STEREOSELECTIVITY OF DNA CATENANE FUSION BY RESOLVASE

COMMUNICATIONS between distant sites on DNA often depend on the way in which the sites are connected(1,2). For example, site-specific recomb ination catalysed by Tn3 resolvase is most efficient when the 114-base -pair res recombination sites are directly repeated in the same DNA mo lecule(3). In vitro a supercoiled plasmid substrate containing two dir ectly repeated res sites gives a resolution product in which the two r ecombinant circles are topologically linked as a simple (two-noded) ca tenane (Fig. 1a). Resolvase is highly selective in forming this produc t rather than unlinked circles or more complex catenanes. It does not catalyse recombination between sites on separate supercoiled molecules , or between inverted sites in the same supercoiled molecule(3-5). Tn3 resolution removes four negative supercoils from the substrate, an en ergetically favourable change which may drive the reaction(6): in rela xed or nicked circular substrates, resolution is incomplete and slower . Resolvase can catalyse fusion of the circles of a nicked or relaxed catenane, giving a single unknotted circular product(6,7). The fusion is the precise topological reversal of resolution, introducing four ne gative supercoils into a related catenane substrate(6), and should the refore not proceed if the catenane is already negatively supercoiled. Here we study recombination between res sites in non-supercoiled DNA c ircles linked into simple catenanes. We used (+2) and (-2) catenanes, which differ only in the direction in which one circle is threaded thr ough the other (Fig. 2a). Although stereoselectivity is a feature of e nzyme catalysis, it is not obvious how resolvase can distinguish betwe en these subtly different catenane diastereomers. A model for the inte rtwining of the res site DNA in the catalytically active complex(4,7) predicts that only the (-2) catenane will recombine, giving unknotted and 4-noded knot circular products. We have confirmed this prediction for the Tn3 and Tn21 resolvases.