Fluorometric assays for DNA topoisomerases and topoisomerase-targeted drugs: quantitation of catalytic activity and DNA cleavage.
We have introduced the novel application of a simple ethidium fluorescence assay, using covalently closed circular DNA, for the study of topoisomerase-targeted drugs. With the specificity of camptothecin for eukaryotic topoisomerases I and of VM26 for eukaryotic topoisomerases II, the two classes of enzymes can be assayed independently in crude extracts and during purification. These assays are fast, sensitive, and quantitative, have a large sample capacity, and eliminate the need for radioactive materials, filters, and agarose gels. We have demonstrated the use of this fluorescence assay to measure the inhibition of the relaxation and supercoiling activities of purified mammalian topoisomerases I and II and bacterial gyrase by nonintercalating drugs. Similarly, the production of drug-induced topoisomerase-mediated cleavable complexes was readily quantitated with both nonintercalating and intercalating drugs. When inhibition and cleavage with VM-26 were measured concurrently as a function of topoisomerase II concentration, a clear inverse relationship between topoisomerase II inhibition and cleavable complex production was observed. When the physiologically relevant salt K+L-glutamate- was used, quantitative relaxation by topoisomerase II was observed up to twice the salt concentration obtained with KCl. The enantiomer K+D-glutamate- gave exactly the same results, indicating that the enhancing role of glutamate- is non-stereospecific.
Andrea
J E
JE
SynPhar Laboratories Inc., Edmonton, Alberta, Canada.
Adachi
K
K
Morgan
A R
AR
D000677:Amsacrine / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002166:Camptothecin / Q000494:pharmacology; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004996:Ethidium / Q000378:metabolism*; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009675:Novobiocin / Q000494:pharmacology; D013713:Teniposide / Q000494:pharmacology; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors
D000970:Antineoplastic Agents; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D000677:Amsacrine; D009675:Novobiocin; D013713:Teniposide; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D004996:Ethidium; D002166:Camptothecin