Single molecule studies of the monomeric restriction endonuclease BcnI-DNA interactions

Orthodox restriction endonucleases (REases) are homodimeric enzymes that symmetrically recognize palindromic 4-8 bp DNA sequences and cleave both strands in a concerted manner. In contrast, BcnI is a monomeric REase, which possess a single active site and forms an asymmetric complex with its pseudopalindromic recognition site 5′-CC/SGG-3′ (where S is C or G, and ‘/’ marks the cleavage position) in one of the two possible orientations. Bulk kinetic studies revealed that the BcnI double-stranded DNA hydrolysis reaction can be divided into five steps: (i) rapid association of BcnI with the DNA substrate; (ii) rapid cleavage of the first DNA strand; (iii) a slow switch in BcnI orientation on the DNA without dissociation into bulk solution; (iv) rapid cleavage of the second DNA strand; and (v) the rate limiting product release. Experiments employing single molecule techniques revealed that apo-BcnI DNA-binding cleft is flexible, but DNA and divalent metal ions binding bring the catalytic and recognition subdomains into close proximity; BcnI preferentially binds specific DNA in the orientation that places the catalytic centre in the proximity of the strand with the central G nucleotide (5′-CCGGG-3); BcnI employs ‘facilitated diffusion’ for target site location. In summary, we have performed a detailed study of the double-stranded DNA hydrolysis reaction of the monomeric restriction enzyme BcnI employing bulk kinetics and various single molecule techniques