title: Synapsis and catalysis by activated Tn3 resolvase mutants.
abstract: 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.
authors.affiliation: Faculty of Biomedical & Life Sciences, University of Glasgow, Glasgow, Scotland, UK.
authors.forename: Femi J
authors.initials: FJ
authors.lastname: Olorunniji
authors.forename: Jiuya
authors.initials: J
authors.lastname: He
authors.forename: Sandra V C T
authors.initials: SV
authors.lastname: Wenwieser
authors.forename: Martin R
authors.initials: MR
authors.lastname: Boocock
authors.forename: W Marshall
authors.initials: WM
authors.lastname: Stark
mesh_terms: D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011995:Recombination, Genetic; D045564:Transposon Resolvases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D004247:DNA; C473491:Tn3 resolvase; D045564:Transposon Resolvases