A high-throughput assay for DNA topoisomerases and other enzymes, based on DNA triplex formation.
We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of topoisomerase enzymes that is also capable of monitoring the activity of other enzymes that alter the topology of DNA. The assay utilises intermolecular triplex formation to resolve supercoiled and relaxed forms of DNA, the principle being the greater efficiency of a negatively supercoiled plasmid to form an intermolecular triplex with an immobilised oligonucleotide than the relaxed form. The assay provides a number of advantages over the standard gel-based methods, including greater speed of analysis, reduced sample handling, better quantitation and improved reliability and accuracy of output data. The assay is performed in microtitre plates and can be adapted to high-throughput screening of libraries of potential inhibitors of topoisomerases including bacterial DNA gyrase.
Burrell
Matthew R
MR
Department of Biological Chemistry, John Innes Centre, Norwich, UK.
Burton
Nicolas P
NP
Maxwell
Anthony
A
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D027081:DNA Gyrase / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D057166:High-Throughput Screening Assays; D006801:Humans; D020128:Inhibitory Concentration 50; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D059005:Topoisomerase II Inhibitors
D004278:DNA, Superhelical; D004791:Enzyme Inhibitors; D009838:Oligodeoxyribonucleotides; D059005:Topoisomerase II Inhibitors; C051802:triplex DNA; D004247:DNA; D004264:DNA Topoisomerases, Type I; D027081:DNA Gyrase