SAMHD1 allosteric activation by dGTP and GTP at physiological concentrations.
A, R-SAMHD1 (1 μm) was incubated with 1 mm dATP (substrate) and physiological concentrations of either dGTP or GTP: 70 nm dGTP or 0.32 mm GTP for macrophage reactions (*, p = 0.0143) and 1.5 μm dGTP or 1.75 mm GTP for T cell reactions (*, p = 0.0451). SAMHD1 was also incubated with physiological concentrations of both nucleotides (dGTP + GTP). B, R-SAMHD1 was incubated with 1 mm dATP and increasing concentrations of dGTP or GTP. C, R-SAMHD1 was incubated with 1 mm dATP and 500 μm dGTP or GTP for the indicated time points. D, R-SAMHD1 was incubated with 250 μm dGTP or GTP and increasing concentrations of dATP. E–H, R-SAMHD1 was incubated with 250 μm dGTP (E) or GTP (D), 250 μm dTTP, and 0.125 μCi/μl [α-32P]dTTP in the presence or absence (No) of 33-mer ssDNA or ssRNA (1 μm). Reaction products were separated by TLC to detect [α-32P]dTTP hydrolysis (PPP), and densitometry was used to quantify product formation in the presence of dGTP (G) or GTP (H). NE, no-enzyme control; CIP, calf intestinal phosphatase control; P, monophosphates; NA, nucleic acids.