Abstract
A facilitated diffusion mechanism has been proposed to account for the kinetic efficiency with which restriction endonucleases are able to locate DNA recognition sites. Such a mechanism involves the initial formation 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 sliding along the DNA, hopping to nearby sites, or intersegment transfer over larger distances. Previous analyses of the manner in which restriction enzymes cleave DNA substrates did rule out the latter mechanism. To discriminate between protein sliding or scanning and protein hopping, 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 sites, whereas the centrally located site was not efficiently cleaved. These 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 restriction endonuclease recognizes cleavage sites with high fidelity, without skipping of potential sites.
Highlights
It is unlikely that site-specific DNA-binding proteins locate their target site by simple three-dimensional diffusion or trialand-error mechanism
Diffusion mechanisms along the DNA helix contour have been proposed to account for the kinetic efficiency with which this protein is able to locate its recognition and cleavage sites
Kinetic analyses of the lac repressor-operator system demonstrated that sliding was the dominant mechanism under the experimental conditions used [12], but different experimental approaches have led to the suggestion that intersegment transfer may be involved [13]
Summary
It is unlikely that site-specific DNA-binding proteins locate their target site by simple three-dimensional diffusion or trialand-error mechanism (reviewed in Ref. 1). Knowledge on the DNA search mechanism of restriction enzymes is based primarily on kinetic studies, combined with a detailed analysis of the cleavage pattern with circular and linear DNA templates containing one or more cleavage sites. These experiments demonstrated that the EcoRI enzyme has more leeway to find the cleavage sites if the sites are centrally located on a linear DNA rather than near an end [9, 10].
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