Analyses of the first chemical step in Flp site-specific recombination: Synapsis may not be a pre-requisite for strand cleavage.
The site-specific recombination reaction mediated by the Flp recombinase occurs within a protein-DNA complex containing four monomers of Flp and two DNA substrates. The reaction requires that the strand-exchange region (also called the spacer or overlap region) of the recombining partners be perfectly homologous. A single Flp monomer bound to its recognition sequence is sufficient to orient the scissile phosphodiester adjacent to it for the phosphoryl transfer reaction that induces strand breakage. Cleavage is inhibited when two to three spacer positions adjacent to the reactive phosphodiester are non-complementary. This requirement for Watson-Crick base-pairing can be overcome under conditions that promote formation of a Flp-Flp dimer across the spacer sequence. Synapsis between two Flp-occupied DNA substrates does not appear to be a pre-requisite for triggering strand cleavage. The reaction is likely initiated when a functional Flp dimer is established across the spacer within a single recombination target site. In the absence of a compatible partner, the cleavage reaction is quickly reversed by resealing the nick. Therefore accumulation of strand breakages is avoided. Coordinated partner cleavages within a synaptic complex can lead to strand joining across partners, thus leading the system towards recombination. Our results are consistent with the generally accepted view that homology between recombining partners is not tested till after strand cleavage has occurred.
Voziyanov
Y
Y
Department of Microbiology, University of Texas at Austin, Austin, TX 78712 USA.
Lee
J
J
Whang
I
I
Lee
J
J
Jayaram
M
M
D001665:Binding Sites; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003839:Deoxyadenosines / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019426:Integrases; D008958:Models, Molecular; D009153:Mutagens / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011995:Recombination, Genetic / Q000235:genetics*; D017421:Sequence Analysis
D003839:Deoxyadenosines; D009153:Mutagens; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases; C058120:cordycepin