B. Berkhout et J. Vanwamel, ACCURATE SCANNING OF THE BSSHII ENDONUCLEASE IN SEARCH FOR ITS DNA CLEAVAGE SITE, The Journal of biological chemistry, 271(4), 1996, pp. 1837-1840
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.