Kinetics of the interaction between DNA and the type IC restriction enzyme EcoR124II.

Abstract

Optical waveguide mode spectroscopy was used to determine the binding constants characterizing the interaction of EcoR124II, a type IC restriction modification enzyme from Salmonella typhimurium, with DNA. The DNA is immobilized on the surface of an optical waveguide, and the enzyme is introduced in bulk solution flowing over the DNA under controlled hydrodynamic conditions. The binding kinetics of the protein to the DNA can be directly observed and the number of bound protein molecules per base pair determined to a high accuracy. Dissociation of the protein 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 and dissociation ("nonspecific" binding), quantified by association and dissociation rate coefficients ka and kd, were the same for both types, but the DNA carrying the recognition site showed an additional process, "irreversible" association (i.e. dissociation was not observed on the time scale of the experiments) of the protein, quantified by a rate coefficient ks. Some inferences regarding the mechanism of base pair searching are made from the measured ka, kd, and ks values.