Synapsis and catalysis by activated Tn3 resolvase mutants.

The serine recombinase Tn3 resolvase catalyses recombination between two 114 bp res sites, each of which contains binding sites for three resolvase dimers. We have analysed the in vitro properties of resolvase variants with 'activating' mutations, which can catalyse recombination at binding site I of res when the rest of res is absent. Site I x site I recombination promoted by these variants can be as fast as res x res recombination promoted by wild-type resolvase. Activated variants have reduced topological selectivity and no longer require the 2-3' interface between subunits that is essential for wild-type resolvase-mediated recombination. They also promote formation of a stable synapse comprising a resolvase tetramer and two copies of site I. Cleavage of the DNA strands by the activated mutants is slow relative to the rate of synapsis. Stable resolvase tetramers were not detected in the absence of DNA or bound to a single site I. Our results lead us to conclude that the synapse is assembled by sequential binding of resolvase monomers to site I followed by interaction of two site I-dimer complexes. We discuss the implications of our results for the mechanisms of synapsis and regulation in recombination by wild-type resolvase.

Implications of structures of synaptic tetramers of gamma delta resolvase for the mechanism of recombination.

The structures of two mutants of the site-specific recombinase, gammadelta resolvase, that form activated tetramers have been determined. One, at 3.5-A resolution, forms a synaptic intermediate of resolvase that is covalently linked to two cleaved DNAs, whereas the other is of an unliganded structure determined at 2.1-A resolution. Comparisons of the four known tetrameric resolvase structures show that the subunits interact through the formation of a common core of four helices. The N-terminal halves of these helices superimpose well on each other, whereas the orientations of their C termini are more variable. The catalytic domains of resolvase in the unliganded structure are arranged asymmetrically, demonstrating that their positions can move substantially while preserving the four-helix core that forms the tetramer. These results suggest that the precleavage synaptic tetramer of gammadelta resolvase, whose structure is not known, may be formed by a similar four-helix core, but differ in the relative orientations of its catalytic and DNA-binding domains.

Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation.

Catalysis of DNA recombination by Tn3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites (res). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent 'accessory' binding sites II and III. 'Hyperactive' resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli. Some single mutants can resolve a res x site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I x site I plasmid. Site I x site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2-3' interface that abolish activity of wild-type resolvase. Activating mutations may allow regulatory mechanisms of the wild-type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.