Stereoselectivity of DNA catenane fusion by resolvase.

Abstract

Communications between distant sites on DNA often depend on the way in which the sites are connected. For example, site-specific recombination catalysed by Tn3 resolvase is most efficient when the 114-base-pair res recombination sites are directly repeated in the same DNA molecule. In vitro a supercoiled plasmid substrate containing two directly repeated res sites gives a resolution product in which the two recombinant circles are topologically linked as a simple (two-noded) catenane (Fig. 1a). Resolvase is highly selective in forming this product 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. Tn3 resolution removes four negative supercoils from the substrate, an energetically favourable change which may drive the reaction: in relaxed 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. The fusion is the precise topological reversal of resolution, introducing four negative supercoils into a relaxed catenane substrate, and should therefore not proceed if the catenane is already negatively supercoiled. Here we study recombination between res sites in non-supercoiled DNA circles linked into simple catenanes. We used (+2) and (-2) catenanes, which differ only in the direction in which one circle is threaded through the other (Fig. 2a). Although stereoselectivity is a feature of enzyme catalysis, it is not obvious how resolvase can distinguish between these subtly different catenane diastereomers. A model for the intertwining of the res site DNA in the catalytically active complex 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.