A Novel Decatenation Assay for DNA Topoisomerases using a Singly-Linked Catenated Substrate

作者：Nidda F. Waraich, Shruti Jain, Sean D. Colloms, W. Marshall Stark, Nicolas P. Burton, Anthony Maxwell · 发表于：BioTechniques · 年份：2020 · DOI：10.2144/btn-2020-0059 · 被引用次数：7 · 研究领域：Cancer therapeutics and mechanisms、DNA and Nucleic Acid Chemistry、Gyrotron and Vacuum Electronics Research

Decatenation is a crucial in vivo reaction of DNA topoisomerases in DNA replication and is frequently used in in vitro drug 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 Tn3 resolvase 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.