ANALYSES OF THE FIRST CHEMICAL STEP IN FLP SITE-SPECIFIC RECOMBINATION - SYNAPSIS MAY NOT BE A PREREQUISITE FOR STRAND CLEAVAGE

The site-specific recombination reaction mediated by the Flp recombina se occurs within a protein-DNA complex containing four monomers of Flp and two DNA substrates. The reaction requires that the strand-exchang e region (also called the spacer or overlap region) of the recombining partners be perfectly homologous. A single Flp monomer bound to its r ecognition sequence is sufficient to orient the scissile phosphodieste r adjacent to it for the phosphoryl transfer reaction that induces str and breakage. Cleavage is inhibited when two to three spacer positions adjacent to the reactive phosphodiester are non-complementary This re quirement for Watson-Crick base-pairing can be overcome under conditio ns that promote formation of a Flp-Flp dimer across the spacer sequenc e. Synapsis between two Flp-occupied DNA substrates does not appear to be a pre-requisite for triggering strand cleavage. The reaction is li kely initiated when a functional Flp dimer is established across the s pacer within a single recombination target site. In the absence of a c ompatible partner, the cleavage reaction is quickly reversed by reseal ing the nick. Therefore accumulation of strand breakages is avoided. C oordinated partner cleavages within a synaptic complex can lead to str and joining across partners, thus leading the system towards recombina tion. Our results are consistent with the generally accepted view that homology between recombining partners is not tested till after strand cleavage has occurred. (C) 1996 Academic Press Limited