Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as sites, 5`-GCTGGTGG-3`. In vitro, homologous pairing between duplex DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is stimulated by the presence of a sequence in the donor linear double-stranded DNA. We show that this stimulation is due to two factors: 1) the enhanced production of -specific single-stranded DNA fragments and 2) their preferential use in the RecA protein-promoted pairing step. Furthermore, under conditions of limiting Mg concentration, joint molecule formation does not occur, even though DNA unwinding and -specific single-stranded DNA fragment production are observed. Also, under these conditions, -specific fragments derived from both the upstream and downstream regions of the DNA strand containing and from cleavage of the non--containing DNA strand are detected. Finally, the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD) in this in vitro reaction is shown to parallel their in vivo phenotypes with respect to stimulation of recombination. Thus we suggest that, in addition to its ability to regulate the degradative activities of RecBCD enzyme, itself may be a preferred site for initiation of homologous pairing in this concerted process.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. S. Dillingham, M. R. Webb, and S. C. Kowalczykowski Bipolar DNA Translocation Contributes to Highly Processive DNA Unwinding by RecBCD Enzyme J. Biol. Chem., November 4, 2005; 280(44): 37069 - 37077. [Abstract] [Full Text] [PDF]

				M. Spies, M. S. Dillingham, and S. C. Kowalczykowski Translocation by the RecB Motor Is an Absolute Requirement for {chi}-Recognition and RecA Protein Loading by RecBCD Enzyme J. Biol. Chem., November 4, 2005; 280(44): 37078 - 37087. [Abstract] [Full Text] [PDF]

				A. Kulkarni and D. A. Julin Specific inhibition of the E.coli RecBCD enzyme by Chi sequences in single-stranded oligodeoxyribonucleotides Nucleic Acids Res., July 14, 2004; 32(12): 3672 - 3682. [Abstract] [Full Text] [PDF]

				Z.-C. Tu, K. C. Ray, S. A. Thompson, and M. J. Blaser Campylobacter fetus Uses Multiple Loci for DNA Inversion within the 5' Conserved Regions of sap Homologs J. Bacteriol., November 15, 2001; 183(22): 6654 - 6661. [Abstract] [Full Text] [PDF]

				A. Quiberoni, I. Biswas, M. El Karoui, L. Rezaiki, P. Tailliez, and A. Gruss In Vivo Evidence for Two Active Nuclease Motifs in the Double-Strand Break Repair Enzyme RexAB of Lactococcus lactis J. Bacteriol., July 1, 2001; 183(13): 4071 - 4078. [Abstract] [Full Text] [PDF]

				D. A. Arnold and S. C. Kowalczykowski Facilitated Loading of RecA Protein Is Essential to Recombination by RecBCD Enzyme J. Biol. Chem., April 14, 2000; 275(16): 12261 - 12265. [Abstract] [Full Text] [PDF]

				A. Kuzminov Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage lambda Microbiol. Mol. Biol. Rev., December 1, 1999; 63(4): 751 - 813. [Abstract] [Full Text] [PDF]

				J. J. Churchill, D. G. Anderson, and S. C. Kowalczykowski The RecBC enzyme loads RecA protein onto ssDNA asymmetrically and independently of chi , resulting in constitutive recombination activation Genes & Dev., April 1, 1999; 13(7): 901 - 911. [Abstract] [Full Text]

				D. A. Arnold, P. R. Bianco, and S. C. Kowalczykowski The Reduced Levels of chi  Recognition Exhibited by the RecBC1004D Enzyme Reflect Its Recombination Defect in Vivo J. Biol. Chem., June 26, 1998; 273(26): 16476 - 16486. [Abstract] [Full Text] [PDF]

				R. Friedman-Ohana and A. Cohen Heteroduplex joint formation in Escherichia coli recombination is initiated by pairing of a 3'-ending strand PNAS, June 9, 1998; 95(12): 6909 - 6914. [Abstract] [Full Text] [PDF]

				R. Friedman-Ohana, I. Karunker, and A. Cohen Chi-Dependent Intramolecular Recombination in Escherichia coli Genetics, February 1, 1998; 148(2): 545 - 558. [Abstract] [Full Text] [PDF]

				R. B. Tracy, J. K. Baumohl, and S. C. Kowalczykowski The preference for GT-rich DNA by the yeast Rad51 protein defines a set of universal pairing sequences Genes & Dev., December 15, 1997; 11(24): 3423 - 3431. [Abstract] [Full Text] [PDF]

				P. R. Bianco and S. C. Kowalczykowski The recombination hotspot Chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5'-GCTGGTGG-3' PNAS, June 24, 1997; 94(13): 6706 - 6711. [Abstract] [Full Text] [PDF]

				H.-W. Chen, B. Ruan, M. Yu, J.-d. Wang, and D. A. Julin The RecD Subunit of the RecBCD Enzyme from Escherichia coli Is a Single-stranded DNA-dependent ATPase J. Biol. Chem., April 11, 1997; 272(15): 10072 - 10079. [Abstract] [Full Text] [PDF]

				D G Anderson and S C Kowalczykowski The recombination hot spot chi is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme. Genes & Dev., March 1, 1997; 11(5): 571 - 581. [Abstract] [PDF]

				R B Tracy and S C Kowalczykowski In vitro selection of preferred DNA pairing sequences by the Escherichia coli RecA protein. Genes & Dev., August 1, 1996; 10(15): 1890 - 1903. [Abstract] [PDF]

				A. F. Taylor and G. R. Smith Strand Specificity of Nicking of DNA at Chi Sites by RecBCD Enzyme J. Biol. Chem., October 13, 1995; 270(41): 24459 - 24467. [Abstract] [Full Text] [PDF]