title: The Flp recombinase cleaves Holliday junctions in trans.
abstract: The Flp site-specific recombinase is encoded by the 2 micrometers plasmid Saccharomyces cerevisiae and is a member of the integrase family of recombinases. Like all members of the integrase family studied, Flp mediates recombination in two steps. First, a pair of strand exchanges creates a Holliday-like intermediate; second, this intermediate is resolved to recombinant products by a second pair of strand exchanges. Evidence derived from experiments using linear substrates indicates that Flp's active site is composed of two Flp protomers. One binds to the Flp recognition target site (FRT site) and activates the scissile phosphodiester bond for cleavage. Another molecule of Flp bound elsewhere in the synaptic complex (in trans) donates the nucleophilic tyrosine that executes cleavage and thereby becomes covalently attached to the 3' phosphoryl group at the cleavage site. It has previously been shown that Flp efficiently resolves synthetic, Holliday-like (chi) structures to linear products. In this paper, we examined whether resolution of chi structures by Flp also occurs via the trans cleavage mechanism. We used in vitro complementation studies of mutant Flp proteins as well as nicked chi structures to show that Flp resolves chi structures by trans cleavage. We propose a model for Flp-mediated recombination that incorporates trans cleavage at both the initial and resolution steps of strand exchange.
authors: Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
authors: J E
authors: JE
authors: Dixon
authors: A C
authors: AC
authors: Shaikh
authors: P D
authors: PD
authors: Sadowski
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase