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.
Red-Mediated Recombination
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
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.
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