Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Mar;177(5):1326–1335. doi: 10.1128/jb.177.5.1326-1335.1995

DNA helicases in recombination and repair: construction of a delta uvrD delta helD delta recQ mutant deficient in recombination and repair.

V M Mendonca 1, H D Klepin 1, S W Matson 1
PMCID: PMC176740  PMID: 7868608

Abstract

DNA helicases play pivotal roles in homologous recombination and recombinational DNA repair. They are involved in both the generation of recombinogenic single-stranded DNA ends and branch migration of synapsed Holliday junctions. Escherichia coli helicases II (uvrD), IV (helD), and RecQ (recQ) have all been implicated in the presynaptic stage of recombination in the RecF pathway. To probe for functional redundancy among these helicases, mutant strains containing single, double, and triple deletions in the helD, uvrD, and recQ genes were constructed and examined for conjugational recombination efficiency and DNA repair proficiency. We were unable to construct a strain harboring a delta recQ delta uvrD double deletion in a recBC sbcB(C) background (RecF pathway), suggesting that a delta recQ deletion mutation was lethal to the cell in a recBC sbcB(C) delta D background. However, we were able to construct a triple delta recQ delta uvrD Delta helD mutant in the recBC sbcB(C) background. This may be due to the increased mutator frequency in delta uvrD mutants which may have resulted in the fortuitous accumulation of a suppressor mutation(s). The triple helicase mutant recBC sbcB(C) delta uvrD delta recQ delta helD severely deficient in Hfr-mediated conjugational recombination and in the repair of methylmethane sulfonate-induced DNA damage. This suggests that the presence of at least one helicase--helicase II, RecQ helicase, or helicase IV--is essential for homologous recombination and recombinational DNA repair in a recBC sbcB(C) background. The triple helicase mutant was recombination and repair proficient in a rec+ background. Genetic analysis of the various double mutants unmasked additional functional redundancies with regard to conjugational recombination and DNA repair, suggesting that mechanisms of recombination depend both on the DNA substrates and on the genotype of the cell.

Full Text

The Full Text of this article is available as a PDF (283.6 KB).

Selected References

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

  1. Appleyard R K. Segregation of New Lysogenic Types during Growth of a Doubly Lysogenic Strain Derived from Escherichia Coli K12. Genetics. 1954 Jul;39(4):440–452. doi: 10.1093/genetics/39.4.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arthur H. M., Lloyd R. G. Hyper-recombination in uvrD mutants of Escherichia coli K-12. Mol Gen Genet. 1980;180(1):185–191. doi: 10.1007/BF00267368. [DOI] [PubMed] [Google Scholar]
  3. Barbour S. D., Nagaishi H., Templin A., Clark A. J. Biochemical and genetic studies of recombination proficiency in Escherichia coli. II. Rec+ revertants caused by indirect suppression of rec- mutations. Proc Natl Acad Sci U S A. 1970 Sep;67(1):128–135. doi: 10.1073/pnas.67.1.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bernstein H., Hopf F. A., Michod R. E. The molecular basis of the evolution of sex. Adv Genet. 1987;24:323–370. doi: 10.1016/s0065-2660(08)60012-7. [DOI] [PubMed] [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  6. CLARK A. J., MARGULIES A. D. ISOLATION AND CHARACTERIZATION OF RECOMBINATION-DEFICIENT MUTANTS OF ESCHERICHIA COLI K12. Proc Natl Acad Sci U S A. 1965 Feb;53:451–459. doi: 10.1073/pnas.53.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clark A. J., Volkert M. R., Margossian L. J. A role for recF in repair of UV damage to DNA. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):887–892. doi: 10.1101/sqb.1979.043.01.096. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Cohen A., Clark A. J. Synthesis of linear plasmid multimers in Escherichia coli K-12. J Bacteriol. 1986 Jul;167(1):327–335. doi: 10.1128/jb.167.1.327-335.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hall S. D., Kane M. F., Kolodner R. D. Identification and characterization of the Escherichia coli RecT protein, a protein encoded by the recE region that promotes renaturation of homologous single-stranded DNA. J Bacteriol. 1993 Jan;175(1):277–287. doi: 10.1128/jb.175.1.277-287.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Howard-Flanders P., Boyce R. P., Theriot L. Three loci in Escherichia coli K-12 that control the excision of pyrimidine dimers and certain other mutagen products from DNA. Genetics. 1966 Jun;53(6):1119–1136. doi: 10.1093/genetics/53.6.1119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jasin M., Schimmel P. Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments. J Bacteriol. 1984 Aug;159(2):783–786. doi: 10.1128/jb.159.2.783-786.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Kowalczykowski S. C. In vitro reconstitution of homologous recombination reactions. Experientia. 1994 Mar 15;50(3):204–215. doi: 10.1007/BF01924003. [DOI] [PubMed] [Google Scholar]
  16. Kusano K., Sunohara Y., Takahashi N., Yoshikura H., Kobayashi I. DNA double-strand break repair: genetic determinants of flanking crossing-over. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):1173–1177. doi: 10.1073/pnas.91.3.1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kushner S. R. In vivo studies of temperature-sensitive recB and recC mutants. J Bacteriol. 1974 Dec;120(3):1213–1218. doi: 10.1128/jb.120.3.1213-1218.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kushner S. R., Nagaishi H., Clark A. J. Indirect suppression of recB and recC mutations by exonuclease I deficiency. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1366–1370. doi: 10.1073/pnas.69.6.1366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kushner S. R., Nagaishi H., Clark A. J. Isolation of exonuclease VIII: the enzyme associated with sbcA indirect suppressor. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3593–3597. doi: 10.1073/pnas.71.9.3593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lloyd R. G., Benson F. E., Shurvinton C. E. Effect of ruv mutations on recombination and DNA repair in Escherichia coli K12. Mol Gen Genet. 1984;194(1-2):303–309. doi: 10.1007/BF00383532. [DOI] [PubMed] [Google Scholar]
  21. Lloyd R. G., Buckman C. Identification and genetic analysis of sbcC mutations in commonly used recBC sbcB strains of Escherichia coli K-12. J Bacteriol. 1985 Nov;164(2):836–844. doi: 10.1128/jb.164.2.836-844.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lloyd R. G., Buckman C. Overlapping functions of recD, recJ and recN provide evidence of three epistatic groups of genes in Escherichia coli recombination and DNA repair. Biochimie. 1991 Feb-Mar;73(2-3):313–320. doi: 10.1016/0300-9084(91)90218-p. [DOI] [PubMed] [Google Scholar]
  23. Lloyd R. G., Picksley S. M., Prescott C. Inducible expression of a gene specific to the RecF pathway for recombination in Escherichia coli K12. Mol Gen Genet. 1983;190(1):162–167. doi: 10.1007/BF00330340. [DOI] [PubMed] [Google Scholar]
  24. Lovett S. T., Clark A. J. Genetic analysis of the recJ gene of Escherichia coli K-12. J Bacteriol. 1984 Jan;157(1):190–196. doi: 10.1128/jb.157.1.190-196.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Lovett S. T., Luisi-DeLuca C., Kolodner R. D. The genetic dependence of recombination in recD mutants of Escherichia coli. Genetics. 1988 Sep;120(1):37–45. doi: 10.1093/genetics/120.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Luisi-DeLuca C., Lovett S. T., Kolodner R. D. Genetic and physical analysis of plasmid recombination in recB recC sbcB and recB recC sbcA Escherichia coli K-12 mutants. Genetics. 1989 Jun;122(2):269–278. doi: 10.1093/genetics/122.2.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Matson S. W., Bean D. W., George J. W. DNA helicases: enzymes with essential roles in all aspects of DNA metabolism. Bioessays. 1994 Jan;16(1):13–22. doi: 10.1002/bies.950160103. [DOI] [PubMed] [Google Scholar]
  29. Mendonca V. M., Kaiser-Rogers K., Matson S. W. Double helicase II (uvrD)-helicase IV (helD) deletion mutants are defective in the recombination pathways of Escherichia coli. J Bacteriol. 1993 Aug;175(15):4641–4651. doi: 10.1128/jb.175.15.4641-4651.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Modrich P. Methyl-directed DNA mismatch correction. J Biol Chem. 1989 Apr 25;264(12):6597–6600. [PubMed] [Google Scholar]
  31. Müller B., West S. C. Processing of Holliday junctions by the Escherichia coli RuvA, RuvB, RuvC and RecG proteins. Experientia. 1994 Mar 15;50(3):216–222. doi: 10.1007/BF01924004. [DOI] [PubMed] [Google Scholar]
  32. Nakayama H., Nakayama K., Nakayama R., Irino N., Nakayama Y., Hanawalt P. C. Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway. Mol Gen Genet. 1984;195(3):474–480. doi: 10.1007/BF00341449. [DOI] [PubMed] [Google Scholar]
  33. Nakayama K., Irino N., Nakayama H. The recQ gene of Escherichia coli K12: molecular cloning and isolation of insertion mutants. Mol Gen Genet. 1985;200(2):266–271. doi: 10.1007/BF00425434. [DOI] [PubMed] [Google Scholar]
  34. Selby C. P., Sancar A. ABC excinuclease incises both 5' and 3' to the CC-1065-DNA adduct and its incision activity is stimulated by DNA helicase II and DNA polymerase I. Biochemistry. 1988 Sep 20;27(19):7184–7188. doi: 10.1021/bi00419a004. [DOI] [PubMed] [Google Scholar]
  35. Siegel E. C. Ultraviolet-sensitive mutator strain of Escherichia coli K-12. J Bacteriol. 1973 Jan;113(1):145–160. doi: 10.1128/jb.113.1.145-160.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Smith G. R. Homologous recombination in E. coli: multiple pathways for multiple reasons. Cell. 1989 Sep 8;58(5):807–809. doi: 10.1016/0092-8674(89)90929-x. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Templin A., Kushner S. R., Clark A. J. Genetic analysis of mutations indirectly suppressing recB and recC mutations. Genetics. 1972 Oct;72(2):105–115. [PMC free article] [PubMed] [Google Scholar]
  39. 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 Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES