Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Dec;178(23):6782–6789. doi: 10.1128/jb.178.23.6782-6789.1996

Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12.

A A Al-Deib 1, A A Mahdi 1, R G Lloyd 1
PMCID: PMC178576  PMID: 8955297

Abstract

The RecG protein of Escherichia coli is a structure-specific DNA helicase that targets strand exchange intermediates in genetic recombination and drives their branch migration along the DNA. Strains carrying null mutations in recG show reduced recombination and DNA repair. Suppressors of this phenotype, called srgA, were located close to metB and shown to be alleles of priA. Suppression depends on the RecA, RecBCD, RecF, RuvAB, and RuvC recombination proteins. Nine srgA mutations were sequenced and shown to specify mutant PriA proteins with single amino acid substitutions located in or close to one of the conserved helicase motifs. The mutant proteins retain the ability to catalyze primosome assembly, as judged by the viability of recG srgA and srgA strains and their ability to support replication of plasmids based on the ColE1 replicon. Multicopy priA+ plasmids increase substantially the recombination- and repair-deficient phenotype of recG strains and confer similar phenotypes on recG srgA double mutants but not on ruvAB or wild-type strains. The multicopy effect is eliminated by K230R, C446G, and C477G substitutions in PriA. It is concluded that the 3'-5' DNA helicase/translocase activity of PriA inhibits recombination and that this effect is normally countered by RecG.

Full Text

The Full Text of this article is available as a PDF (245.5 KB).

Selected References

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

  1. Asai T., Bates D. B., Kogoma T. DNA replication triggered by double-stranded breaks in E. coli: dependence on homologous recombination functions. Cell. 1994 Sep 23;78(6):1051–1061. doi: 10.1016/0092-8674(94)90279-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Asai T., Kogoma T. Roles of ruvA, ruvC and recG gene functions in normal and DNA damage-inducible replication of the Escherichia coli chromosome. Genetics. 1994 Aug;137(4):895–902. doi: 10.1093/genetics/137.4.895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Asai T., Sommer S., Bailone A., Kogoma T. Homologous recombination-dependent initiation of DNA replication from DNA damage-inducible origins in Escherichia coli. EMBO J. 1993 Aug;12(8):3287–3295. doi: 10.1002/j.1460-2075.1993.tb05998.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Foster P. L., Trimarchi J. M., Maurer R. A. Two enzymes, both of which process recombination intermediates, have opposite effects on adaptive mutation in Escherichia coli. Genetics. 1996 Jan;142(1):25–37. doi: 10.1093/genetics/142.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Harris R. S., Ross K. J., Rosenberg S. M. Opposing roles of the holliday junction processing systems of Escherichia coli in recombination-dependent adaptive mutation. Genetics. 1996 Mar;142(3):681–691. doi: 10.1093/genetics/142.3.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Itoh T., Tomizawa J. Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc Natl Acad Sci U S A. 1980 May;77(5):2450–2454. doi: 10.1073/pnas.77.5.2450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kogoma T., Cadwell G. W., Barnard K. G., Asai T. The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair. J Bacteriol. 1996 Mar;178(5):1258–1264. doi: 10.1128/jb.178.5.1258-1264.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kogoma T. Recombination by replication. Cell. 1996 May 31;85(5):625–627. doi: 10.1016/s0092-8674(00)81229-5. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Krishnan B. R., Blakesley R. W., Berg D. E. Linear amplification DNA sequencing directly from single phage plaques and bacterial colonies. Nucleic Acids Res. 1991 Mar 11;19(5):1153–1153. doi: 10.1093/nar/19.5.1153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kuzminov A. Collapse and repair of replication forks in Escherichia coli. Mol Microbiol. 1995 May;16(3):373–384. doi: 10.1111/j.1365-2958.1995.tb02403.x. [DOI] [PubMed] [Google Scholar]
  13. Lee E. H., Masai H., Allen G. C., Jr, Kornberg A. The priA gene encoding the primosomal replicative n' protein of Escherichia coli. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4620–4624. doi: 10.1073/pnas.87.12.4620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lee M. S., Marians K. J. Escherichia coli replication factor Y, a component of the primosome, can act as a DNA helicase. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8345–8349. doi: 10.1073/pnas.84.23.8345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lloyd R. G., Buckman C. Conjugational recombination in Escherichia coli: genetic analysis of recombinant formation in Hfr x F- crosses. Genetics. 1995 Mar;139(3):1123–1148. doi: 10.1093/genetics/139.3.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lloyd R. G., Buckman C. Genetic analysis of the recG locus of Escherichia coli K-12 and of its role in recombination and DNA repair. J Bacteriol. 1991 Feb;173(3):1004–1011. doi: 10.1128/jb.173.3.1004-1011.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lloyd R. G. Conjugational recombination in resolvase-deficient ruvC mutants of Escherichia coli K-12 depends on recG. J Bacteriol. 1991 Sep;173(17):5414–5418. doi: 10.1128/jb.173.17.5414-5418.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Lloyd R. G., Low B., Godson G. N., Birge E. A. Isolation and characterization of an Escherichia coli K-12 mutant with a temperature-sensitive recA- phenotype. J Bacteriol. 1974 Oct;120(1):407–415. doi: 10.1128/jb.120.1.407-415.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lloyd R. G., Sharples G. J. Dissociation of synthetic Holliday junctions by E. coli RecG protein. EMBO J. 1993 Jan;12(1):17–22. doi: 10.1002/j.1460-2075.1993.tb05627.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lloyd R. G., Thomas A. A molecular model for conjugational recombination in Escherichia coli K12. Mol Gen Genet. 1984;197(2):328–336. doi: 10.1007/BF00330981. [DOI] [PubMed] [Google Scholar]
  22. Magee T. R., Kogoma T. Requirement of RecBC enzyme and an elevated level of activated RecA for induced stable DNA replication in Escherichia coli. J Bacteriol. 1990 Apr;172(4):1834–1839. doi: 10.1128/jb.172.4.1834-1839.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mahdi A. A., Sharples G. J., Mandal T. N., Lloyd R. G. Holliday junction resolvases encoded by homologous rusA genes in Escherichia coli K-12 and phage 82. J Mol Biol. 1996 Apr 5;257(3):561–573. doi: 10.1006/jmbi.1996.0185. [DOI] [PubMed] [Google Scholar]
  24. Mandal T. N., Mahdi A. A., Sharples G. J., Lloyd R. G. Resolution of Holliday intermediates in recombination and DNA repair: indirect suppression of ruvA, ruvB, and ruvC mutations. J Bacteriol. 1993 Jul;175(14):4325–4334. doi: 10.1128/jb.175.14.4325-4334.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marians K. J. Prokaryotic DNA replication. Annu Rev Biochem. 1992;61:673–719. doi: 10.1146/annurev.bi.61.070192.003325. [DOI] [PubMed] [Google Scholar]
  26. Masai H., Asai T., Kubota Y., Arai K., Kogoma T. Escherichia coli PriA protein is essential for inducible and constitutive stable DNA replication. EMBO J. 1994 Nov 15;13(22):5338–5345. doi: 10.1002/j.1460-2075.1994.tb06868.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nurse P., DiGate R. J., Zavitz K. H., Marians K. J. Molecular cloning and DNA sequence analysis of Escherichia coli priA, the gene encoding the primosomal protein replication factor Y. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4615–4619. doi: 10.1073/pnas.87.12.4615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nurse P., Zavitz K. H., Marians K. J. Inactivation of the Escherichia coli priA DNA replication protein induces the SOS response. J Bacteriol. 1991 Nov;173(21):6686–6693. doi: 10.1128/jb.173.21.6686-6693.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Parsons C. A., Stasiak A., Bennett R. J., West S. C. Structure of a multisubunit complex that promotes DNA branch migration. Nature. 1995 Mar 23;374(6520):375–378. doi: 10.1038/374375a0. [DOI] [PubMed] [Google Scholar]
  30. Rosenberg S. M., Hastings P. J. The split-end model for homologous recombination at double-strand breaks and at Chi. Biochimie. 1991 Apr;73(4):385–397. doi: 10.1016/0300-9084(91)90105-a. [DOI] [PubMed] [Google Scholar]
  31. Sandler S. J., Samra H. S., Clark A. J. Differential suppression of priA2::kan phenotypes in Escherichia coli K-12 by mutations in priA, lexA, and dnaC. Genetics. 1996 May;143(1):5–13. doi: 10.1093/genetics/143.1.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shah R., Bennett R. J., West S. C. Genetic recombination in E. coli: RuvC protein cleaves Holliday junctions at resolution hotspots in vitro. Cell. 1994 Dec 2;79(5):853–864. doi: 10.1016/0092-8674(94)90074-4. [DOI] [PubMed] [Google Scholar]
  33. Sharples G. J., Chan S. N., Mahdi A. A., Whitby M. C., Lloyd R. G. Processing of intermediates in recombination and DNA repair: identification of a new endonuclease that specifically cleaves Holliday junctions. EMBO J. 1994 Dec 15;13(24):6133–6142. doi: 10.1002/j.1460-2075.1994.tb06960.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sharples G. J., Whitby M. C., Ryder L., Lloyd R. G. A mutation in helicase motif III of E. coli RecG protein abolishes branch migration of Holliday junctions. Nucleic Acids Res. 1994 Feb 11;22(3):308–313. doi: 10.1093/nar/22.3.308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shurvinton C. E., Lloyd R. G., Benson F. E., Attfield P. V. Genetic analysis and molecular cloning of the Escherichia coli ruv gene. Mol Gen Genet. 1984;194(1-2):322–329. doi: 10.1007/BF00383535. [DOI] [PubMed] [Google Scholar]
  36. Smith G. R. Conjugational recombination in E. coli: myths and mechanisms. Cell. 1991 Jan 11;64(1):19–27. doi: 10.1016/0092-8674(91)90205-d. [DOI] [PubMed] [Google Scholar]
  37. Triggs-Raine B. L., Loewen P. C. Physical characterization of katG, encoding catalase HPI of Escherichia coli. Gene. 1987;52(2-3):121–128. doi: 10.1016/0378-1119(87)90038-2. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. West S. C. The RuvABC proteins and Holliday junction processing in Escherichia coli. J Bacteriol. 1996 Mar;178(5):1237–1241. doi: 10.1128/jb.178.5.1237-1241.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Whitby M. C., Lloyd R. G. Branch migration of three-strand recombination intermediates by RecG, a possible pathway for securing exchanges initiated by 3'-tailed duplex DNA. EMBO J. 1995 Jul 17;14(14):3302–3310. doi: 10.1002/j.1460-2075.1995.tb07337.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Whitby M. C., Ryder L., Lloyd R. G. Reverse branch migration of Holliday junctions by RecG protein: a new mechanism for resolution of intermediates in recombination and DNA repair. Cell. 1993 Oct 22;75(2):341–350. doi: 10.1016/0092-8674(93)80075-p. [DOI] [PubMed] [Google Scholar]
  43. Whitby M. C., Vincent S. D., Lloyd R. G. Branch migration of Holliday junctions: identification of RecG protein as a junction specific DNA helicase. EMBO J. 1994 Nov 1;13(21):5220–5228. doi: 10.1002/j.1460-2075.1994.tb06853.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Zavitz K. H., Marians K. J. ATPase-deficient mutants of the Escherichia coli DNA replication protein PriA are capable of catalyzing the assembly of active primosomes. J Biol Chem. 1992 Apr 5;267(10):6933–6940. [PubMed] [Google Scholar]
  46. Zavitz K. H., Marians K. J. Dissecting the functional role of PriA protein-catalysed primosome assembly in Escherichia coli DNA replication. Mol Microbiol. 1991 Dec;5(12):2869–2873. doi: 10.1111/j.1365-2958.1991.tb01846.x. [DOI] [PubMed] [Google Scholar]
  47. Zavitz K. H., Marians K. J. Helicase-deficient cysteine to glycine substitution mutants of Escherichia coli replication protein PriA retain single-stranded DNA-dependent ATPase activity. Zn2+ stimulation of mutant PriA helicase and primosome assembly activities. J Biol Chem. 1993 Feb 25;268(6):4337–4346. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES