Gated rotation mechanism of site-specific recombination by ΦC31 integrase

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 109; no. 48; pp. 19661 - 19666
• Main Authors: Olorunniji, Femi J., Buck, Dorothy E., Colloms, Sean D., McEwan, Andrew R., Smith, Margaret C. M., Stark, W. Marshall, Rosser, Susan J.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 27-11-2012; National Acad Sciences
• Subjects: Bacteriophages; Bacteriophages - enzymology; Bacteriophages - genetics; Biological Sciences; Catenanes; DNA; DNA, Viral - genetics; Gels; Hybridity; Integrases - genetics; Integrases - metabolism; Ligation; Plasmids; Recombination, Genetic; Rotation; Synapses; Topology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1210964109

Integrases, such as that of the Streptomyces temperate bacteriophage ΦC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ΦC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a "subunit rotation" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining ait sites, and that many rounds of subunit rotation can occur before the strands are religated. We have 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.