Gated rotation mechanism of site-specific recombination by ΦC31 integrase

Integrases, such as that of the Streptomyces temperate bacteriophage ΦC31, promote site-specific recombination between DNAsequences in the bacteriophage and bacterial genomes to integrateor excise the phage DNA. ΦC31 integrase belongs to the serinerecombinase family, a large group of structurally related enzymeswith diverse biological functions. It has been proposed that serineintegrases use a "subunit rotation" mechanism to exchange DNAstrands after double-strand DNA cleavage at the two recombiningatt sites, and that many rounds of subunit rotation can occur beforethe strands are religated.Wehave analyzed the mechanism of ΦC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid "phes" recombination sites, each of which comprises a ΦC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive "360° rotation" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory "gating" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.