Topoisomerase 2 assay kit

TOPO-II activity was assayed in vitro through the Topoisomerase II assay kit (no. TG1001, TopoGEN Inc., Port Orange, FL, USA), following the instructions of the manufacturer. Nuclear extracts containing TOPO-II activity were obtained from cells and the ability to decatenate kDNA was analysed in the presence of DMSO and of the tested complexes, as previously shown [53 (link)]. Briefly, decatenation assay was performed with 50 ng kDNA in a 10 mL reaction mixture containing 50 mM Tris–HCl (pH 8.0); 150 mM NaCl; 10 mM MgCl₂; 0.5 mM dithiothreitol; 2 mM ATP; 30 mg/mL BSA with DMSO; or 5, 15, 30 µM of compounds and 0.5 mg of cell nuclear extract. Reactions were incubated at 37 °C for 30 min and stopped by adding 5 mL of stop buffer (5% Sarkosyl 0.125% bromophenol blue and 25% glycerol). Samples were loaded directly onto a 1% agarose gel containing ethidium bromide (0.5 mg/mL). TOPO-II activity was measured by the appearance of decatenated minicircular products and determined as the percentage of DMSO by ImageJ software (Image J, USA National Institutes of Health, Bethesda, MD, USA).

Topoisomerase II assay kit (TG1001-2, TopoGEN) was used to measure decatenation activity of TOP2A on relaxed supercoiled DNA template in generated stable clones. Briefly, extracts were prepared from TOP2A overexpressing and control cells, combined with DNA and buffer and incubated at 37C for 30 minutes. Stop buffer was added to the reaction and an agarose gel was run on the reactions along with appropriate decatenated and linear DNA markers.

pRep41 plasmids expressing FLAG-tagged Top2-WT or -2A proteins under the inducible nmt promoter were constructed and transformed in the S. pombe WT strain. The resulting strains were cultured at 26 °C for 24 h in the absence of thiamine, and then Top2-FLAG proteins were immunoprecipitated using anti-FLAG M2–agarose beads (Sigma) as described previously (10 (link)). Top2 decatenation assays were performed using the Topoisomerase II Assay Kit (TopoGEN, Inc., TG1001-1). Kinetoplast DNA (153 ng) was incubated with the Top2 IP fraction (∼5 ng) at 37 °C for 1–30 min in reaction buffer (50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 10 mm MgCl₂, 0.5 mm DTT, 30 μg/ml BSA, and 2 mm ATP). The reaction was terminated with stop buffer (1% sarkosyl, 0.025% bromophenol blue, and 5% glycerol). Samples were run on a 1% agarose gel in 1× Tris borate-EDTA at 135 V for 25 min and stained with ethidium bromide. Intensity of DNA bands was measured using ImageJ (National Institutes of Health).