REPERCUSSIONS OF DNA TRACKING BY THE TYPE IC RESTRICTION-ENDONUCLEASEECOR124I ON LINEAR, CIRCULAR AND CATENATED SUBSTRATES

Type I restriction endonucleases such as EcoR124I cleave DNA at undefi ned loci, distant from their recognition sequences, by a mechanism tha t involves the enzyme tracking along the DNA between recognition and c leavage sites. This mechanism was examined on plasmids that carried re cognition sites for EcoR124I and recombination sites for resolvase, th e latter to create DNA catenanes. Supercoiled substrates with either o ne or two restriction sites were linearized by EcoR124I at similar rat es, although the two-site molecule underwent further cleavage more rea dily than the one-site DNA. The catenane from the plasmid with one Eco R124I site, carrying the site on the smaller of the two rings, was cle aved by EcoR124I exclusively in the small ring, and this underwent mul tiple cleavage akin to the two-site plasmid. Linear substrates derived from the plasmids were cleaved by EcoR124I at very slow rates. The co mmunication between recognition and cleavage sites therefore cannot st em from random looping. Instead, it must follow the DNA contour betwee n the sites. On a circular DNA, the translocation of non-specific DNA past the specifically bound protein should increase negative supercoil ing in one domain and decrease it in the other. The ensuing topologica l barrier may be the trigger for DNA cleavage.