Investigating How the Restriction Endonuclease BcnI Flips on DNA

Abstract

Restriction endonucleases (REases) are enzymes that cleave DNA at or near a site specified by a particular nucleotide sequence. in most cases, REases are remarkably specific, and these enzymes are indispensable laboratory tools used for genetic modification, manipulation and analysis. Type II REases are the most heavily utilized, with the Type IIP subtype accounting for the vast majority of enzymes used in molecular biology. Most Type IIP REases are homodimers that cleave palindromic DNA sequences, but there are a few exceptions. Notably, BcnI, an REase identified in Bacillus centrosporus, is active as a monomer and cleaves the pseudopalindromic sequence 5’-CC/SGG-5’ (where S stands for C or G). The BcnI monomer is capable of cleaving both versions of its restriction site, and it is thought to complete both cuts in a single binding event. In order for this to happen, BcnI must rotate about an axis perpendicular to the DNA strand after making the first cut to be correctly oriented to cleave the second strand. This “flipping” cannot be achieved without changing the mode of contact with the DNA backbone. Flipping on the DNA is a critical part of the mechanism of action for HIV reverse transcriptase, allowing this multifunctional protein to adopt the appropriate orientation for the substrate it is interacting with. Two models for flipping have been put forward - “hopping”, in which electrostatic interactions between the protein and DNA backbone are disturbed and the protein enters a pseudobound state, and “tumbling”, in which most electrostatic interactions remain intact and the protein-DNA complex remains compact. We have studied the activity of BcnI under various conditions using a multiplexed single-molecule DNA cleavage assay. Our results suggest that, unlike HIV-RT, BcnI uses the tumbling model for flipping on the DNA.