A PROTEIN DISSOCIATION STEP LIMITS TURNOVER IN FLP RECOMBINASE-MEDIATED SITE-SPECIFIC RECOMBINATION

When two ongoing FLP-mediated recombination reactions are mixed, forma tion of cross-products is subject to a lag of several minutes, and the subsequent rate of cross-product formation is greatly reduced relativ e to normal reaction progress curves. The lag reflects the formation o f a stable complex containing multiple FLP monomers and two FLP recomb ination target-containing DNA recombination products, a process comple ted within 5-10 min after addition of FLP recombinase to a reaction mi xture. The reaction products are sequestered within this complex for a n extended period of time, unavailable for further reaction. The lengt h of the lag increases with increasing FLP protein concentration and i s not affected by the introduction of unreacted (non FLP-bound) substr ate. The results provide evidence that disassembly of FLP complexes fr om products occurs in a minimum of two steps. At least one FLP protein monomer is released from reaction complexes in a discrete step that l eaves the reaction products sequestered. The recombination products ar e released in a form free to react with other FLP recombination target -containing DNA molecules only after at least one additional disassemb ly step. One or both of these disassembly steps are rate limiting for reaction turnover under conditions often used to monitor FLP-mediated recombination in vitro.