Abstract
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.
Highlights
The FLP recombinase (Mr 48,794) is encoded by the 2-micron plasmid of the yeast Saccharomyces cerevisiae, and promotes a site-specific recombination reaction at sequences within the same plasmid [1, 2]
Experimental Design—The FLP-mediated recombination reaction is relatively slow, even when the FLP recombinase is present in stoichiometric excess relative to available FLP recombination target (FRT) sites
To provide a measure of the disassembly of FLP-FRT complexes following recombination, we designed an experiment to determine the rate at which recombination products were made available for subsequent reactions
Summary
The FLP recombinase (Mr 48,794) is encoded by the 2-micron plasmid of the yeast Saccharomyces cerevisiae, and promotes a site-specific recombination reaction at sequences within the same plasmid [1, 2]. If the spacer is replaced with a symmetric (palindromic) sequence, two reacting FRTs can align in either of two orientations, resulting in a new but predictable set of reaction products. Recent experiments suggest that a pair of cleavage and strand exchange reactions may be carried out by a complex containing three tightly bound FLP monomers.. Recent experiments suggest that a pair of cleavage and strand exchange reactions may be carried out by a complex containing three tightly bound FLP monomers.2 These strong protein-protein interactions led us to ask about the pathway for disassembly of an FLP-FRT complex. A second possibility would involve the release of FLP protomers from the protein-DNA complex after an FLP recombinase-catalyzed recombination reaction as a prerequisite to further reaction.
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