A method has been developed for determination of the absolute structure of DNA catenanes. The catenated DNA is partially denatured before being thickened with a coating of RecA protein and spread for electron microscopy. This treatment allows visualization of the orientation of each ring as well as identification of the overlying and underlying DNAs at crossing points. These determinations define the topology of a catenane, providing a powerful means for testing mechanisms of catenane-producing enzymes in DNA recombination and replication. The technique was used to show that the single interlock of the catenated products of site-specific recombination mediated by Tn3 resolvase is exclusively of negative sign. The unique topology of the products indicates that resolvase fixes the sum of the number of supercoils between recombination sites at synapsis and the number of such supercoils lost or gained during strand exchange. The data strongly suggest that there are in fact three negative supercoils between synapsed sites; one supercoil is dissolved in the cross-over mechanism, whereas the other two are metamorphosed into the unique catenane interlock.
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