Topoisomerase IIα-Mediated Cleavage of Plasmid DNA

Plasmid DNA cleavage reactions were performed using the procedure of Fortune and Osheroff.⁶⁶ Reaction mixtures contained 75 nM of wild-type or mutant TOP2A and the 5 nM negatively supercoiled pBR322 DNA in 20 μL of 10 mM Tris–HCl, pH 7.9, 100 mM KCl, 1 mM EDTA, 5 mM MgCl₂, and 2.5% glycerol. Unless stated otherwise, assays were started by the addition of the enzyme, and DNA cleavage mixtures were incubated for 6 min at 37 °C. DNA cleavage reactions were carried out in the absence of the compound (1% DMSO solution as a control), or in the presence of the etoposide, DNA cleavage complexes were trapped by the addition of 2 μL of 5% SDS followed by 2 μL of 250 mM Na₂EDTA, pH 8.0. Proteinase K was added (2 μL of a 0.8 mg/mL solution), and reaction mixtures were incubated for 30 min at 37 °C to digest TOP2A. Samples were mixed with 2 μL of Nucleic Acid Sample Loading Buffer (Bio-Rad, Hercules, CA), heated for 2 min at 45 °C, and subjected to electrophoresis in 1% agarose gels in 40 mM Tris–acetate, pH 8.3, and 2 mM EDTA containing 0.5 μg/mL ethidium bromide. Double-stranded DNA cleavage was monitored by the conversion of the negatively supercoiled plasmid DNA to linear molecules. DNA bands were visualized by UV light and quantified using a Bio-Rad ChemiDoc MP Imaging System and Image Lab Software (Hercules, CA). The results were analyzed and plotted using GraphPad Prism 8 (La Jolla, CA). Relative DNA cleavage was calculated by setting DNA cleavage levels in the presence of DMSO to 1. Statistical analysis was performed using a one-way ANOVA followed by a Tukey’s Post-Test Analysis.