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.
Highlights
In site-specific recombination, the DNA double helix is cleaved by breaking both strands at two precisely defined positions, and the broken ends are rejoined to new partners
We made nicked-site I substrates for activated resolvase (Figure 2A) by annealing a full-length top strand oligonucleotide with two shorter strands, such that the ‘bottom strand’ has a nick at or close to the position of the scissile phosphodiester
Resolvase might cleave the bottom strand if the nick is close to but not at the scissile phosphodiester (Figure 2B)
Summary
In site-specific recombination, the DNA double helix is cleaved by breaking both strands at two precisely defined positions, and the broken ends are rejoined to new partners. The process is catalysed by a recombinase enzyme. Serine recombinases [2] exchange DNA strands between two ‘crossover sites’ (typically ∼30 bp long). Each crossover site binds a recombinase dimer, and two crossover sites are brought together by dimer–dimer interactions to form a synaptic tetramer. Double-strand cleavage at specific phosphodiester bonds close to the centre of each site results in an intermediate with recombinase subunits attached to each 5 DNA end via a phosphodiester with the active site serine. Following a rotation-like exchange of the positions of the cleaved half-sites, the strands are rejoined to form recombinant sites [1,3] (Figure 1)
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