Skip to main content
PMC home page
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
. 1994 Apr 12;91(8):2980–2984. doi: 10.1073/pnas.91.8.2980

Reversible inactivation of the Escherichia coli RecBCD enzyme by the recombination hotspot chi in vitro: evidence for functional inactivation or loss of the RecD subunit.

D A Dixon 1, J J Churchill 1, S C Kowalczykowski 1
PMCID: PMC43498  PMID: 8159691

Abstract

Genetic recombination in Escherichia coli is stimulated by a RecBCD enzyme-mediated event at DNA sequences known as Chi (chi) sites (5'-GCTGGTGG-3'). Previously, it was shown that chi acts to regulate the nuclease activity of RecBCD; here, we demonstrate that, under appropriate conditions, interaction with chi sites can also result in an inactivation of helicase activity of RecBCD. The unwinding of double-stranded DNA-containing chi sites, under conditions of limiting Mg2+ ion, results in the reversible inactivation of RecBCD; addition of excess Mg2+ to the reaction reactivates all activities of RecBCD. Inactivation is the consequence of a chi-dependent modification of RecBCD that appears to result from an inability of the chi-modified RecBCD to reinitiate unwinding of intact DNA molecules. This characteristic behavior of RecBCD and chi is displayed by the reconstituted RecBC (i.e., without the RecD subunit), except that it is not dependent on chi interaction. This biochemical similarity between the chi-modified RecBCD and RecBC enzymes implies that recognition of chi results in a dissociation or functional inactivation of RecD subunit and lends support to the hypothesis that interaction with chi results in ejection of the RecD subunit.

Full text

PDF
2980

Images in this article

2982Image
on p.2982
2984Image
on p.2984

Selected References

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

  1. Amundsen S. K., Taylor A. F., Chaudhury A. M., Smith G. R. recD: the gene for an essential third subunit of exonuclease V. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5558–5562. doi: 10.1073/pnas.83.15.5558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boehmer P. E., Emmerson P. T. Escherichia coli RecBCD enzyme: inducible overproduction and reconstitution of the ATP-dependent deoxyribonuclease from purified subunits. Gene. 1991 Jun 15;102(1):1–6. doi: 10.1016/0378-1119(91)90529-k. [DOI] [PubMed] [Google Scholar]
  3. Boehmer P. E., Emmerson P. T. The RecB subunit of the Escherichia coli RecBCD enzyme couples ATP hydrolysis to DNA unwinding. J Biol Chem. 1992 Mar 5;267(7):4981–4987. [PubMed] [Google Scholar]
  4. Chaudhury A. M., Smith G. R. A new class of Escherichia coli recBC mutants: implications for the role of RecBC enzyme in homologous recombination. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7850–7854. doi: 10.1073/pnas.81.24.7850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cheng K. C., Smith G. R. Distribution of Chi-stimulated recombinational exchanges and heteroduplex endpoints in phage lambda. Genetics. 1989 Sep;123(1):5–17. doi: 10.1093/genetics/123.1.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dabert P., Ehrlich S. D., Gruss A. Chi sequence protects against RecBCD degradation of DNA in vivo. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):12073–12077. doi: 10.1073/pnas.89.24.12073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dixon D. A., Kowalczykowski S. C. Homologous pairing in vitro stimulated by the recombination hotspot, Chi. Cell. 1991 Jul 26;66(2):361–371. doi: 10.1016/0092-8674(91)90625-9. [DOI] [PubMed] [Google Scholar]
  8. Dixon D. A., Kowalczykowski S. C. The recombination hotspot chi is a regulatory sequence that acts by attenuating the nuclease activity of the E. coli RecBCD enzyme. Cell. 1993 Apr 9;73(1):87–96. doi: 10.1016/0092-8674(93)90162-j. [DOI] [PubMed] [Google Scholar]
  9. Dower N. A., Stahl F. W. Chi activity during transduction-associated recombination. Proc Natl Acad Sci U S A. 1981 Nov;78(11):7033–7037. doi: 10.1073/pnas.78.11.7033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eggleston A. K., Kowalczykowski S. C. Biochemical characterization of a mutant recBCD enzyme, the recB2109CD enzyme, which lacks chi-specific, but not non-specific, nuclease activity. J Mol Biol. 1993 Jun 5;231(3):605–620. doi: 10.1006/jmbi.1993.1313. [DOI] [PubMed] [Google Scholar]
  11. Eichler D. C., Lehman I. R. On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli. J Biol Chem. 1977 Jan 25;252(2):499–503. [PubMed] [Google Scholar]
  12. Hermanns U., Wackernagel W. The recBC enzyme of Escherichia coli K12: premature cessation of catalytic activities in vitro and reactivation by potassium ions. Eur J Biochem. 1977 Jun 15;76(2):425–432. doi: 10.1111/j.1432-1033.1977.tb11611.x. [DOI] [PubMed] [Google Scholar]
  13. Lam S. T., Stahl M. M., McMilin K. D., Stahl F. W. Rec-mediated recombinational hot spot activity in bacteriophage lambda. II. A mutation which causes hot spot activity. Genetics. 1974 Jul;77(3):425–433. doi: 10.1093/genetics/77.3.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Mackay V., Linn S. Selective inhibition of the dnase activity of the recBC enzyme by the DNA binding protein from Escherichia coli. J Biol Chem. 1976 Jun 25;251(12):3716–3719. [PubMed] [Google Scholar]
  16. Masterson C., Boehmer P. E., McDonald F., Chaudhuri S., Hickson I. D., Emmerson P. T. Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits. J Biol Chem. 1992 Jul 5;267(19):13564–13572. [PubMed] [Google Scholar]
  17. Palas K. M., Kushner S. R. Biochemical and physical characterization of exonuclease V from Escherichia coli. Comparison of the catalytic activities of the RecBC and RecBCD enzymes. J Biol Chem. 1990 Feb 25;265(6):3447–3454. [PubMed] [Google Scholar]
  18. Ponticelli A. S., Schultz D. W., Taylor A. F., Smith G. R. Chi-dependent DNA strand cleavage by RecBC enzyme. Cell. 1985 May;41(1):145–151. doi: 10.1016/0092-8674(85)90069-8. [DOI] [PubMed] [Google Scholar]
  19. Roman L. J., Eggleston A. K., Kowalczykowski S. C. Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme. J Biol Chem. 1992 Feb 25;267(6):4207–4214. [PubMed] [Google Scholar]
  20. Roman L. J., Kowalczykowski S. C. Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay. Biochemistry. 1989 Apr 4;28(7):2863–2873. doi: 10.1021/bi00433a018. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Ruyechan W. T., Wetmur J. G. Studies on the cooperative binding of the Escherichia coli DNA unwinding protein to single-stranded DNA. Biochemistry. 1975 Dec 16;14(25):5529–5534. doi: 10.1021/bi00696a023. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Smith G. R., Kunes S. M., Schultz D. W., Taylor A., Triman K. L. Structure of chi hotspots of generalized recombination. Cell. 1981 May;24(2):429–436. doi: 10.1016/0092-8674(81)90333-0. [DOI] [PubMed] [Google Scholar]
  25. Stahl F. W., Crasemann J. M., Stahl M. M. Rec-mediated recombinational hot spot activity in bacteriophage lambda. III. Chi mutations are site-mutations stimulating rec-mediated recombination. J Mol Biol. 1975 May 15;94(2):203–212. doi: 10.1016/0022-2836(75)90078-9. [DOI] [PubMed] [Google Scholar]
  26. Stahl F. W., Thomason L. C., Siddiqi I., Stahl M. M. Further tests of a recombination model in which chi removes the RecD subunit from the RecBCD enzyme of Escherichia coli. Genetics. 1990 Nov;126(3):519–533. doi: 10.1093/genetics/126.3.519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Taylor A. F., Schultz D. W., Ponticelli A. S., Smith G. R. RecBC enzyme nicking at Chi sites during DNA unwinding: location and orientation-dependence of the cutting. Cell. 1985 May;41(1):153–163. doi: 10.1016/0092-8674(85)90070-4. [DOI] [PubMed] [Google Scholar]
  28. Taylor A. F., Smith G. R. RecBCD enzyme is altered upon cutting DNA at a chi recombination hotspot. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5226–5230. doi: 10.1073/pnas.89.12.5226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Taylor A., Smith G. R. Unwinding and rewinding of DNA by the RecBC enzyme. Cell. 1980 Nov;22(2 Pt 2):447–457. doi: 10.1016/0092-8674(80)90355-4. [DOI] [PubMed] [Google Scholar]
  30. Thaler D. S., Sampson E., Siddiqi I., Rosenberg S. M., Thomason L. C., Stahl F. W., Stahl M. M. Recombination of bacteriophage lambda in recD mutants of Escherichia coli. Genome. 1989;31(1):53–67. doi: 10.1139/g89-013. [DOI] [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