Figure 5.

Qualitative RNase H assay. The DNA/RNA P/T substrate is shown on top. The cleavage sites determined for SFV and PFV PR-RT are indicated by arrows. 320 nM of DY-647 labeled P/T substrate was incubated with 50 nM of SFVmac or PFV PR-RT in 50 mM Tris/HCl, pH8.0, 80 mM KCl, 6 mM MgCl₂ for the times indicated on top of the gel. Reaction products were analyzed on a 15% polyacrylamide sequencing gel and visualized by detection of the fluorescence emission of the RNA template strand at 670 nm upon excitation at 633 nm. The cleavage sites are indicated on the left. The first nucleotide of the RNA hybridized to the 3'-OH nucleotide of the DNA primer is denoted -1. The partially hydrolyzed RNA on the right was used for the determination of the cleavage sites. Numbers on the right indicate the length of the RNA in nucleotides.