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J. Bacteriol. doi:10.1128/JB.01052-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Effects of single-strand DNases ExoI, RecJ, ExoVII, and SbcCD on homologous recombination of recBCD⁺ strains of Escherichia coli and roles of SbcB15 and XonA2 mutant enzymes of ExoI

Genetics, Department of Biology and Environmental Sciences, Carl von Ossietzky University Oldenburg, D-26111 Oldenburg, Germany

^* To whom correspondence should be addressed. Email: wilfried.wackernagel{at}uni-oldenburg.de.

	Abstract

To assess contributions of single-strand (ss) DNases to recombination in a recBCD⁺ background we studied 31 strains with all combinations of null alleles of exonuclease I (xon), exonuclease VII (xseA), RecJ DNase (recJ), and SbcCD DNase (sbcCD) and exonuclease I mutant alleles xonA2 and sbcB15. The quadruple mutants xse recJ sbcCD xon and xse recJ sbcCD sbcB15 were cold-sensitive while that with xonA2 was not. UV sensitivity increased with ssDNase deficiencies. Most triple and quadruple mutants were highly sensitive. Absence of ssDNases hardly affected P1 transductional recombinant formation and conjugational recombinant production was decreased (up to 94%) in several cases. Strains with sbcB15 were generally like wildtype. We identified the sbcB15 mutation to cause an A183V exchange in the exo motif III and xonA2 as a stop codon eliminating the terminal 8 aa. Purified enzymes had 1.6% (SbcB15) and 0.9% (XonA2) of the specific activity of Xon⁺, respectively, with altered activity profiles. In gel shift assays SbcB15 associated relatively stably with 3' DNA overhangs giving protection against Xon⁺. In addition to their postsynaptic roles in the RecBCD pathway ExoI and RecJ are proposed to have presynaptic roles of DNA end-blunting. Blunting may be specifically required during conjugation to make DNA with overhangs RecBCD targets for initiation of recombination. Evidence is provided that SbcB15 protein known to activate the RecF pathway in recBC strains contributes independently of RecF to recombination in recBCD⁺ cells. DNA end binding by SbcB15 can explain also other specific phenotypes of strains with sbcB15.

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