RNase H activities of the full length PFV PR-RT and RNase H-(Q591-N751). (A) Quantitative RNase H activity assay. Steady state kinetic measurements of the velocity (pmol/s) of the RNase H activities were performed with 1 nM PR-RT (closed circles) and 10 nM RNase H-(Q591-N751) (open circles), respectively, using increasing amounts of a fluorescently labeled DNA/RNA hybrid substrate as indicated. For PFV PR-RT the following values were obtained: KM = 5.3 nM (± 0.8), vmax = 0.05 pmol/s (± 0.002). The values were obtained by the fit program GraFit using the Michaelis-Menten equation v = vmax [S]/(KM + [S]) for the fitting procedure. The curve shows the best fit to the data. Due to its low activity, the kinetic parameters for the RNase H domain could not be determined. (B) Qualitative RNase H assay. The 20/27mer DNA/RNA primer/template substrate is shown on top of the figure. The cleavage sites are indicated by arrows in the sequence on top and at the corresponding band on the gel. The first nucleotide of the RNA hybridized to the 3'-OH nucleotide of the DNA strand is designated -1. 240 nM of the substrate were incubated with 30 nM PFV PR-RT or 20 μM of the RNase H domain for the times indicated on top of the gel. Reaction products were separated on a 15% sequencing gel and visualized by phosphoimaging.