Junction mobility and resolution of Holliday structures by Flp site-specific recombinase. Testing partner...

Absolute homology between partner substrates within the strand exchange region (spacer) is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Recent experiments suggest that 3-base pair homology adjacent to the points of exchange at each end of the spacer is utilized in a base complementarity-dependent strand joining reaction. Homology of the central 2 base pairs of the spacer is also critical, but how homology is tested at these two positions is unknown. We have addressed the role of homology-dependent branch migration in Flp recombination by assaying strand cleavage and resolution in a set of synthetic Holliday junctions in which the branch point is freely or partially mobile through the spacer, or is immobilized at each position within the spacer or immediately flanking it. A strong bias in the direction of Holliday resolution is observed only when the branch point is located just outside the spacer (at the junction of the Flp binding element and the spacer). A significantly smaller bias is noticed when the branch point is frozen immediately adjacent to this position within the spacer. Resolution in these cases is most often mediated by exchange of the scissile phosphodiesters at the branch point or proximal to it, and rarely by exchange of the scissile phosphodiesters distal to it. In light of these and previous results, we discuss possible checkpoints for testing partner compatibility during Flp recombination.