PabRNase HII specifically cuts the RNA-DNA/DNA duplex when the RNA is completely annealed to the DNA template. (A) Structures of substrates S7, S8, and S9. The thick line and the closed circle represent the RNA portion and the fluorescent label, respectively. (B) The indicated amounts of PabRNase HII were incubated with the S7 substrate (lanes 2 to 5), the S8 substrate (lanes 6 to 9), and the S9 substrate (lanes 10 to 13). An 11-nt ladder was used (lane 1). Fluorescently labeled products were visualized with a Mode Imager Typhoon 9400 (GE Healthcare), and quantification was performed using ImageQuant 5.2 software. (C) Graphical representation of sites and extents of cleavage at mismatches in RNA-DNA/DNA substrates. Cleavage sites are denoted by different bars. Deoxyribonucleotides and ribonucleotides are shown by uppercase and lowercase letters, respectively.