ATPase assays

ATPase measurements were conducted using the established enzyme-coupled PK/LDH ATPase assay (41). In this method, ATP regeneration is coupled to the oxidation of NADH. As NADH is converted to NAD, the corresponding decrease in absorption at 340 nM correlates 1:1 to the corresponding hydrolysis of ATP. ATPase rates were calculated using an NADH standard curve equating 340 nm absorption with NADH concentration. Measurements were conducted using a CLARIOStar Omega plate reader. DNA-stimulated ATPase activity was measured at 37°C using 100 nM gyrase tetramer in the presence of saturating DNA (100 ng/ul) over a range of 0–2 mM ATP. The 75 ul reactions volumes contained either 50 or 300 mM potassium glutamate, 50 mM Tris-pH 7.9, 5 mM MgCl₂, 0.1 mg/ml BSA, 5 mM βME, 2 mM phosphoenolpyruvate, 0.2 mM freshly-made NADH and 1.5 U/ml pyruvate kinase/lactic dehydrogenase mix (Sigma). Three sample replicates were measured for each condition. ATPase data were fit to a pseudo Michaelis–Menten model (V = k_cat*[ATP]/(K_M + [ATP])) in Mathematica. Error bars in the data (Supplemental Figure S6A and B) are reported as standard deviations and errors for the Michaelis–Menten parameters as the deviation of those parameters from the fitted data.