Most type II restriction endonuclease are homodimers; they bind to the palindromic DNA sequence and cleaves the DNA within the recognition site. Several models have been proposed to explain those cleavage mechanisms. Unlike the other type II restriction endonuclease, BcnI is a monomer with a single active site and interacts with the five base pair long pseudo-palindromic DNA sequence making a double strand break within the target site while forming an asymmetric complex. Previous studies have shown that BcnI cleaves the DNA in a single binding event. Restriction endonuclease BcnI cleaves one DNA strand then flips and cleaves the second strand. To study this flipping transition on a single molecular level we used total internal reflection fluorescence (TIRF) microscopy. This technique allows us to investigate multiple cleavage events simultaneously in real time. The dwell time distributions analysis of those cleavage events provides information regarding the kinetic intermediate steps and rate of the cleavage mechanism. The fitting parameters of the dwell time distributions using the Gamma probability distributions yield the approximate number of intermediate steps and average rate of the steps. Testing BcnI activity under different environmental conditions reveals that the activity, and the number of significant rates limiting steps of the enzyme is higher for higher ionic strength. The presence of high screening increases the probability of flipping of the enzyme and hence the probability of cleavage. The results from our smTIRF experiments indicate that the protein flipping mechanism for BcnI is most likely described by the tumbling model.
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