Rate and selectively of synapsis of res recombination sites by Tn3 resolvase

Abstract

Site-specific recombination catalysed by Tn3 resolvase requires the formation of an intermediate synaptic complex containing two res recombination sites and several resolvase subunits. Synaptic complexes were observed directly by chemical crosslinking of resolvase subunits followed by agarose gel electrophoresis. The highest yield of synaptic complex was from a "standard" substrate, a supercoiled plasmid with res sites in direct repeat, but complexes were also made between sites in inverted repeat, or in nicked or linear molecules, or in separate molecules. The substrate selectivity for synapsis is less stringent than for recombination; thus recombination selectivity is dependent on steps after synapsis. The stability of the synapse after its formation might be a key factor, since unproductive synapses are less stable than productive ones. In a standard substrate, synapsis is fast relative to the rate of recombination. Crosslinking in active reaction mixtures yields synaptic complexes derived from both the substrate and the catenane recombination product. Although catalysis of strand exchange is at binding site I of res, a pair of isolated site 1's do not synapse, whereas a synaptic complex is formed from a plasmid carrying two copies of res binding sites II and III. Our data are consistent with a model in which the formation of the synaptic intermediate is driven, and its structure defined, by the initial interaction of these accessory sites.