Figure 4

Effect of Y146S/Y154S mutation on the dNTPase and nuclease activity of SAMHD1 in vitro. (A) The region of the ¹H NMR spectrum with the signal of the H8 proton of the guanine base. The peak is shifted from 8.04 ppm to 7.92 ppm upon hydrolysis of dGTP to deoxyguanosine. The three spectra were collected 4, 160 and 344 min after addition of 2 μM wild type (WT) 109–626 SAMHD1 to a sample containing 2 mM dGTP. (B) Concentration of deoxyguanosine released in SAMHD1-catalyzed dGTP hydrolysis as a function of time. (C) The nuclease activity of wild type and Y146S/Y154S SAMHD1 proteins was measure using a nuclease activity assay. Briefly the different proteins were incubated with a single stranded DNA (ssDNA) containing a 5′ FAM label and a 3′ BHQ1 black hole quencher. The fluorescence of the ssDNA substrate containing a 5′ FAM label and a 3′ BHQ1 black hole quencher is increased more than 6 fold after the ssDNA is cleaved. Plots of total FAM fluorescence measured as a function of time reveal that Y146S/Y154S mutation has only a modest effect on SAMHD1 nuclease activity.