FIG. 5.

RH assay using “generic” RNA-DNA hybrid template (adapted from reference 18). (A) Diagram illustrating the RNA-DNA hybrid template and the expected Ty1 RH cut sites. (B) Comparison of WT and M520I RH cleavages. (C) Comparison of WT and S469G RH cleavages. (D) Comparison of WT and S469G RH cleavages in the presence of heparin. (B to D) RH assays were performed in 10 mM Tris-HCl (pH 7.8), 80 mM NaCl, 5 mM DTT, 5 mM MgCl₂, and increasing concentrations of MnCl₂ (Mn/Mg ratios of 0, 0.0002, 0.001, 0.002, 0.01, 0.02, 0.1, 0.2, and 1). C, uncut substrate. A ladder was generated by subjecting uncut template to 0.1 M sodium carbonate for 4 min at 100°C. Hydrolysis was initiated by the addition of equal activities (normalized by RT activity) of WT or mutant enzymes at 22°C for 1 h in a final volume of 30 μl.