DNA splicing by an active site mutant of Flp recombinase. Possible catalytic cooperativity between the inactive protein and its DNA substrate.

摘要: Abstract Each strand transfer catalyzed by the Flp recombinase is composite of two transesterification reactions. The active nucleophilic species in reactions are catalytic site tyrosine (Tyr-343) and 5'-hydroxyl from Flp-nicked DNA substrate, respectively. A "half recombination site" capable undergoing this pair transesterifications presence Flp. When substrate a half-site containing chiral phosphorothioate at exchange point, reaction yields product which phosphate chirality retained. mutant that lacks tyrosine, Flp(Y343F), incapable mediating full-recombination but can execute half-site. efficiency about 2% wild type reaction. activity Flp(Y343F) critically dependent on length spacer. Furthermore, reaction, cleavage steps cannot be uncoupled. These results strongly suggest direct attack spacer phosphodiester normal point.