Nicked-site substrates for a serine recombinase reveal enzyme–DNA communications and an essential tethering role of covalent enzyme–DNA linkages

Olorunniji, F. J., McPherson, A. L., Pavlou, H. J., McIlwraith, M. J., Brazier, J. A., Cosstick, R. and Stark, W. M. (2015) Nicked-site substrates for a serine recombinase reveal enzyme–DNA communications and an essential tethering role of covalent enzyme–DNA linkages. Nucleic Acids Research, (doi: 10.1093/nar/gkv521) (PMID:25990737) (PMCID:PMC4499144))

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Abstract

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase–DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:McQue, Mrs Arlene and Stark, Professor Marshall and Olorunniji, Dr Femi
Authors: Olorunniji, F. J., McPherson, A. L., Pavlou, H. J., McIlwraith, M. J., Brazier, J. A., Cosstick, R., and Stark, W. M.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Nucleic Acids Research
Publisher:Oxford University Press
ISSN:0305-1048
ISSN (Online):1362-4962
Copyright Holders:Copyright © 2015 The Authors
First Published:First published in Nucleic Acids Research
Publisher Policy:Reproduced under a Creative Commons License

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