RECOMBINATION BY RESOLVASE TO ANALYZE DNA COMMUNICATIONS BY THE SFII RESTRICTION-ENDONUCLEASE

The SfiI endonuclease differs from other type II restriction enzymes b y cleaving DNA concertedly at two copies of its recognition site, its optimal activity being with two sites on the same DNA molecule. The na ture of this communication event between distant DNA sites was analyse d on plasmids with recognition sites for SfiI interspersed with recomb ination sites for resolvase. These were converted by resolvase to cate nanes carrying one SfiI site on each ring. The catenanes were cleaved by SfiI almost as readily as a single ring with two sites, in contrast to the slow reactions on DNA rings with one SfiI site. Interactions b etween SfiI sites on the same DNA therefore cannot follow the DNA cont our and, instead, must stem from their physical proximity. In buffer l acking Mg2+, where SfiI is inactive while resolvase is active, the add ition of SfiI to a plasmid with target sites for both proteins blocked recombination by resolvase, due to the restriction enzyme bridging it s sites and thus isolating the sites for resolvase into separate loops . The extent of DNA looping by SfiI matched its extent of DNA cleavage in the presence of Mg2+.