BPS2026 – A real-time assay for restriction endonuclease-mediated DNA cleavage

Restriction endonucleases (REases) are enzymes that cleave DNA at a location specified by a short recognition sequence. These enzymes serve a crucial role in bacterial defence against bacteriophage infections. Over 5,000 unique REases have been identified in a broad range of species, many of them commercially available. Although they are widely used in biomedical research, there are relatively little published data available on their reaction kinetics, and there are few straightforward experimental assays that provide access to detailed information about the rate of DNA cleavage by REases. To fill that gap, we have developed a fluorescence-based assay in which the cleavage of a fluorogenic DNA substrate can be tracked in real time using a fluorescence spectrophotometer. The data we collect can be analyzed by generating a Michaelis-Menten plot from the initial velocity of a set of reactions carried out with varying substrate concentrations, or we can use reaction progress kinetic analysis to generate a similar plot from a reaction that has run to completion. Our assay permits us to examine the impact of a wide range of experimental parameters, including salt concentration, pH, temperature, and the structure and sequence of the DNA substrate. Using this assay, we are investigating the kinetic mechanism of DNA cleavage by EcoRV, one of the first discovered and most heavily studied of the Type IIP REases. Like most enzymes of this type, EcoRV is a homodimer that cleaves within a palindromic DNA sequence (5'-GAT|ATC-3'), and requires Mg 2+ as a cofactor. Our preliminary studies of this enzyme suggest that the standard Michaelis-Menten model does not accurately describe its activity, and that substrate and/or product inhibition may play a role in the kinetics of DNA cleavage. Further study will facilitate the development and testing of alternative models for REase-mediated DNA cleavage.