An Escherichiacoli strain, BJ5183, that shows highly efficient conservative (two-progeny) DNA double-strand break repair of restriction breaks

Abstract

We examined the mode of recombination in an Escherichia coli strain, BJ5183, which has been frequently used in recovery and cloning of eukaryotic DNA. One of the important criteria in characterizing a homologous recombination mechanism is whether it produces two recombinant DNA molecules or only one recombinant DNA molecule out of two parental DNA molecules. Our previous work transferring plasmid molecules with a restriction break into Escherichia coli cells distinguished two modes in recombination stimulated by a double-strand break. In a recBC sbcA mutant strain, where recET genes on the Rac prophage are responsible for recombination (RecE pathway), recombination is often conservative, in the sense that it generates two recombinants out of two parental DNAs. In a recBC sbcBC mutant strain, in which recA and recF genes are responsible (RecF pathway), recombination is non-conservative, in the sense that it generates only one recombinant out of two parental DNAs. Unexpectedly, BJ5183, described as recBC sbcBC, showed very efficient conservative (two-progeny) double-strand break repair. Moreover, this recombination was not eliminated by disruption of its recA gene, which is essential to the RecF pathway. Our polymerase chain reaction analysis detected a recET gene homologue in this strain. This region was easily replaced by a recT::Tn 10 through general transduction and the resulting recT-negative derivative was defective in the conservative double-strand break repair. These results led us to conclude that, in strain BJ5183, the action of recET homologue is responsible for the conservative double-strand break repair as in the RecE pathway. BJ5183 carries a mutation in the endA gene, which codes for Endonuclease I. An endA mutation conferred a higher double-strand break-repair activity to a recBC sbcA mutant strain.