Tn3 resolvase catalyses multiple recombination events without intermediate rejoining of DNA ends

Abstract

Resolvases and DNA invertases catalyse site-specific recombination by a concerted cut-and-religate mechanism. Topological data strongly suggest a rotational movement of the DNA half-sites during recombination: in an “iterative” mode of reaction, after cleavage of all four strands of the two recombining sites, the recombinase-linked half-sites seem to rotate through multiple steps of 180° prior to final religation. However, current structural data provide no clear support for the postulated corresponding rotation of enzyme subunits within an active tetramer. A key issue is whether repetition of apparent 180° rotation steps requires rejoining of the DNA strands and resetting of the catalytic machinery, or if multiple rotation steps can take place in the fully cleaved intermediate. We present evidence that a resolvase-catalysed DNA knotting reaction, brought about by apparent 360° rotation, can proceed without rejoining of the DNA strands in the recombinant (180° rotation) configuration. This behaviour is not compatible with a mechanism requiring a fixed arrangement of the catalytic subunits, and strongly suggests that recombination is coupled to disruption of the dimer interface between two subunits bound at each crossover site. We also show that an artificial supercoiled plasmid containing two res sites, with a single mismatched base-pair in one of the crossover sites, is a substrate for “suicidal” reactions in which resolvase remains covalently linked to two half-sites.