Figure 1.

dNTP hydrolysis activity of feline SAMHD1 protein. A, sequence comparison between human and feline SAMHD1 proteins in HD active site, two allosteric sites (A1 and A2), and C-T phosphorylation site. Red residues, histidine/aspartate residues and residues known to be important for the dNTPase catalytic activity; blue residues, A1 site interacting with dGTP/GTP regulator; green residues, A2 site interacting with dNTPs; and black residue, threonine phosphorylation site (Thr-592 in hSAMHD1). Numbers indicate the first and last amino acid positions of the specified regions for each protein. NCBI reference sequences NM_015474.3 for hSAMHD1 and XM_003983547.2 for fSAMHD1. B, TLC-based assay for dGTP-dependent dTTP hydrolysis. [α-³²P]dTTP substrate was incubated with 1 μg purified WT hSAMHD1 (H) and fSAMHD1 (F) in the presence (+) and absence (−) of dGTP for 30 min at 37 °C and heat-killed for 10 min at 70 °C, which was analyzed by TLC (32). PPPi, triphosphate product. Percentages of the cleaved dTTP substrate obtained from triplicated assays were plotted with the error bars representing S.D. C, HPLC-based dNTPase assay for hSAMHD1. dGTP was incubated with (blue line) and without (red line) hSAMHD1 protein for 30 min at 37 °C, and the dG product was detected after separation from dGTP substrate by HPLC. dCMP was added to each reaction as an internal loading control. D, HPLC-based dNTPase assay for fSAMHD1 protein. dGTP was incubated with WT fSAMHD1 (blue line) and fSAMHD1 HD mutant protein (red line). This is a representative data set from triplicates.