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Journal of Bacteriology, September 1999, p. 5402-5408, Vol. 181, No. 17
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
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Roles of RuvC and RecG in Phage
Red-Mediated Recombination
and
lacking recBCD function either by mutation or by expression
of
gam were examined. The substrates for recombination
were nonreplicating
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.
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