Malachite green reagent

RecA ATPase activity was measured using coupled spectrophotometric enzyme assay. The reaction was performed in 100 μL assay buffer (6 mmol/l MgCl₂, 20 mmol/l KCl and 100 mmol/l Tris–HCl, pH 7.4) 1 mmol/l ATP and different concentrations of RecA (0–3.0 μM), RecA_K70A (0–3.0 μM), and RecA_K70R (0–3.0 μM), or vehicle (DMSO) and incubated at 37°C for 3 h. At the end of the incubation, the ATPase activity of RecA, RecA_K70A, and RecA_K70R were assessed by malachite green reagent (Sigma-Aldrich) (0.0812% wt/vol malachite green, 2.32% wt/vol polyvinyl alcohol and 5.72% wt/vol ammonium molybdate in 6 mol/L HCl, and argon water mixed in a ratio of 2:1:1:2, vol/vol/vol/v). Reactions were analyzed in triplicate at an absorbance of 620 nm. The kinetic analysis of the RecA, RecA_K70A, and RecA_K70R ATPase activity was carried out using a nonlinear regression fit of the experimental points to the Michaelis–Menten equation. Commercially available Salmon Sperm ssDNA was obtained from Sigma-Aldrich.

DNaseI, Glucose-1-phosphate, dTTP, dTDP-D-glucose, malachite green reagent, NADPH, phenylmethanesulfonyl fluoride (PMSF), pyrophosphatase, UTP and UDP-glucose were obtained from Sigma-Aldrich (St. Louis MO), dTDP-4-keto-6-deoxy-glucose came from Carbosynth (Berkshire, UK), novobiocin and ampicillin were obtained from Duchefa Biochemie (Haarlem, The Netherlands), while restriction endonucleases were purchased from Promega (Madison, WI).

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.