Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 May 1;90(9):3875–3879. doi: 10.1073/pnas.90.9.3875

Biochemical interaction of the Escherichia coli RecF, RecO, and RecR proteins with RecA protein and single-stranded DNA binding protein.

K Umezu 1, N W Chi 1, R D Kolodner 1
PMCID: PMC46408  PMID: 8483906

Abstract

The Escherichia coli RecF, RecO, and RecR proteins were analyzed for their effect on RecA-mediated pairing of single-stranded circular DNA and homologous linear duplex DNA substrates. As shown by other workers, joint molecule formation by RecA was inhibited by E. coli single-stranded DNA binding protein (SSB) when it was added to single-stranded DNA before RecA. This inhibitory effect was overcome by the addition of RecO and RecR or RecF, RecO, and RecR. Both the rate and extent of joint molecule formation were restored to the maximal level observed when SSB was added after RecA. RecF, RecO, and RecR proteins had no effect on the conversion of joint molecules to final products and only appeared to stimulate an early step in the pairing reaction. The stimulatory effect of RecF, RecO, and RecR was not seen without SSB or when SSB was added after RecA. RecF protein by itself inhibited reactions in mixtures containing RecA and SSB, and this inhibition was overcome by the addition of RecO and RecR. These data suggest that RecO and RecR, and possibly RecF, help RecA overcome inhibition by SSB and utilize SSB-single-stranded-DNA complexes as substrates.

Full text

PDF
3875

Images in this article

Selected References

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

  1. Clark A. J. rec genes and homologous recombination proteins in Escherichia coli. Biochimie. 1991 Apr;73(4):523–532. doi: 10.1016/0300-9084(91)90124-j. [DOI] [PubMed] [Google Scholar]
  2. Connolly B., Parsons C. A., Benson F. E., Dunderdale H. J., Sharples G. J., Lloyd R. G., West S. C. Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6063–6067. doi: 10.1073/pnas.88.14.6063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cox M. M., Lehman I. R. Enzymes of general recombination. Annu Rev Biochem. 1987;56:229–262. doi: 10.1146/annurev.bi.56.070187.001305. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Griffin T. J., 4th, Kolodner R. D. Purification and preliminary characterization of the Escherichia coli K-12 recF protein. J Bacteriol. 1990 Nov;172(11):6291–6299. doi: 10.1128/jb.172.11.6291-6299.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Griffith J., Shores C. G. RecA protein rapidly crystallizes in the presence of spermidine: a valuable step in its purification and physical characterization. Biochemistry. 1985 Jan 1;24(1):158–162. doi: 10.1021/bi00322a022. [DOI] [PubMed] [Google Scholar]
  7. Horii Z., Clark A. J. Genetic analysis of the recF pathway to genetic recombination in Escherichia coli K12: isolation and characterization of mutants. J Mol Biol. 1973 Oct 25;80(2):327–344. doi: 10.1016/0022-2836(73)90176-9. [DOI] [PubMed] [Google Scholar]
  8. Iwasaki H., Takahagi M., Nakata A., Shinagawa H. Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration. Genes Dev. 1992 Nov;6(11):2214–2220. doi: 10.1101/gad.6.11.2214. [DOI] [PubMed] [Google Scholar]
  9. Iwasaki H., Takahagi M., Shiba T., Nakata A., Shinagawa H. Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure. EMBO J. 1991 Dec;10(13):4381–4389. doi: 10.1002/j.1460-2075.1991.tb05016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kolodner R., Fishel R. A., Howard M. Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli. J Bacteriol. 1985 Sep;163(3):1060–1066. doi: 10.1128/jb.163.3.1060-1066.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kowalczykowski S. C. Biochemical and biological function of Escherichia coli RecA protein: behavior of mutant RecA proteins. Biochimie. 1991 Feb-Mar;73(2-3):289–304. doi: 10.1016/0300-9084(91)90216-n. [DOI] [PubMed] [Google Scholar]
  12. Kowalczykowski S. C., Clow J., Somani R., Varghese A. Effects of the Escherichia coli SSB protein on the binding of Escherichia coli RecA protein to single-stranded DNA. Demonstration of competitive binding and the lack of a specific protein-protein interaction. J Mol Biol. 1987 Jan 5;193(1):81–95. doi: 10.1016/0022-2836(87)90629-2. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Lavery P. E., Kowalczykowski S. C. Properties of recA441 protein-catalyzed DNA strand exchange can be attributed to an enhanced ability to compete with SSB protein. J Biol Chem. 1990 Mar 5;265(7):4004–4010. [PubMed] [Google Scholar]
  15. Lloyd R. G., Evans N. P., Buckman C. Formation of recombinant lacZ+ DNA in conjugational crosses with a recB mutant of Escherichia coli K12 depends on recF, recJ, and recO. Mol Gen Genet. 1987 Aug;209(1):135–141. doi: 10.1007/BF00329848. [DOI] [PubMed] [Google Scholar]
  16. Lovett S. T., Kolodner R. D. Identification and purification of a single-stranded-DNA-specific exonuclease encoded by the recJ gene of Escherichia coli. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2627–2631. doi: 10.1073/pnas.86.8.2627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Madiraju M. V., Clark A. J. Evidence for ATP binding and double-stranded DNA binding by Escherichia coli RecF protein. J Bacteriol. 1992 Dec;174(23):7705–7710. doi: 10.1128/jb.174.23.7705-7710.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Madiraju M. V., Templin A., Clark A. J. Properties of a mutant recA-encoded protein reveal a possible role for Escherichia coli recF-encoded protein in genetic recombination. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6592–6596. doi: 10.1073/pnas.85.18.6592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mahdi A. A., Lloyd R. G. Identification of the recR locus of Escherichia coli K-12 and analysis of its role in recombination and DNA repair. Mol Gen Genet. 1989 Apr;216(2-3):503–510. doi: 10.1007/BF00334397. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Meyer R. R., Laine P. S. The single-stranded DNA-binding protein of Escherichia coli. Microbiol Rev. 1990 Dec;54(4):342–380. doi: 10.1128/mr.54.4.342-380.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Moreau P. L. Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli. J Bacteriol. 1988 Jun;170(6):2493–2500. doi: 10.1128/jb.170.6.2493-2500.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Norris D., Kolodner R. Interaction of a Saccharomyces cerevisiae strand exchange stimulatory factor with DNA. Biochemistry. 1990 Aug 28;29(34):7911–7917. doi: 10.1021/bi00486a019. [DOI] [PubMed] [Google Scholar]
  25. Sawitzke J. A., Stahl F. W. Phage lambda has an analog of Escherichia coli recO, recR and recF genes. Genetics. 1992 Jan;130(1):7–16. doi: 10.1093/genetics/130.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sharp P. M., Li W. H. The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987 Feb 11;15(3):1281–1295. doi: 10.1093/nar/15.3.1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sharples G. J., Benson F. E., Illing G. T., Lloyd R. G. Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination. Mol Gen Genet. 1990 Apr;221(2):219–226. doi: 10.1007/BF00261724. [DOI] [PubMed] [Google Scholar]
  28. Shibata T., Osber L., Radding C. M. Purification of recA protein from Escherichia coli. Methods Enzymol. 1983;100:197–209. doi: 10.1016/0076-6879(83)00056-7. [DOI] [PubMed] [Google Scholar]
  29. Smith G. R. Homologous recombination in procaryotes. Microbiol Rev. 1988 Mar;52(1):1–28. doi: 10.1128/mr.52.1.1-28.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thresher R. J., Christiansen G., Griffith J. D. Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA. J Mol Biol. 1988 May 5;201(1):101–113. doi: 10.1016/0022-2836(88)90442-1. [DOI] [PubMed] [Google Scholar]
  31. Tsaneva I. R., Müller B., West S. C. ATP-dependent branch migration of Holliday junctions promoted by the RuvA and RuvB proteins of E. coli. Cell. 1992 Jun 26;69(7):1171–1180. doi: 10.1016/0092-8674(92)90638-s. [DOI] [PubMed] [Google Scholar]
  32. Tsang S. S., Muniyappa K., Azhderian E., Gonda D. K., Radding C. M., Flory J., Chase J. W. Intermediates in homologous pairing promoted by recA protein. Isolation and characterization of active presynaptic complexes. J Mol Biol. 1985 Sep 20;185(2):295–309. doi: 10.1016/0022-2836(85)90405-x. [DOI] [PubMed] [Google Scholar]
  33. Umezu K., Nakayama K., Nakayama H. Escherichia coli RecQ protein is a DNA helicase. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5363–5367. doi: 10.1073/pnas.87.14.5363. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES