dUTPase activity assay

Malachite Green phosphate assay was used to measure the dUTPase activity as previously described¹². Briefly, Pi released was quantified in 200 μl assay volume of reaction buffer containing 75 mM HEPES pH 7.5, 250 mM NaCl, 5 mM MgCl₂ and 0.01 U of inorganic pyrophosphatase (Thermo scientific), along with 0.075 μg of the corresponding Dut. The reactions were started by addition of dUTP at different concentrations (12.5, 25, 50, 100, 200 and 400 μM final concentration) and stopped samples were taken at 0, 2, 4, 6, 8 and 10 min by adding 50 μl of malachite green development solution to stop the reaction. After 10 min incubation at room temperature, the Pi production was calculated based on the absorbance at 630 nm and against a previously determined standard curve for Pi. Reactions showed linearity over the time-course of the reaction and the initial velocity was calculated following this procedure using GraphPad Prism software.