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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, FJ, McPherson, AL, Pavlou, HJ, McIlwraith, MJ, Brazier, JA, Cosstick, R and Stark, WM (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 0305-1048

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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: Article
Uncontrolled Keywords: 05 Environmental Sciences, 06 Biological Sciences, 08 Information and Computing Sciences
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH426 Genetics
R Medicine > RM Therapeutics. Pharmacology
Divisions: Pharmacy & Biomolecular Sciences
Publisher: Oxford University Press (OUP)
Related URLs:
Date Deposited: 06 Mar 2020 11:39
Last Modified: 06 Mar 2020 11:45
DOI or Identification number: 10.1093/nar/gkv521
URI: http://researchonline.ljmu.ac.uk/id/eprint/12420

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