DNA splicing by an active site mutant of Flp recombinase. Possible catalytic cooperativity between the inactive protein and its DNA substrate.
Each strand transfer catalyzed by the Flp recombinase is the composite of two transesterification reactions. The active nucleophilic species in the two reactions are the catalytic site tyrosine (Tyr-343) of Flp and the 5'-hydroxyl from the Flp-nicked DNA substrate, respectively. A "half recombination site" is capable of undergoing this pair of transesterifications in the presence of Flp. When the substrate is a half-site containing a chiral phosphorothioate at the exchange point, the Flp reaction yields a product in which the phosphate chirality is retained. A mutant of Flp that lacks the active site tyrosine, Flp(Y343F), is incapable of mediating strand transfer in a full-recombination site but can execute strand transfer in a half-site. The efficiency of this reaction is about 2% of that of the wild type reaction. The activity of Flp(Y343F) is critically dependent on the length of the half-site spacer. Furthermore, in this reaction, the strand cleavage and strand exchange steps cannot be uncoupled. These results strongly suggest a direct attack by the 5'-hydroxyl of the half-site spacer on the phosphodiester at the normal strand transfer point.
Serre
M C
MC
Department of Microbiology, University of Texas, Austin 78712.
Zheng
L
L
Jayaram
M
M
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D013379:Substrate Specificity; D014443:Tyrosine
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014443:Tyrosine; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase