KINETICS OF THE INTERACTION BETWEEN DNA AND THE TYPE-IC RESTRICTION ENZYME ECOR124II

Optical waveguide mode spectroscopy was used to determine the binding constants characterizing the interaction of EcoR124II, a type IC restr iction modification enzyme from Salmonella typhimurium, with DNA. The DNA is immobilized on the surface of an optical waveguide, and the enz yme is introduced in bulk solution flowing over the DNA under controll ed hydrodynamic conditions. The binding kinetics of the protein to the DNA can be directly observed and the number of bound protein molecule s per base pair determined to a high accuracy. Dissociation of the pro tein was measured by switching flowing protein to protein-free buffer. Binding to two different kinds of DNA, with and without the specific sequence recognized by EcoR124II, was investigated. Protein binding an d dissociation (''nonspecific'' binding), quantified by association an d dissociation rate coefficients k(a) and k(d), were the same for both types, but the DNA carrying the recognition site showed an additional process, ''irreversible'' association (i.e. dissociation was not obse rved on the time scale of the experiments) of the protein, quantified by a rate coefficient k(s). Some inferences regarding the mechanism of base pair searching are made from the measured k(a), k(d), and k(s) v alues.