Genetic recombination of bacterial plasmid DNA: Analysis of the effect of recombination-deficient mutations on plasmid recombination

Abstract

The recombination of two plasmids, pRDK101 and pACYC184, was examined in wild-type Escherichia coli and in various E. coli strains containing different recombination-deficient mutations. Recombination-induced oligomer formation from monomeric plasmids was found to be dependent on the functions of the recA, recB, recC and recF genes. Intramolecular recombination of tetrameric plasmids to form lower-order oligomers and monomers also required the functions of the recA and recF genes but did not require the function of the recB and recC genes. In all cases where recA, recB, recC and recF mutations appeared to block the formation or reduction of plasmid circular oligomers, these effects could be reversed by the presence of an sbcA mutation. Further studies on recombination in vivo were carried out utilizing two tetracycline-sensitive derivatives of the compatible plasmids pACYC184 and pBR322. Recombination events between these two plasmids could be quantitated by measuring the production of tetracycline-resistant cells during the growth of transformants. Plasmid recombination was found to be reduced by recA and recF mutations, and the effect of these two mutations was reversed by the presence of an sbcA mutation. Plasmid recombination measured by this genetic assay was stimulated by recB recC mutations, and electrophoretic analysis of the recombination products demonstrated that they were primarily circular monomers. A role for the recB recC gene product, exonuclease V, in the resolution of recombination intermediates is discussed.