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. ORCID: https://orcid.org/0000-0002-4952-584X, 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 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
URI	https://reading-clone.eprints-hosting.org/id/eprint/40713
Identification Number/DOI	10.1093/nar/gkv521
Refereed	Yes
Divisions	Life Sciences > School of Chemistry, Food and Pharmacy > School of Pharmacy > Medicinal Chemistry Research Group
Publisher	Oxford University Press
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