Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages.
Bottom Line: 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.
Affiliation: Institute of Molecular, Cell and Systems Biology, University of Glasgow, Bower Building, Glasgow G12 8QQ, UK.Show MeSH
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Mentions: Binding/synapsis assays were based on previously described methods (7). Briefly, substrates containing site I (the Tn3 crossover site) were assembled by annealing equimolar amounts of 50 nt (or 70 nt) top and bottom strand oligonucleotides, one of which was first 32P-labelled at the 5′ end. The nicked-site substrates used in single-strand cleavage assays were assembled by annealing a 50 nt or a 70 nt site I top strand oligonucleotide with equimolar amounts of two shorter bottom strand oligonucleotides, such that a single-strand break (nick) is present near or at the scissile position on the bottom strand, and the assembled double-stranded substrate has blunt ends. The appropriate oligonucleotide was 32P-labelled at its 5′ end with T4 polynucleotide kinase. The top strand of the 70 bp site is 5′-ggcaagcttgcgtgactcaacTGTCTGATAATTTATAAATTATCAGACAtagtgggatggtctgcagcgg-3′; the central AT dinucleotide of site I is in bold, and the nucleotides flanking site I are in lower case. The 50 bp site is 10 bp shorter at each end (lacking the nucleotides in italics). The positions of bottom strand nicks are described in the main text. The 5′ end at the nick is phosphorylated unless stated otherwise (see Figure 2A).
Affiliation: Institute of Molecular, Cell and Systems Biology, University of Glasgow, Bower Building, Glasgow G12 8QQ, UK.