Figure 2

Thermal stability and temperature dependence of P.a . RNase H2 activity. A. Aliquots of P.a. RNase H2 were pre-incubated at 95°C for the indicated times and then tested for activity with the heteroduplex substrate S-rC 14-1-15 (labeled with ³²P). Cleavage reactions were allowed to proceed for 20 minutes at 70°C. Reaction products were separated by denaturing PAGE and visualized by phosphorimaging. Percent cleavage of the substrate (Y-axis) is shown plotted against pre-incubation time at 95°C (X-axis). Assays were run in triplicate for each pre-incubation time point. B. ³²P-labeled S-rC 14-1-15 was incubated in the absence or presence of 0.25 mU of recombinant P.a. RNase H2 for 20 minutes at 30°C, 40°C, 50°C, 60°C, or 70°C. Reactions were stopped with the addition of EDTA and cleavage products were separated by denaturing PAGE and visualized by phosphorimaging. A gel image is shown. Cleavage reactions were run in triplicate and no enzyme controls were run once. C. The phosphor gel image from panel B above was quantified and triplicate data points averaged. The percent cleavage of substrate (Y-axis) is shown plotted against reaction temperature (X-axis).