FIG 2.

Metal-dependent RNase H activity. (A) Reaction mixtures (10 μl) containing 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 20 nM (200 fmol) ³²P-RNA:DNA hybrid duplex (depicted at the bottom), 0.4 nM (4 fmol) wild-type RnhC, and 5 mM the indicated divalent cation (as the chloride salt) were incubated at 37°C for 20 min. Divalent cation was omitted from a control reaction in lane −. (B) Reaction mixtures (10 μl) containing 50 mM Tris-HCl (pH 7.5); 50 mM NaCl; 10 mM MgCl₂; 1 mM DTT; 20 nM (200 fmol) RNA:DNA hybrid duplex; and 0.4 nM (4 fmol) wild-type RnhC, RnhC-E49Q, or RnhC-D73N were incubated at 37°C for 20 min. RnhC was omitted from a control reaction in lane −. The reactions were quenched with an equal volume of 90% formamide, 50 mM EDTA, 0.3% bromophenol blue. The reaction products were analyzed by electrophoresis through a 40-cm 18% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate, 1 mM EDTA. The products were visualized by autoradiography.