Binding of T4 endonuclease V to deoxyribonucleic acid irradiated with ultraviolet light.
Endonuclease V of bacteriophage T4 binds to UV-irradiated deoxyribonucleic acid (DNA) but not to unirradiated DNA. We have developed an assay 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. We have used it 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. From our data we conclude that (1) T4 endonuclease V binds to UV-irradiated DNA but not to DNA that has been previously incised by the endonuclease, (2) equilibrium between the free and complexed form of the enzyme is attained under our reaction conditions, (3) dissociation of enzyme--DNA complexes is retarded by sodium cyanide, and (4) retention of enzyme--DNA complexes on nitrocellulose filters is enhanced by high concentrations of saline--citrate.
D003486:Cyanides / Q000494:pharmacology; D004274:DNA, Recombinant / Q000528:radiation effects*; D004278:DNA, Superhelical / Q000528:radiation effects*; D003851:Deoxyribonucleases / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D004706:Endodeoxyribonucleases; D004720:Endonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011485:Protein Binding; D013604:T-Phages / Q000201:enzymology*; D014466:Ultraviolet Rays
D003486:Cyanides; D004274:DNA, Recombinant; D004278:DNA, Superhelical; D003851:Deoxyribonucleases; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C026044:deoxyribonuclease V