Assembly and orientation of Flp recombinase active sites on two-, three- and four-armed DNA substrates: implications for a recombination mechanism.
The normal recombination reaction catalyzed by the Flp (pronounced flip) site-specific recombinase between two full-site DNA substrates requires the action of four recombinase monomers in concert. Each monomer of the recombinase harbors an incomplete active site, and is hence chemically incompetent. In order to organize the strand cleavage pocket, it must accept the catalytic tyrosine (Tyr 343) from a second Flp monomer. We address the issue of the potential modes of assembling the shared active site in substrates containing two, three or four Flp binding arms. In normal full-sites (two Flp binding arms), strand cleavage occurs within a substrate and not across substrates. Flp is able to resolve a Y structure (three Flp binding arms) into linear plus hairpin recombinants. Strand cleavage by Flp in a Y structure and in a Holliday structure (four Flp binding arms) follows the trans rather than the cis mode. Within the context of two normal full-sites, all of the strand cutting patterns are best accommodated by a single-cleavage mode, namely the trans-horizontal mode. The assembly and orientation of a Flp active site is determined by whether two Flp-bound DNA arms have the stacking flexibility to accommodate the relevant protein-protein interactions. These results provide support for a model in which pairs of monomers 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 cleavage/joining events at either end of the strand exchange region.
Lee
J
J
Department of Microbiology, University of Texas at Austin, 78712, USA.
Whang
I
I
Jayaram
M
M
D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D013379:Substrate Specificity
D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase