Fig. 4.

Decapping by aprataxin liberates GMP. (A) Reaction mixtures (10 μL) containing 50 mM Tris⋅HCl (pH 8.0); 40 mM NaCl; 5 mM EDTA; 1 pmol 5′ ³²P-labeled pDNAppG; and 0, 0.25, 0.5, 1, 2, 4, 8, or 16 pmol of S. pombe aprataxin as indicated were incubated at 30 °C for 10 min. The reaction products were analyzed by PEI-cellulose TLC (using 0.45 M ammonium sulfate as the mobile phase) in parallel with cold GTP, GDP, and GMP standards. An autoradiogram of the TLC plate is shown. (B) Extents of ³²P-GMP release from DNAppG are plotted as a function of input aprataxin. Each datum is the average of three independent enzyme titration experiments ± SEM.