Mechanism of site-specific recombination. Logic of assembling recombinase catalytic site from fractional active sites.
The active nucleophilic species in the strand cleavage and strand exchange steps of site-specific recombination by the Flp protein are the active site tyrosine (Tyr-343) of Flp and the 5'-hydroxyl of Flp-nicked DNA, respectively. The target phosphodiester, activated by Flp, can be cleaved by an exogenous nucleophile derived, for example, from H2O2. Flp variants that are defective in the phosphate activation step and cannot sustain Tyr-343-mediated cleavage also fail to elicit H2O2-mediated cleavage. An Flp mutant lacking Tyr-343, (Flp(Y343F)), can carry out both the strand cleavage and strand exchange reactions in the presence of a age and strand exchange reactions in the presence of a tyrosine analog. These results are consistent with a cis-activation/trans-nucleophilic attack paradigm for strand breakage and strand union. The proposed model conceptually unifies the chemistry and enzymology of the two partial reactions of recombination. The mechanism of Flp action has strong implications for phosphoryl transfer reactions in other site-specific DNA recombination systems and in RNA splicing.
Lee
J
J
Department of Microbiology, University of Texas, Austin 78712.
Jayaram
M
M
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae; D014443:Tyrosine / Q000378:metabolism
D005656:Fungal Proteins; D014443:Tyrosine; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase