Loading stop buffer

Gyrase activity (TopoGEN Inc.) was measured by supercoiling of relaxed pHOT-1 DNA, following the manufacturer’s protocol. Briefly, 1 μl of gyrase test solution, 4 μl of 5× gyrase “assay” buffer (TopoGEN Inc.), and 250 ng of relaxed pHOT-1 DNA (TopoGEN Inc.) were combined in molecular biology–grade dH₂O at a final assay volume of 20 μl and incubated at 37°C for 60 min. After incubation, 4 μl of 5× loading “stop” buffer (TopoGEN Inc.) was added and mixed by vortex. The reaction products were separated on a 1% tris-borate EDTA (TBE)/agarose gel [no ethidium bromide (EtBr)] and stained with EtBr, and the bands were imaged. Enzyme activity was quantified by measuring the intensity of the supercoiled pHOT-1 bands by densitometry using ImageJ (fig. S12).

Top1 activity (TopoGEN Inc.) was measured by relaxation of supercoiled pHOT-1 DNA, following the manufacturer’s protocol. Briefly, 1 μl of test solution, 2 μl of 10× Top1 “reaction” buffer (TopoGEN Inc.), and 125 ng of supercoiled pHOT-1 DNA (TopoGEN Inc.) were combined in molecular biology–grade dH₂O at a final assay volume of 20 μl and incubated at 37°C for 30 min. After incubation, 4 μl of 5× loading stop buffer (TopoGEN Inc.) was added and mixed by vortex. The reaction products were separated on a 1% TBE/agarose gel (no EtBr) and stained with EtBr, and the bands were imaged. Enzyme activity was quantified by measuring the intensity of the relaxed pHOT-1 bands by densitometry using ImageJ (fig. S13).