Abstract
DNA segment exchange by site-specific serine recombinases (SRs) is thought to proceed by rigid-body rotation of the two halves of the synaptic complex, following the cleavages that create the two pairs of exchangeable ends. It remains unresolved how the amount of rotation occurring between cleavage and religation is controlled. We report single-DNA experiments for Bxb1 integrase, a model SR, where dynamics of individual synapses were observed, using relaxation of supercoiling to report on cleavage and rotation events. Relaxation events often consist of multiple rotations, with the number of rotations per relaxation event and rotation velocity sensitive to DNA sequence at the center of the recombination crossover site, torsional stress and salt concentration. Bulk and single-DNA experiments indicate that the thermodynamic stability of the annealed, but cleaved, crossover sites controls ligation efficiency of recombinant and parental synaptic complexes, regulating the number of rotations during a breakage-religation cycle. The outcome is consistent with a 'controlled rotation' model analogous to that observed for type IB topoisomerases, with religation probability varying in accord with DNA base-pairing free energies at the crossover site. Significantly, we find no evidence for a special regulatory mechanism favoring ligation and product release after a single 180° rotation.