Orientation of Cys654 and Cys709 in PFV RNase H. (A) Enlargement of α-helices A and D of PFV RNase H. The blow-up shows the structural elements αA and αD, harboring residues C654 and C709, respectively. (B) Qualitative RNase H activity assay. Comparison of RNase H mutant C654S (RH_C654S) with the wt RNase H (RHwt) and the full length PR-RT. Reaction products were separated on a denaturing sequencing gel and visualized by phosphoimaging. The 27/20-mer RNA/DNA substrate, 32P-labeled at the 5’ end of the RNA, is shown on top of the gel. Arrows and numbers indicate the cleavage sites identified on the sequencing gel.