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Journal of Bacteriology, September 1999, p. 5402-5408, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Roles of RuvC and RecG in Phage lambda  Red-Mediated Recombination

Anthony R. Poteete,* Anita C. Fenton, and Kenan C. Murphy

Department of Molecular Genetics & Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

Received 22 March 1999/Accepted 30 May 1999

The recombination properties of Escherichia coli strains expressing the red genes of bacteriophage lambda  and lacking recBCD function either by mutation or by expression of lambda  gam were examined. The substrates for recombination were nonreplicating lambda  chromosomes, introduced by infection; Red-mediated recombination was initiated by a double-strand break created by the action of a restriction endonuclease in the infected cell. In one type of experiment, two phages marked with restriction site polymorphisms were crossed. Efficient formation of recombinant DNA molecules was observed in ruvC+ recG+, ruvC recG+, ruvC+ recG, and ruvC recG hosts. In a second type of experiment, a 1-kb nonhomology was inserted between the double-strand break and the donor chromosome's restriction site marker. In this case, recombinant formation was found to be partially dependent upon ruvC function, especially in a recG mutant background. In a third type of experiment, the recombining partners were the host cell chromosome and a 4-kb linear DNA fragment containing the cat gene, with flanking lac sequences, released from the infecting phage chromosome by restriction enzyme cleavage in the cell; the formation of chloramphenicol-resistant bacterial progeny was measured. Dependence on RuvC varied considerably among the three types of cross. However, in all cases, the frequency of Red-mediated recombination was higher in recG than in recG+. These observations favor models in which RecG tends to push invading 3'-ended strands back out of recombination intermediates.

* Corresponding author. Mailing address: Dept. of Molecular Genetics & Microbiology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655. Phone: (508) 856-3708. Fax: (508) 856-5920. E-mail: tpoteete{at}ummed.edu.

Journal of Bacteriology, September 1999, p. 5402-5408, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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