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The Flp recombinase cleaves Holliday junctions in trans
J E Dixon1, A C Shaikh, P D Sadowski
1Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
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.
Insights
The Flp recombinase resolves complex DNA structures using a trans cleavage mechanism, where one protein molecule acts on another. This mechanism is crucial for both initial strand exchange and final resolution in Flp-mediated recombination.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Flp recombinase, from the 2 micrometers plasmid of Saccharomyces cerevisiae, is an integrase family member.
- Flp-mediated recombination involves two steps: formation of a Holliday-like intermediate and its resolution.
- Previous studies indicated Flp's active site involves two protomers and a trans cleavage mechanism for initial strand exchange.
Purpose of the Study:
- To investigate whether Flp resolves Holliday-like (chi) structures via the trans cleavage mechanism.
- To elucidate the mechanism of chi structure resolution by Flp recombinase.
Main Methods:
- In vitro complementation studies using mutant Flp proteins.
- Experiments utilizing nicked chi structures.
Main Results:
- Flp was shown to resolve chi structures through a trans cleavage mechanism.
- Evidence supports the involvement of trans cleavage in both initial strand exchange and resolution steps.
Conclusions:
- Flp recombinase resolves chi structures via trans cleavage.
- A model for Flp-mediated recombination incorporating trans cleavage at both stages of strand exchange is proposed.