Contributions of supercoiling to Tn3 resolvase and phage Mu Gin site-specific recombination

Members of the resolvase/invertase family of site-specific recombinases require supercoiled substrates containing two recombination sites. To dissect the roles of supercoiling in recombination by the Tn3 and γδ resolvases and the phage Mu Gin invertase, we used substrates that provided some but not all of the topological features of the standard substrate. We divided the Tn3 resolvase reaction into two stages, synapsis and postsynapsis. We found the contributions of supercoiling to each stage were distinct, since substrate catenation in the absence of supercoiling or low levels of substrate supercoiling were sufficient for synapsis but not postsynapsis. Using structural and functional topological analyses, we verified that the resolvase synaptic complexes with nicked catenanes were recombination intermediates. The requirement for supercoiling was even less stringent for the γδ resolvase, which recombined nicked catenanes about half as well as it did supercoiled substrates. Gin recombination of catenanes occurred even if the recombinational enhancer was on a nicked ring, as long as both crossover sites were on a supercoiled ring. Therefore, supercoiling is required at the Gin crossover sites but not at the enhancer. We conclude that solely conformational effects of supercoiling are required for resolvase synapsis and the function of the Gin enhancer, but that a torsional effect, probably double helix unwinding, is needed for Tn3 resolvase postsynapsis and at the Gin recombination sites.

• View at Publisher
• View at Pubmed
• View at Scopus
• View at Web of Science

Free Full Text ( Final Version , 1mb )