title: DNA splicing by an active site mutant of Flp recombinase. Possible catalytic cooperativity between the inactive protein and its DNA substrate.
abstract: 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.
authors.affiliation: Department of Microbiology, University of Texas, Austin 78712.
authors.forename: M C
authors.initials: MC
authors.lastname: Serre
authors.forename: L
authors.initials: L
authors.lastname: Zheng
authors.forename: M
authors.initials: M
authors.lastname: Jayaram
mesh_terms: 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
chemical_list: D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014443:Tyrosine; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase