Assays for the Preferential Binding of Human Topoisomerase I to Supercoiled DNA

To assay the preferential binding of eukaryotic type IB topoisomerases to supercoiled DNA, two methods are described that make use of a catalytically inactive mutant form of the enzyme. In the gel shift assay, the preference for binding to supercoiled plasmid DNA is detected in the presence of linear and nicked forms of the same DNA by a reduction in the mobility of the supercoiled plasmid during electrophoresis in agarose. The more quantitative filter binding assay compares the ability of nicked and supercoiled forms of the circular DNA to compete for the binding of a ³ H-labeled nicked DNA to the topoisomerase where the enzyme–DNA complexes are quantitated by the retention of the labeled DNA on a nitrocellulose membrane.