Figure 5.
Activity with N6-methyl-dATP is evolutionary conserved among vertebrates. MutT homologues (MTH1 and NUDT1) from different animal species as well as E. coli MutT and NUDT1 from the plant A. thaliana were screened for hydrolysis activity with N6-methyl-dATP. Enzyme (1.25 nm) was incubated with 50 μm N6-methyl-dATP in MTH1 reaction buffer (pH 7.5) with 0.4 units/ml of PPase for 20 min at 22 °C in triplicates. Pi was detected using Biomol Green (Enzo Life Sciences). Absorbance at 630 nm was read after 20 min. A Pi standard curve was included on the plate and used to convert the assay signal to produced PPi. Data are shown as hydrolyzed N6-methyl-dATP (μm) divided by concentration of NUDT1 enzyme (μm) per second. The graph shows the mean ± S.D. from an experiment performed in triplicate.