Mechanism of Tn3 resolvase recombination in vivo

Abstract

To determine the physiologically important features of site-specific recombination by Escherichia coli Tn3 resolvase we analyzed the salient properties of the reaction in vivo. A two-plasmid system in which one plasmid served as substrate while the other encoded both resolvase and a thermolabile repressor of resolvase transcription provided controlled, synchronous recombination. Recombination proceeded rapidly and was promoted by (-) DNA supercoiling. The structures of the in vivo recombination products were predominantly the same as those previously identified in vitro. By examination of the products of successive rounds of recombination of a four-site substrate, we ruled out a tracking mechanism for site alignment. Inversion and plasmid fusion occurred in vivo at a much lower rate than resolution but ultimately reached a higher extent than found in vitro. We propose that inversion and fusion exploit topologically interlinked substrates that occur at low levels in vivo. This proposal is supported by the unexpected specificity of fusion. Our data imply that supercoiled DNA, the resolvase synaptic complex, and the mechanism of strand exchange are fundamentally similar in vivo and in vitro, but that the repertoire of resolvase substrates and products is expanded in vivo by the action of other enzymes that alter DNA topology.