Figure 1: Tetramer SAMHD1 is induced by dGTP and possesses a more potent dNTPase activity than the dimer.
(a) SAMHD1c′ (SAMHD1 109–626), but not SAMHD1c (SAMHD1 120–626), forms tetramers. Purified recombinant SAMHD1c′ and SAMHD1c proteins were incubated with 50 μM dGTP and separated by size-exclusion chromatography. The elution profiles were monitored by ultraviolet absorbance at A280 nm and compared with known molecular size markers. (b) Both tetramer and monomer peaks contained predominantly SAMHD1c′ proteins, as analysed by SDS–PAGE and Coomassie Brilliant Blue staining. (c) Analytical ultracentrifugation confirmed the existence of the SAMHD1c′ homotetramer. Molecular weights were determined by equilibrium ultracentrifugation at 72,600 g at 16 °C for 4 h in 20 mM Tris–HCl, pH 8.0, with 200 mM NaCl, 5 mM MgCl2, 25 μM dATP and 25 μM dGTP at protein concentrations of 1 mg ml−1. The main peak is centred at 280,000 Da. (d) dGTP, but not dATP, dCTP or dTTP (50 uM), induces the formation of SAMHD1c′ tetramer. (e) SAMHD1c′ has a more potent dNTPase activity than SAMHD1c. Purified recombinant SAMHD1c′ and SAMHD1c proteins were incubated with 1 mM dGTP for the indicated time points at 25 °C. The amount of dG generated at each indicated time point, as described in Methods, is shown here. The s.e. from triplicate samples (n=3) is shown. Student’s t-test was performed with Microsoft Excel to generate P-values.
