Quantification of SAMHD1 Enzymatic Activity

In vitro SAMHD1 activity was measured as described [42 (link)]. Briefly, 1 μM his-tagged human SAMHD1 and 1.5 μM PPase from E.coli were incubated at room temperature in 20 μL reaction buffer (50 mM Tris, 150 mM NaCl, 1.25 mM MgCl₂, 0.5 mM TCEP, 0.05% Brij-35) and different concentrations of GTP, dGTP and CNDAC-TP in a clear 384-well plate (Corning, 3700, New York, USA). Reactions were stopped by addition of 20 μL EDTA (20 mM in water). Subsequently, 10 μL malachite green reagent (Sigma-Aldrich, MAK307, Missouri, USA) were added. Absorbance was recorded at 620 nm after incubating the samples for 60 min at room temperature. For normalization, background subtraction of controls containing the same substrate and PPase concentrations but no SAMHD1 was performed.