17. Muniyappa K., Shaner S. L., Tsang S. S., Radding C. M. Mechanism of the concerted action of recA protein and helix-destabilizing proteins in homologous recombination. Proc Natl Acad Sci U S A. 1984 May;81(9):2757–2761. doi: 10.1073/pnas.81.9.2757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Müller B., Koller T., Stasiak A. Characterization of the DNA binding activity of stable RecA-DNA complexes. Interaction between the two DNA binding sites within RecA helical filaments. J Mol Biol. 1990 Mar 5;212(1):97–112. doi: 10.1016/0022-2836(90)90307-8. [DOI] [PubMed] [Google Scholar]
  19. Pugh B. F., Cox M. M. General mechanism for RecA protein binding to duplex DNA. J Mol Biol. 1988 Sep 20;203(2):479–493. doi: 10.1016/0022-2836(88)90014-9. [DOI] [PubMed] [Google Scholar]
  20. Pugh B. F., Cox M. M. recA protein binding to the heteroduplex product of DNA strand exchange. J Biol Chem. 1987 Jan 25;262(3):1337–1343. [PubMed] [Google Scholar]
  21. Radding C. M. Helical interactions in homologous pairing and strand exchange driven by RecA protein. J Biol Chem. 1991 Mar 25;266(9):5355–5358. [PubMed] [Google Scholar]
  22. Rao B. J., Radding C. M. Formation of base triplets by non-Watson-Crick bonds mediates homologous recognition in RecA recombination filaments. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6161–6165. doi: 10.1073/pnas.91.13.6161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rao B. J., Radding C. M. Homologous recognition promoted by RecA protein via non-Watson-Crick bonds between identical DNA strands. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6646–6650. doi: 10.1073/pnas.90.14.6646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Roca A. I., Cox M. M. The RecA protein: structure and function. Crit Rev Biochem Mol Biol. 1990;25(6):415–456. doi: 10.3109/10409239009090617. [DOI] [PubMed] [Google Scholar]
  25. Rosselli W., Stasiak A. Energetics of RecA-mediated recombination reactions. Without ATP hydrolysis RecA can mediate polar strand exchange but is unable to recycle. J Mol Biol. 1990 Nov 20;216(2):335–352. doi: 10.1016/S0022-2836(05)80325-0. [DOI] [PubMed] [Google Scholar]
  26. Shibata T., DasGupta C., Cunningham R. P., Radding C. M. Homologous pairing in genetic recombination: formation of D loops by combined action of recA protein and a helix-destabilizing protein. Proc Natl Acad Sci U S A. 1980 May;77(5):2606–2610. doi: 10.1073/pnas.77.5.2606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stasiak A., Stasiak A. Z., Koller T. Visualization of RecA-DNA complexes involved in consecutive stages of an in vitro strand exchange reaction. Cold Spring Harb Symp Quant Biol. 1984;49:561–570. doi: 10.1101/sqb.1984.049.01.063. [DOI] [PubMed] [Google Scholar]
  28. Takahashi M., Kubista M., Nordén B. Binding stoichiometry and structure of RecA-DNA complexes studied by flow linear dichroism and fluorescence spectroscopy. Evidence for multiple heterogeneous DNA co-ordination. J Mol Biol. 1989 Jan 5;205(1):137–147. doi: 10.1016/0022-2836(89)90371-9. [DOI] [PubMed] [Google Scholar]
  29. Tsang S. S., Chow S. A., Radding C. M. Networks of DNA and RecA protein are intermediates in homologous pairing. Biochemistry. 1985 Jun 18;24(13):3226–3232. doi: 10.1021/bi00334a023. [DOI] [PubMed] [Google Scholar]
  30. Ullsperger C. J., Cox M. M. Quantitative RecA protein binding to the hybrid duplex product of DNA strand exchange. Biochemistry. 1995 Aug 29;34(34):10859–10866. doi: 10.1021/bi00034a019. [DOI] [PubMed] [Google Scholar]
  31. Zlotnick A., Mitchell R. S., Steed R. K., Brenner S. L. Analysis of two distinct single-stranded DNA binding sites on the recA nucleoprotein filament. J Biol Chem. 1993 Oct 25;268(30):22525–22530. [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES