A novel decatenation assay for DNA topoisomerases using a singly-linked catenated substrate

METHOD SUMMARY We engineered a plasmid such that reaction with Tn3resolvase generates a singly-linked catenane product. Restriction enzyme digestion and caesium chloride density gradient centrifugation enable the purification of the catenane product substantially free from starting plasmid. This product may be utilized in topoisomerase enzyme assays. Decatenation is a crucialin vivoreaction of DNA topoisomerases in DNA replication and is frequently used inin vitrodrug screening. Usually this reaction is monitored using kinetoplast DNA as a substrate, although this assay has several limitations. Here we have engineered a substrate for Tn3resolvase that generates a singly-linked catenane that can readily be purified from the DNA substrate after restriction enzyme digestion and centrifugation. We show that this catenated substrate can be used with high sensitivity in topoisomerase assays and drug-inhibition assays.