ASSEMBLY AND ORIENTATION OF FLP RECOMBINASE-ACTIVE SITES ON 2-ARMED, 3-ARMED AND 4-ARMED DNA SUBSTRATES - IMPLICATIONS FOR A RECOMBINATION MECHANISM

The normal recombination reaction catalyzed by the Flp (pronounced fli p) site-specific recombinase between two full-site DNA substrates requ ires the action of four recombinase monomers in concert. Each monomer of the recombinase harbors an incomplete active site, and is hence che mically incompetent. In order to organize the strand cleavage pocket, it must accept the catalytic tyrosine (Tyr 343) from a second Flp mono mer. We address the issue of the potential modes of assembling the sha red active site in substrates containing two, three or four Flp bindin g arms. In normal full-sites (two Flp binding arms), strand cleavage o ccurs within a substrate and not across substrates. Flp is able to res olve a Y structure (three Flp binding arms) into linear plus hairpin r ecombinants. Strand cleavage by Flp in a Y structure and a Holliday st ructure (four Flp binding arms) follows the trans rather than the cis mode. Within the context of two normal full-sites, all of the strand c utting patterns are best accommodated by a single cleavage mode, namel y the trans-horizontal mode. The assembly and orientation of a Flp act ive site is determined by whether two Flp-bound DNA arms have the stac king flexibility to accommodate the relevant protein-protein interacti ons. These results provide support for a model in which pairs of monom ers bound within each of the two DNA partners contribute to the strand cleavage reactions that initiate and terminate a normal recombination event. Thus all four Flp monomers are required to mediate the cleavag e/joining events at either end of the strand exchange region. (C) 1996 Academic Press Limited