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Genetics. Jan 1992; 130(1): 37–49.
PMCID: PMC1204803

Restriction-Stimulated Homologous Recombination of Plasmids by the Rece Pathway of Escherichia Coli

Abstract

To test the double-strand break (DSB) repair model in recombination by the RecE pathway of Escherichia coli, we constructed chimeric phages that allow restriction-mediated release of linear plasmid substrates of the bioluminescence recombination assay in infected EcoRI(+) cells. Kinetics of DSB repair and expression of recombination products were followed by Southern hybridization and by the bioluminescence recombination assay, respectively. Plasmid recombinants were analyzed with restriction endonucleases. Our results indicate that a DSB can induce more than one type of RecE-mediated recombination. A DSB within the homology induced intermolecular recombination that followed the rules of the DSB repair model: (1) Recombination was enhanced by in vivo restriction. (2) Repair of the break depended on homologous sequences on the resident plasmid. (3) Break-repair was frequently associated with conversion of alleles that were cis to the break. (4) Conversion frequency decreased as the distance from the break increased. (5) Some clones contained a mixture of plasmid recombinants as expected by replication of a heteroduplex in the primary recombinant. The rules of the DSB repair model were not followed when recombination was induced by a DSB outside the homology. Both the cut and the uncut substrates were recipients in conversion events. Recombination events were associated with deletions that spanned the break site, but these deletions did not reach the homology. We propose that a break outside the homology may stimulate a RecE-mediated recombination pathway that does not involve direct participation of DNA ends in the homologous pairing reaction.

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Selected References

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  • Betlach M, Hershfield V, Chow L, Brown W, Goodman H, Boyer HW. 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]
  • Better M, Freifelder D. Studies on the replication of Escherichia coli phage lambda DNA. I. The kinetics of DNA replication and requirements for the generation of rolling circles. Virology. 1983 Apr 15;126(1):168–182. [PubMed]
  • Cohen A, Clark AJ. Synthesis of linear plasmid multimers in Escherichia coli K-12. J Bacteriol. 1986 Jul;167(1):327–335. [PMC free article] [PubMed]
  • Conley EC, Saunders VA, Jackson V, Saunders JR. Mechanism of intramolecular recyclization and deletion formation following transformation of Escherichia coli with linearized plasmid DNA. Nucleic Acids Res. 1986 Nov 25;14(22):8919–8932. [PMC free article] [PubMed]
  • Frischauf AM, Lehrach H, Poustka A, Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. [PubMed]
  • Gillen JR, Willis DK, Clark AJ. Genetic analysis of the RecE pathway of genetic recombination in Escherichia coli K-12. J Bacteriol. 1981 Jan;145(1):521–532. [PMC free article] [PubMed]
  • Holmes DS, Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. [PubMed]
  • Kobayashi I, Murialdo H, Crasemann JM, Stahl MM, Stahl FW. Orientation of cohesive end site cos determines the active orientation of chi sequence in stimulating recA . recBC-mediated recombination in phage lambda lytic infections. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5981–5985. [PMC free article] [PubMed]
  • Kushner SR, Nagaishi H, Clark AJ. Isolation of exonuclease VIII: the enzyme associated with sbcA indirect suppressor. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3593–3597. [PMC free article] [PubMed]
  • LURIA SE, BURROUS JW. Hybridization between Escherichia coli and Shigella. J Bacteriol. 1957 Oct;74(4):461–476. [PMC free article] [PubMed]
  • Nickoloff JA, Singer JD, Hoekstra MF, Heffron F. Double-strand breaks stimulate alternative mechanisms of recombination repair. J Mol Biol. 1989 Jun 5;207(3):527–541. [PubMed]
  • 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. [PubMed]
  • Orr-Weaver TL, Szostak JW. Yeast recombination: the association between double-strand gap repair and crossing-over. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4417–4421. [PMC free article] [PubMed]
  • Ponticelli AS, Schultz DW, Taylor AF, Smith GR. Chi-dependent DNA strand cleavage by RecBC enzyme. Cell. 1985 May;41(1):145–151. [PubMed]
  • Radding CM, Shreffler DC. Regulation of lambda exonuclease. II. Joint regulation of exonuclease and a new lambda antigen. J Mol Biol. 1966 Jul;18(2):251–261. [PubMed]
  • Resnick MA. The repair of double-strand breaks in DNA; a model involving recombination. J Theor Biol. 1976 Jun;59(1):97–106. [PubMed]
  • Rosamond J, Telander KM, Linn S. Modulation of the action of the recBC enzyme of Escherichia coli K-12 by Ca2+. J Biol Chem. 1979 Sep 10;254(17):8646–8652. [PubMed]
  • Silberstein Z, Maor S, Berger I, Cohen A. Lambda Red-mediated synthesis of plasmid linear multimers in Escherichia coli K12. Mol Gen Genet. 1990 Sep;223(3):496–507. [PubMed]
  • Smith GE, Summers MD. The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl-paper. Anal Biochem. 1980 Nov 15;109(1):123–129. [PubMed]
  • Smith GR. Mechanism and control of homologous recombination in Escherichia coli. Annu Rev Genet. 1987;21:179–201. [PubMed]
  • Smith GR. Homologous recombination in procaryotes. Microbiol Rev. 1988 Mar;52(1):1–28. [PMC free article] [PubMed]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
  • Stahl FW, Kobayashi I, Stahl MM. In phage lambda, cos is a recombinator in the red pathway. J Mol Biol. 1985 Jan 20;181(2):199–209. [PubMed]
  • Stahl FW, McMilin KD, Stahl MM, Crasemann JM, Lam S. The distribution of crossovers along unreplicated lambda bacteriophage chromosomes. Genetics. 1974 Jul;77(3):395–408. [PMC free article] [PubMed]
  • Stahl FW, Kobayashi I, Thaler D, Stahl MM. Direction of travel of RecBC recombinase through bacteriophage lambda DNA. Genetics. 1986 Jun;113(2):215–227. [PMC free article] [PubMed]
  • Symington LS, Morrison P, Kolodner R. Intramolecular recombination of linear DNA catalyzed by the Escherichia coli RecE recombination system. J Mol Biol. 1985 Dec 5;186(3):515–525. [PubMed]
  • Taylor A, Smith GR. Unwinding and rewinding of DNA by the RecBC enzyme. Cell. 1980 Nov;22(2 Pt 2):447–457. [PubMed]
  • Taylor AF, Smith GR. Substrate specificity of the DNA unwinding activity of the RecBC enzyme of Escherichia coli. J Mol Biol. 1985 Sep 20;185(2):431–443. [PubMed]
  • Taylor AF, Schultz DW, Ponticelli AS, Smith GR. RecBC enzyme nicking at Chi sites during DNA unwinding: location and orientation-dependence of the cutting. Cell. 1985 May;41(1):153–163. [PubMed]
  • Thaler DS, Stahl MM, Stahl FW. Tests of the double-strand-break repair model for red-mediated recombination of phage lambda and plasmid lambda dv. Genetics. 1987 Aug;116(4):501–511. [PMC free article] [PubMed]
  • Thaler DS, Stahl MM, Stahl FW. Double-chain-cut sites are recombination hotspots in the Red pathway of phage lambda. J Mol Biol. 1987 May 5;195(1):75–87. [PubMed]
  • Thaler DS, Sampson E, Siddiqi I, Rosenberg SM, Thomason LC, Stahl FW, Stahl MM. Recombination of bacteriophage lambda in recD mutants of Escherichia coli. Genome. 1989;31(1):53–67. [PubMed]

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