FIG 1.
SAMHD1 does not efficiently hydrolyze NRTI-TPs. SAMHD1 (5 μM) was incubated at 37°C with NRTI-TP or dNTP (500 μM), in the presence of dGTP (100 μM) and MgCl2 (10 mM). Reactions proceeded for 3, 6, or 20 h for NRTI-TPs and for 5, 15, or 30 min for dNTPs. Reactions were terminated by 10-fold dilution into 25 mM Tris (pH 8.0)-12.5% acetonitrile, and mixtures were analyzed by anion-exchange HPLC (DNAPac PA100 column). (A) Representative chromatograms for dATP hydrolysis. dG, deoxyribosylguanine. (B and C) Data from at least duplicate experiments for dNTP (B) or NRTI-TP (C) hydrolysis, plotted as percent hydrolysis over time (with GraphPad Prism 5). ddATP, dideoxyadenosine triphosphate; ddGTP, dideoxyguanosine triphosphate. (D) Chromatogram for dATP hydrolysis after 30 min of incubation with the SAMHD1 hydrolase active-site D207A mutant, dGTP, and MgCl2.