A rapid method for the measurement of the unwinding angle of intercalating agents and the superhelix density of circular DNAs.
By incubating covalently-closed circular DNA in the presence of calf thymus topoisomerase and unwinding ligands (intercalating drugs and proteins) DNAs of different superhelix density can be produced. These changes in superhelix density can be monitored by an ethidium fluorescence assay, since the level of ethidium binding varies with the superhelix density of a DNA. Thus the equivalence point, in a titration between an unwinding ligand and a supercoiled DNA, can be found. This forms the basis for an extremely rapid method for measuring unwinding angles and superhelix densities. Results are presented which agree well with those reported by previous authors using different techniques. The present method compares very favourably with others when evaluated in terms of rapidity, cost of materials, cost of equipment, accuracy and also applicability.
D000166:Acridines / Q000494:pharmacology; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004279:DNA, Viral; D004996:Ethidium / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D008024:Ligands; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D010957:Plasmids; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013096:Spermine / Q000031:analogs & derivatives
D000166:Acridines; D004270:DNA, Circular; D004278:DNA, Superhelical; D004279:DNA, Viral; D006657:Histones; D008024:Ligands; D013096:Spermine; D004264:DNA Topoisomerases, Type I; D004996:Ethidium