The catalytic residues of Tn3 resolvase

Olorunniji, F.J. and Stark, W.M. (2009) The catalytic residues of Tn3 resolvase. Nucleic Acids Research, 37(22), pp. 7590-7602. (doi: 10.1093/nar/gkp797) (PMID:19789272) (PMCID:PMC2794168)

Abstract

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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Stark, Professor Marshall and Olorunniji, Dr Femi
• Authors: Olorunniji, F.J., and Stark, W.M.
• Subjects: Q Science > QH Natural history > QH301 Biology; Q Science > QH Natural history > QH426 Genetics
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: Nucleic Acids Research
• Publisher: Oxford University Press
• ISSN: 0305-1048
• ISSN (Online): 1362-4962
• Published Online: 29 September 2009
• Copyright Holders: Copyright © 2009 The Authors
• First Published: First published in Nucleic Acids Research 37(22):7590-7602
• Publisher Policy: Reproduced in accordance with the copyright policy of the publisher

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
44881	1	The mechanism of DNA strand exchange by serine recombinases	William Stark	Biotechnology and Biological Sciences Research Council (BBSRC)	BB/E022200/1	Institute of Molecular Cell and Systems Biology