Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1998 Feb;148(2):545–557. doi: 10.1093/genetics/148.2.545

Chi-dependent intramolecular recombination in Escherichia coli.

R Friedman-Ohana 1, I Karunker 1, A Cohen 1
PMCID: PMC1459820  PMID: 9504905

Abstract

Homologous recombination in Escherichia coli is enhanced by a cis-acting octamer sequence named Chi (5'-GCTGGTGG-3') that interacts with RecBCD. To gain insight into the mechanism of Chi-enhanced recombination, we recruited an experimental system that permits physical monitoring of intramolecular recombination by linear substrates released by in vivo restriction from infecting chimera phage. Recombination of the released substrates depended on recA, recBCD and cis-acting Chi octamers. Recombination proficiency was lowered by a xonA mutation and by mutations that inactivated the RuvABC and RecG resolution enzymes. Activity of Chi sites was influenced by their locations and by the number of Chi octamers at each site. A single Chi site stimulated recombination, but a combination of Chi sites on the two homologs was synergistic. These data suggest a role for Chi at both ends of the linear substrate. Chi was lost in all recombinational exchanges stimulated by a single Chi site. Exchanges in substrates with Chi sites on both homologs occurred in the interval between the sites as well as in the flanking interval. These observations suggest that the generation of circular products by intramolecular recombination involves Chi-dependent processing of one end by RecBCD and pairing of the processed end with its duplex homolog.

Full Text

The Full Text of this article is available as a PDF (262.0 KB).

Selected References

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

  1. Anderson D. G., Kowalczykowski S. C. The recombination hot spot chi is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme. Genes Dev. 1997 Mar 1;11(5):571–581. doi: 10.1101/gad.11.5.571. [DOI] [PubMed] [Google Scholar]
  2. Bedale W. A., Inman R. B., Cox M. M. A reverse DNA strand exchange mediated by recA protein and exonuclease I. The generation of apparent DNA strand breaks by recA protein is explained. J Biol Chem. 1993 Jul 15;268(20):15004–15016. [PubMed] [Google Scholar]
  3. Betlach M., Hershfield V., Chow L., Brown W., Goodman H., Boyer H. W. A restriction endonuclease analysis of the bacterial plasmid controlling the ecoRI restriction and modification of DNA. Fed Proc. 1976 Jul;35(9):2037–2043. [PubMed] [Google Scholar]
  4. 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]
  5. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheng K. C., Smith G. R. Cutting of chi-like sequences by the RecBCD enzyme of Escherichia coli. J Mol Biol. 1987 Apr 20;194(4):747–750. doi: 10.1016/0022-2836(87)90252-x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Cheng K. C., Smith G. R. Recombinational hotspot activity of Chi-like sequences. J Mol Biol. 1984 Dec 5;180(2):371–377. doi: 10.1016/s0022-2836(84)80009-1. [DOI] [PubMed] [Google Scholar]
  9. Clark A. J. Recombination deficient mutants of E. coli and other bacteria. Annu Rev Genet. 1973;7:67–86. doi: 10.1146/annurev.ge.07.120173.000435. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Dabert P., Smith G. R. Gene replacement with linear DNA fragments in wild-type Escherichia coli: enhancement by Chi sites. Genetics. 1997 Apr;145(4):877–889. doi: 10.1093/genetics/145.4.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dixon D. A., Kowalczykowski S. C. Role of the Escherichia coli recombination hotspot, chi, in RecABCD-dependent homologous pairing. J Biol Chem. 1995 Jul 7;270(27):16360–16370. doi: 10.1074/jbc.270.27.16360. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Emmerson P. T. Recombination deficient mutants of Escherichia coli K12 that map between thy A and argA. Genetics. 1968 Sep;60(1):19–30. doi: 10.1093/genetics/60.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Feng W. Y., Hays J. B. DNA structures generated during recombination initiated by mismatch repair of UV-irradiated nonreplicating phage DNA in Escherichia coli: requirements for helicase, exonucleases, and RecF and RecBCD functions. Genetics. 1995 Aug;140(4):1175–1186. doi: 10.1093/genetics/140.4.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
  18. Glassberg J., Meyer R. R., Kornberg A. Mutant single-strand binding protein of Escherichia coli: genetic and physiological characterization. J Bacteriol. 1979 Oct;140(1):14–19. doi: 10.1128/jb.140.1.14-19.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  20. Howard-Flanders P., Theriot L. Mutants of Escherichia coli K-12 defective in DNA repair and in genetic recombination. Genetics. 1966 Jun;53(6):1137–1150. doi: 10.1093/genetics/53.6.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kolodner R., Hall S. D., Luisi-DeLuca C. Homologous pairing proteins encoded by the Escherichia coli recE and recT genes. Mol Microbiol. 1994 Jan;11(1):23–30. doi: 10.1111/j.1365-2958.1994.tb00286.x. [DOI] [PubMed] [Google Scholar]
  22. Konforti B. B., Davis R. W. ATP hydrolysis and the displaced strand are two factors that determine the polarity of RecA-promoted DNA strand exchange. J Mol Biol. 1992 Sep 5;227(1):38–53. doi: 10.1016/0022-2836(92)90680-i. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Kuzminov A., Schabtach E., Stahl F. W. Chi sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease. EMBO J. 1994 Jun 15;13(12):2764–2776. doi: 10.1002/j.1460-2075.1994.tb06570.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. LURIA S. E., BURROUS J. W. Hybridization between Escherichia coli and Shigella. J Bacteriol. 1957 Oct;74(4):461–476. doi: 10.1128/jb.74.4.461-476.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Miesel L., Roth J. R. Evidence that SbcB and RecF pathway functions contribute to RecBCD-dependent transductional recombination. J Bacteriol. 1996 Jun;178(11):3146–3155. doi: 10.1128/jb.178.11.3146-3155.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Myers R. S., Kuzminov A., Stahl F. W. The recombination hot spot chi activates RecBCD recombination by converting Escherichia coli to a recD mutant phenocopy. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6244–6248. doi: 10.1073/pnas.92.14.6244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Myers R. S., Stahl F. W. Chi and the RecBC D enzyme of Escherichia coli. Annu Rev Genet. 1994;28:49–70. doi: 10.1146/annurev.ge.28.120194.000405. [DOI] [PubMed] [Google Scholar]
  31. Nussbaum A., Cohen A. Use of a bioluminescence gene reporter for the investigation of red-dependent and gam-dependent plasmid recombination in Escherichia coli K12. J Mol Biol. 1988 Sep 20;203(2):391–402. doi: 10.1016/0022-2836(88)90007-1. [DOI] [PubMed] [Google Scholar]
  32. Nussbaum A., Shalit M., Cohen A. Restriction-stimulated homologous recombination of plasmids by the RecE pathway of Escherichia coli. Genetics. 1992 Jan;130(1):37–49. doi: 10.1093/genetics/130.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Razavy H., Szigety S. K., Rosenberg S. M. Evidence for both 3' and 5' single-strand DNA ends in intermediates in chi-stimulated recombination in vivo. Genetics. 1996 Feb;142(2):333–339. doi: 10.1093/genetics/142.2.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rinken R., Thomas B., Wackernagel W. Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding. J Bacteriol. 1992 Aug;174(16):5424–5429. doi: 10.1128/jb.174.16.5424-5429.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Schultz D. W., Swindle J., Smith G. R. Clustering of mutations inactivating a Chi recombinational hotspot. J Mol Biol. 1981 Mar 5;146(3):275–286. doi: 10.1016/0022-2836(81)90388-0. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Silberstein Z., Shalit M., Cohen A. Heteroduplex strand-specificity in restriction-stimulated recombination by the RecE pathway of Escherichia coli. Genetics. 1993 Mar;133(3):439–448. doi: 10.1093/genetics/133.3.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Silberstein Z., Tzfati Y., Cohen A. Primary products of break-induced recombination by Escherichia coli RecE pathway. J Bacteriol. 1995 Apr;177(7):1692–1698. doi: 10.1128/jb.177.7.1692-1698.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Smith G. R., Amundsen S. K., Chaudhury A. M., Cheng K. C., Ponticelli A. S., Roberts C. M., Schultz D. W., Taylor A. F. Roles of RecBC enzyme and chi sites in homologous recombination. Cold Spring Harb Symp Quant Biol. 1984;49:485–495. doi: 10.1101/sqb.1984.049.01.055. [DOI] [PubMed] [Google Scholar]
  41. Stahl F. W., Stahl M. M., Malone R. E., Crasemann J. M. Directionality and nonreciprocality of Chi-stimulated recombination in phage lambda. Genetics. 1980 Feb;94(2):235–248. doi: 10.1093/genetics/94.2.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stahl F. W., Stahl M. M. Recombination pathway specificity of Chi. Genetics. 1977 Aug;86(4):715–725. doi: 10.1093/genetics/86.4.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. 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]
  46. Taylor A. F., Smith G. R. Strand specificity of nicking of DNA at Chi sites by RecBCD enzyme. Modulation by ATP and magnesium levels. J Biol Chem. 1995 Oct 13;270(41):24459–24467. doi: 10.1074/jbc.270.41.24459. [DOI] [PubMed] [Google Scholar]
  47. West S. C. Enzymes and molecular mechanisms of genetic recombination. Annu Rev Biochem. 1992;61:603–640. doi: 10.1146/annurev.bi.61.070192.003131. [DOI] [PubMed] [Google Scholar]
  48. Willetts N. S., Clark A. J. Characteristics of some multiply recombination-deficient strains of Escherichia coli. J Bacteriol. 1969 Oct;100(1):231–239. doi: 10.1128/jb.100.1.231-239.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES