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The Flp recombinase cleaves Holliday junctions in trans
Author(s) -
Dixon Julie E.,
Shaikh Arkady C.,
Sadowski Paul D.
Publication year - 1995
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1995.mmi_18030449.x
Subject(s) - biology , holliday junction , recombinase , genetics , recombination , flp frt recombination , computational biology , gene , genetic recombination
The Flp site‐specific recombinase is encoded by the 2 µm plasmid of 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 (χ) structures to linear products. In this paper, we examined whether resolution of χ structures by Flp also occurs via the trans cleavage mechanism. We used in vitro complementation studies of mutant Flp proteins as well as nicked χ structures to show that Flp resolves χ 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.