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))
|
Text
106529.pdf - Published Version 5MB |
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: | Stark, Professor Marshall and Olorunniji, Dr Femi and McQue, Mrs Arlene |
| 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 > School of Cardiovascular & Metabolic Health 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 |
| Copyright Holders: | Copyright © 2015 The Authors |
| First Published: | First published in Nucleic Acids Research |
| Publisher Policy: | Reproduced under a Creative Commons License |
University Staff: Request a correction | Enlighten Editors: Update this record
Funder and Project Information
Funder and Project Information