SAMHD1 dNTP Triphosphohydrolase Activity Assay

To measure dNTP triphosphohydrolase activity of SAMHD1, 1.6 μM recombinant SAMHD1-GST (SAMHD1) was incubated with different 500 μM nucleoside-5'-triphosphate substrates in the presence of 500 μM dCMP, 500 μM GTP and reaction buffer (50 mM Tris-HCl [pH 8], 100 mM KCl, 5 mM MgCl₂, and 0.1% Triton X-100). Reactions were incubated for 2 h at 37°C and terminated by incubation for 10 min at 75°C. Reactions were separated and quantified by anion exchange HPLC method [32 (link)]. Separation was done using two DNAPac PA100 columns equilibrated with running buffer (25 mM Tris–HCl [pH 8] and 0.5% acetonitrile) for 10 min, 30 μL sample was injected and eluted with a linear gradient of 240 mM NH₄Cl for 12 min, run at an isocratic gradient with 240 mM NH₄Cl for 5 min, and column was again equilibrated with running buffer (Beckman Coulter System Gold 126 Solvent Module). Absorbance was measured with a Beckman Coulter System Gold 166 Detector at 254 nm. The amounts of deoxycytidine-5'-monophosphate (dCMP), dGTP and (deoxy)nucleoside-5'-TP analogs were determined by integrating the peak area using 32 Karat 8.0 Software. Data was normalized to dCMP peak area for each sample, used as a sample loading control. Determining changes for different (deoxy)nucleoside-5'-triphosphates of interest was calculated by setting sample without SAMHD1 peak area to 100%.