CONTRIBUTIONS OF SUPERCOILING TO TN3 RESOLVASE AND PHAGE MU GIN SITE-SPECIFIC RECOMBINATION

Members of the resolvase/invertase family of site-specific recombinase s require supercoiled substrates containing two recombination sites. T o dissect the roles of supercoiling in recombination by the Tn3 and ga mma delta resolvases and the phage Mu Gin invertase, we used substrate s that provided some but not all of the topological features of the st andard substrate. We divided the Tn3 resolvase reaction into two stage s, synapsis and postsynapsis. We found the contributions of supercoili ng to each stage were distinct, since substrate catenation in the abse nce of supercoiling or low levels of substrate supercoiling were suffi cient for synapsis but not postsynapsis. Using structural and function al topological analyses, we verified that the resolvase synaptic compl exes with nicked catenanes were recombination intermediates. The requi rement for supercoiling was even less stringent for the gamma delta re solvase, which recombined nicked catenanes about half as well as it di d 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 i s required at the Gin crossover sites but not at the enhancer. We conc lude that solely conformational effects of supercoiling are required f or resolvase synapsis and the function of the Gill enhancer, but that a torsional effect, probably double helix unwinding, is needed for Tn3 resolvase postsynapsis and at the Gin recombination sites. (C) 1996 A cademic Press Limited.