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Nucleic Acids Research Advance Access published online on November 10, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn885
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Synapsis and catalysis by activated Tn3 resolvase mutants

Femi J. Olorunniji1, Jiuya He2, Sandra V.C.T. Wenwieser3, Martin R. Boocock1 and W. Marshall Stark1,*

1Faculty of Biomedical & Life Sciences, University of Glasgow, Bower Building, Glasgow G12 8QQ, Scotland, 2MRC Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, England, UK and 3Villa La Vague, 4 Rue des Giroflées, Monaco

*To whom correspondence should be addressed. Tel: +44 141 330 5116; Fax: +44 141 330 4878; Email: M.Stark{at}bio.gla.ac.uk

Received August 26, 2008. Accepted October 20, 2008.

The serine recombinase Tn3 resolvase catalyses recombination between two 114 bp res sites, each of which contains binding sites for three resolvase dimers. We have analysed the in vitro properties of resolvase variants with ‘activating’ mutations, which can catalyse recombination at binding site I of res when the rest of res is absent. Site I x site I recombination promoted by these variants can be as fast as res x res recombination promoted by wild-type resolvase. Activated variants have reduced topological selectivity and no longer require the 2–3' interface between subunits that is essential for wild-type resolvase-mediated recombination. They also promote formation of a stable synapse comprising a resolvase tetramer and two copies of site I. Cleavage of the DNA strands by the activated mutants is slow relative to the rate of synapsis. Stable resolvase tetramers were not detected in the absence of DNA or bound to a single site I. Our results lead us to conclude that the synapse is assembled by sequential binding of resolvase monomers to site I followed by interaction of two site I-dimer complexes. We discuss the implications of our results for the mechanisms of synapsis and regulation in recombination by wild-type resolvase.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.


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