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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., 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, 43 (12). pp. 6134-6143. ISSN 1362-4962

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To link to this item DOI: 10.1093/nar/gkv521

Abstract/Summary

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:Article
Refereed:Yes
Divisions:Faculty of Life Sciences > School of Chemistry, Food and Pharmacy > School of Pharmacy > Medicinal Chemistry Research Group
ID Code:40713
Publisher:Oxford University Press

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