Geometry of site alignment during int family recombination: Antiparallel synapsis by the Flp recombinase

The Flp site-specific recombinase functions in the copy number amplificatio n of the yeast 2 mu m plasmid. The recombination reaction is catalyzed by f our monomers of Flp bound to two separate, but identical, recombination sit es (FRT sites) and occurs in two sequential pairs of strand exchanges. The relative orientation of the two recombination sites during synapsis was exa mined. Topoisomerase relaxation and nick ligation were used to detect topol ogical nodes introduced by the synapse prior to the chemical steps of recom bination. A single negative supercoil was found to be trapped by Flp in sub strates with inverted FRT sites whereas no trapped supercoils were observed with direct repeats. The topology of products resulting from Flp-mediated recombination adjacent to a well characterised synapse, that of Tn3 resolva se/res, was analyzed. The deletion and inversion reactions yielded the four noded catenane and the three noded knot, respectively, as the simplest and the most abundant products. The linking number change introduced by the Fl p-mediated inversion reaction was determined to be +/-2. The most parsimoni ous explanation of these results is that Flp aligns its recombination sites with antiparallel geometry. The majority of synapses appear to occur witho ut entrapment of additional random plectonemic DNA supercoils between the s ites and no additional crossings are introduced as a result of the chemical steps of recombination. (C) 2000 Academic Press.