A protein dissociation step limits turnover in FLP recombinase-mediated site-specific recombination.
When two ongoing FLP-mediated recombination reactions are mixed, formation of cross-products is subject to a lag of several minutes, and the subsequent rate of cross-product formation is greatly reduced relative to normal reaction progress curves. The lag reflects the formation of a stable complex containing multiple FLP monomers and two FLP recombination target-containing DNA recombination products, a process completed within 5-10 min after addition of FLP recombinase to a reaction mixture. The reaction products are sequestered within this complex for an extended period of time, unavailable for further reaction. The length of the lag increases with increasing FLP protein concentration and is not affected by the introduction of unreacted (non FLP-bound) substrate. The results provide evidence that disassembly of FLP complexes from products occurs in a minimum of two steps. At least one FLP protein monomer is released from reaction complexes in a discrete step that leaves the reaction products sequestered. The recombination products are released in a form free to react with other FLP recombination target-containing DNA molecules only after at least one additional dissassembly 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.
Waite
L L
LL
Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin-Madison 53706, USA.
Cox
M M
MM
D001483:Base Sequence; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D004271:DNA, Fungal; D005656:Fungal Proteins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase