JUNCTION MOBILITY AND RESOLUTION OF HOLLIDAY STRUCTURES BY FLP SITE-SPECIFIC RECOMBINASE - TESTING PARTNER COMPATIBILITY DURING RECOMBINATION

Absolute homology between partner substrates within the strand exchang e region (spacer) is an essential requirement for recombination mediat ed by the yeast site-specific recombinase Flp. Recent experiments sugg est that 3-base pair homology adjacent to the points of exchange at ea ch end of the spacer is utilized in a base complementarity-dependent s trand joining reaction. Homology of the central 2 base pairs of the sp acer is also critical, but how homology is tested at these two positio ns is unknown, We have addressed the role of homology-dependent branch migration in Flp recombination by assaying strand cleavage and resolu tion in a set of synthetic Holliday junctions in which the branch poin t is freely or partially mobile through the spacer, or is immobilized at each position within the spacer or immediately nanking 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 t he Flp, binding element and the spacer). A significantly smaller bias is noticed when the branch point is frozen immediately adjacent to thi s position within the spacer. Resolution in these cases is most often mediated by exchange of the scissile phosphodiesters at the branch poi nt or proximal to it, and rarely by exchange of the scissile phosphodi esters distal to it. In light of these and previous results, we discus s possible checkpoints for testing partner compatibility during Flp re combination.