Determination of the stereostructure of the product of Tn3 resolvase by a general method.

Publisher Website
Google Scholar
Abstract
A method was 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 EM. This treatment allows visualization of the orientation of each ring and identification of the overlying DNA and underlying DNA 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 negative. 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 suggest that there are in fact 3 negative supercoils between synapsed sites; 1 supercoil is dissolved in the cross-over mechanism, whereas the other 2 are metamorphosed into the unique catenane interlock.