Binding of T4 endonuclease V to deoxyribonucleic acid irradiated with ultraviolet light

Abstract

Endonuclease V of bacteriophage T4 binds to UV-irradiated DNA but not to unirradiated DNA. An assay was developed to detect this binding, based on the retention of enzyme-DNA complexes on nitrocellulose filters. The amount of complex retained, ascertained by using radioactive DNA, is a measure of T4 endonuclease V activity. The assay is simple, rapid and specific, which makes it useful for detecting T4 endonuclease V activity both in crude lysates and in purified preparations. It was used to monitor enzyme activity during purification and to study binding of the enzyme to DNA under conditions that minimize the ability of the enzyme to nick DNA. The data showed that T4 endonuclease V binds to UV-irradiated DNA but not to DNA that was previously incised by the endonuclease, equilibrium between the free and complexed form of the enzyme is attained under the reaction conditions, dissociation of enzyme-DNA complexes is retarded by sodium cyanide and retention of enzyme-DNA complexes on nitrocellulose filters is enhanced by high concentrations of saline-citrate.