Nucleic Acids Research Advance Access published online on September 29, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp797
Nucleic Acid Enzymes |
The catalytic residues of Tn3 resolvase
Faculty of Biomedical and Life Sciences, University of Glasgow, Bower Building, Glasgow G12 8QQ, Scotland, UK
*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 5, 2009. Revised September 8, 2009. Accepted September 9, 2009.
To characterize the residues that participate in the catalysis of DNA cleavage and rejoining by the site-specific recombinase Tn3 resolvase, we mutated conserved polar or charged residues in the catalytic domain of an activated resolvase variant. We analysed the effects of mutations at 14 residues on proficiency in binding to the recombination site (‘site I’), formation of a synaptic complex between two site Is, DNA cleavage and recombination. Mutations of Y6, R8, S10, D36, R68 and R71 resulted in greatly reduced cleavage and recombination activity, suggesting crucial roles of these six residues in catalysis, whereas mutations of the other residues had less dramatic effects. No mutations strongly inhibited binding of resolvase to site I, but several caused conspicuous changes in the yield or stability of the synapse of two site Is observed by non-denaturing gel electrophoresis. The involvement of some residues in both synapsis and catalysis suggests that they contribute to a regulatory mechanism, in which engagement of catalytic residues with the substrate is coupled to correct assembly of the synapse.