ACCURATE SCANNING OF THE BSSHII ENDONUCLEASE IN SEARCH FOR ITS DNA CLEAVAGE SITE

A facilitated diffusion mechanism has been proposed to account for the kinetic efficiency with which restriction endonucleases are able to l ocate DNA recognition sites, Such a mechanism involves the initial for mation of a nonspecific complex upon collision of the protein with the DNA, with the subsequent diffusion of the protein along the DNA helix until either a recognition site is located or the protein dissociates into solution. Protein translocation may be facilitated by either sli ding along the DNA, hopping to nearby sites, or intersegment transfer over larger distances, Previous analyses of the manner in which restri ction enzymes cleave DNA substrates did rule out the latter mechanism, To discriminate between protein sliding or scanning and protein hoppi ng, we designed a unique DNA template with three overlapping, mutually exclusive recognition sites for the BssHII endonuclease. Analysis of the cleavage pattern demonstrated efficient usage of both external sit es, whereas the centrally located site was not efficiently cleaved, Th ese results confirm that linear diffusion of the BssHII enzyme occurs by scanning along the DNA, Furthermore, the scanning enzyme was found to stop and cleave at the first site encountered, Thus, a sliding rest riction endonuclease recognizes cleavage sites with high fidelity, wit hout skipping of potential sites.