resolvase by a general method (site-specific recombination/partial denaturation mapping/catenanes,

Le résumé fourni par la source

A method has been developed for determina- tion of the absolute structure of DNA catenanes. The catenated DNA is partially denatured before being thickened with a coat- ing 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 recombina- tion 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 recombina- tion 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 cat- enane interlock. FIG. 1. The two types of singly interlinked catenanes. Molecules are shown in plane projection with the orientation at each node (crossing of 2 DNA duplexes) represented by an arrow. The sign of each node is assigned by convention: If the overlying arrow can be aligned with the underlying one by a clockwise rotation of less than 180?, the node is assigned a value of -1. If a counterclockwise rota- tion can produce alignment, the node is given a value of +1. Note that the two catenane types are identical except for the relative ori- entation of the rings.

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.