Using Single-Turnover Kinetics with Osmotic Stress To Characterize the EcoRV Cleavage Reaction

Abstract

Type II restriction endonucleases require metal ions to specifically cleave DNA at canonical sites. Despite the wealth of structural and biochemical information, the number of Mg2+ ions used for cleavage by EcoRV, in particular, at physiological divalent ion concentrations is still not established. In this work we employ a single-turnover technique that uses osmotic stress in order to probe reaction kinetics between an initial specific EcoRV-DNA complex formed in the absence of Mg2+ and the final cleavage step. With osmotic stress, complex dissociation before cleavage is minimized and the reaction rates are slowed to a convenient timescale of minutes to hours. We find that cleavage occurs by a two-step mechanism that can be characterized by two rate constants. The dependence of these rate constants on Mg2+ concentration and osmotic pressure gives the number of Mg2+ ions and water molecules coupled to each kinetic step of the EcoRV cleavage reaction. Each kinetic step is coupled to the binding 1.5 – 2.5 Mg2+ ions, the uptake of ~30 water molecules, and the cleavage of a DNA single strand. We suggest that each kinetic step reflects an independent, rate limiting conformational change of each monomer of the dimeric enzyme that allows Mg2+ ion binding. This modified single turnover protocol has general applicability for metalloenzymes.