title: Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages.
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.
authors.affiliation: Institute of Molecular, Cell and Systems Biology, University of Glasgow, Bower Building, Glasgow G12 8QQ, UK.
authors.forename: Femi J
authors.initials: FJ
authors.lastname: Olorunniji
authors.affiliation: Institute of Molecular, Cell and Systems Biology, University of Glasgow, Bower Building, Glasgow G12 8QQ, UK.
authors.forename: Arlene L
authors.initials: AL
authors.lastname: McPherson
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Building, Parks Road, Oxford OX1 3PT, UK.
authors.forename: Hania J
authors.initials: HJ
authors.lastname: Pavlou
authors.affiliation: Beatson Institute for Cancer Research, Switchback Road, Bearsden, Glasgow G61 1BD, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McIlwraith
authors.affiliation: Reading School of Pharmacy, University of Reading, Whiteknights, Reading, Berkshire, RG6 6AD, UK.
authors.forename: John A
authors.initials: JA
authors.lastname: Brazier
authors.affiliation: Department of Chemistry, University of Liverpool, Crown Street, Liverpool, L69 7ZD, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Cosstick
authors.affiliation: Institute of Molecular, Cell and Systems Biology, University of Glasgow, Bower Building, Glasgow G12 8QQ, UK Marshall.Stark@glasgow.ac.uk.
authors.forename: W Marshall
authors.initials: WM
authors.lastname: Stark
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D053837:DNA Cleavage; D007700:Kinetics; D011995:Recombination, Genetic; D045564:Transposon Resolvases / Q000378:metabolism*
chemical_list: D004247:DNA; C473491:Tn3 resolvase; D045564:Transposon Resolvases