FIGURE 1.

Nuclease activity of the full-length human SAMHD1 and its domains. A and B, time points of ssDNA and ssRNA cleavage, respectively, by full-length SAMHD1. The 5′- or 3′-³²P-labeled ssDNA or ssRNA substrate was incubated without protein (C lane) or with 100 nm SAMHD1 at 37 °C and analyzed by denaturing gel electrophoresis. L, ladder. C, time point of cleavage of ³²P-labeled ssDNA (40 nt) or ssRNA (39 nt) by full-length SAMHD1 (100 nm), the SAM domain (100 nm), or the HD domain (100 nm). To calculate nuclease activity (as percent of the maximal activity of full-length SAMHD1), the gel images were quantified using ImageQuant TL software (GE Healthcare). D, time points of ssDNA (left panels) and ssRNA (right panels) cleavage by isolated SAM and HD domains. The 5′-³²P-labeled ssDNA or ssRNA substrate was incubated with 0.3 μg of SAM or HD domain (as indicated) at 37 °C. E, HPLC analysis of the reaction products generated by SAMHD1 during hydrolysis of dNTPs. Shown are elution profiles of the reaction products separated by ion-pair reverse-phase HPLC (monitored at 254 nm). The upper two profiles show control samples incubated without enzyme (dGTP and deoxyguanosine (dG) standards; 5 mm each), whereas the lower profile shows the sample incubated with the enzyme for 15 min. F, dGTP triphosphatase activity of full-length SAMHD1 and its isolated SAM and HD domains. G, cleavage of ssDNA by SAMHD1 in the presence of dGTP. H, cleavage products of SAMHD1. Shown is the PNK or TdT labeling of the DNA cleavage products. Lanes 1–3, 5′-³²P-labeled ssDNA6 (40 nt, 0.1 μm) was incubated at 37 °C in the absence (lane 1) or presence of 250 nm (lane 2) of 400 nm (lane 3) SAMHD1 for 25 min without the addition of PNK or TdT. Lanes 4–9, PNK-treated reactions. Unlabeled ssDNA (0.1 μm) was incubated at 37 °C in the absence (lanes 4–7) or presence of 250 nm (lanes 5 and 8) or 400 nm (lanes 6 and 9) SAMHD1 for 20 min, followed by treatment with PNK (30 min) in the 5′-end labeling (forward; lanes 4–6) or phosphate exchange (lanes 7–9) reactions. Lanes 10–12, TdT treatment reactions. Unlabeled ssDNA (0.1 μm) was incubated at 37 °C in the absence (lane 10) or presence of 250 nm (lane 11) or 500 nm (lane 12) SAMHD1 for 20 min, followed by treatment with TdT (30 min) in the 3′-end labeling reaction.