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. 1997 Jul 15;25(14):2766–2772. doi: 10.1093/nar/25.14.2766

Purification and characterization of the RecF protein from Bacillus subtilis 168.

S Ayora 1, J C Alonso 1
PMCID: PMC146836  PMID: 9207023

Abstract

Genetic evidence suggests that the Bacillus subtilis recF gene product is involved in DNA repair and recombination. The RecF protein was overproduced and purified. NH2-terminal protein sequence analysis of RecF was consistent with the deduced amino acid sequence of the recF gene. The RecF protein (predicted molecular mass 42.3 kDa) bound single- and double-stranded DNA in a filter binding and in a gel retarding assay. The RecF-ssDNA or -dsDNA complex formation proceeds in the absence of nucleotide cofactors. RecF-ssDNA interaction is markedly stimulated by divalent cations. The apparent equilibrium constants of the RecF-DNA complexes are approximately 110-130 nM for both ssDNA and dsDNA. The binding reaction shows no cooperativity. The RecF protein does not physically interact with the RecR protein. Under our experimental conditions an ATPase activity was not associated with the purified RecF protein or with the RecF and RecR proteins.

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Selected References

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  1. Alonso J. C., Fisher L. M. Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants. Mol Gen Genet. 1995 Mar 20;246(6):680–686. doi: 10.1007/BF00290713. [DOI] [PubMed] [Google Scholar]
  2. Alonso J. C., Lüder G. Characterization of recF suppressors in Bacillus subtilis. Biochimie. 1991 Feb-Mar;73(2-3):277–280. doi: 10.1016/0300-9084(91)90213-k. [DOI] [PubMed] [Google Scholar]
  3. Alonso J. C., Stiege A. C., Dobrinski B., Lurz R. Purification and properties of the RecR protein from Bacillus subtilis 168. J Biol Chem. 1993 Jan 15;268(2):1424–1429. [PubMed] [Google Scholar]
  4. Alonso J. C., Stiege A. C. Molecular analysis of the Bacillus subtilis recF function. Mol Gen Genet. 1991 Sep;228(3):393–400. doi: 10.1007/BF00260632. [DOI] [PubMed] [Google Scholar]
  5. Ayora S., Rojo F., Ogasawara N., Nakai S., Alonso J. C. The Mfd protein of Bacillus subtilis 168 is involved in both transcription-coupled DNA repair and DNA recombination. J Mol Biol. 1996 Feb 23;256(2):301–318. doi: 10.1006/jmbi.1996.0087. [DOI] [PubMed] [Google Scholar]
  6. Ayora S., Stiege A. C., Alonso J. C. RecR is a zinc metalloprotein from Bacillus subtilis 168. Mol Microbiol. 1997 Feb;23(4):639–647. doi: 10.1046/j.1365-2958.1997.2431611.x. [DOI] [PubMed] [Google Scholar]
  7. Ayora S., Stiege A. C., Lurz R., Alonso J. C. Bacillus subtilis 168 RecR protein-DNA complexes visualized as looped structures. Mol Gen Genet. 1997 Mar 18;254(1):54–62. doi: 10.1007/s004380050390. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Clark A. J., Sandler S. J. Homologous genetic recombination: the pieces begin to fall into place. Crit Rev Microbiol. 1994;20(2):125–142. doi: 10.3109/10408419409113552. [DOI] [PubMed] [Google Scholar]
  10. Eggleston A. K., West S. C. Exchanging partners: recombination in E. coli. Trends Genet. 1996 Jan;12(1):20–26. doi: 10.1016/0168-9525(96)81384-9. [DOI] [PubMed] [Google Scholar]
  11. Gassel M., Alonso J. C. Expression of the recE gene during induction of the SOS response in Bacillus subtilis recombination-deficient strains. Mol Microbiol. 1989 Sep;3(9):1269–1276. doi: 10.1111/j.1365-2958.1989.tb00277.x. [DOI] [PubMed] [Google Scholar]
  12. Gorbalenya A. E., Koonin E. V. Superfamily of UvrA-related NTP-binding proteins. Implications for rational classification of recombination/repair systems. J Mol Biol. 1990 Jun 20;213(4):583–591. doi: 10.1016/S0022-2836(05)80243-8. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Haima P., Bron S., Venema G. The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg. Mol Gen Genet. 1987 Sep;209(2):335–342. doi: 10.1007/BF00329663. [DOI] [PubMed] [Google Scholar]
  15. Hegde S. P., Qin M. H., Li X. H., Atkinson M. A., Clark A. J., Rajagopalan M., Madiraju M. V. Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF-RecO-RecR complex in DNA repair and recombination. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14468–14473. doi: 10.1073/pnas.93.25.14468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hegde S. P., Rajagopalan M., Madiraju M. V. Preferential binding of Escherichia coli RecF protein to gapped DNA in the presence of adenosine (gamma-thio) triphosphate. J Bacteriol. 1996 Jan;178(1):184–190. doi: 10.1128/jb.178.1.184-190.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kowalczykowski S. C., Dixon D. A., Eggleston A. K., Lauder S. D., Rehrauer W. M. Biochemistry of homologous recombination in Escherichia coli. Microbiol Rev. 1994 Sep;58(3):401–465. doi: 10.1128/mr.58.3.401-465.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lohman T. M., Ferrari M. E. Escherichia coli single-stranded DNA-binding protein: multiple DNA-binding modes and cooperativities. Annu Rev Biochem. 1994;63:527–570. doi: 10.1146/annurev.bi.63.070194.002523. [DOI] [PubMed] [Google Scholar]
  19. Madiraju M. V., Clark A. J. Effect of RecF protein on reactions catalyzed by RecA protein. Nucleic Acids Res. 1991 Nov 25;19(22):6295–6300. doi: 10.1093/nar/19.22.6295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Menetski J. P., Kowalczykowski S. C. Interaction of recA protein with single-stranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors. J Mol Biol. 1985 Jan 20;181(2):281–295. doi: 10.1016/0022-2836(85)90092-0. [DOI] [PubMed] [Google Scholar]
  22. Riggs A. D., Bourgeois S., Cohn M. The lac repressor-operator interaction. 3. Kinetic studies. J Mol Biol. 1970 Nov 14;53(3):401–417. doi: 10.1016/0022-2836(70)90074-4. [DOI] [PubMed] [Google Scholar]
  23. Sandler S. J., Chackerian B., Li J. T., Clark A. J. Sequence and complementation analysis of recF genes from Escherichia coli, Salmonella typhimurium, Pseudomonas putida and Bacillus subtilis: evidence for an essential phosphate binding loop. Nucleic Acids Res. 1992 Feb 25;20(4):839–845. doi: 10.1093/nar/20.4.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  25. Umezu K., Chi N. W., Kolodner R. D. Biochemical interaction of the Escherichia coli RecF, RecO, and RecR proteins with RecA protein and single-stranded DNA binding protein. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3875–3879. doi: 10.1073/pnas.90.9.3875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Umezu K., Kolodner R. D. Protein interactions in genetic recombination in Escherichia coli. Interactions involving RecO and RecR overcome the inhibition of RecA by single-stranded DNA-binding protein. J Biol Chem. 1994 Nov 25;269(47):30005–30013. [PubMed] [Google Scholar]
  27. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Webb B. L., Cox M. M., Inman R. B. An interaction between the Escherichia coli RecF and RecR proteins dependent on ATP and double-stranded DNA. J Biol Chem. 1995 Dec 29;270(52):31397–31404. doi: 10.1074/jbc.270.52.31397. [DOI] [PubMed] [Google Scholar]
  29. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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