Figure 2.

Metal-dependent RNase H activity and nucleic acid substrate specificity of RnhA. (A) Substrates. The ³²P-RNA:DNA, ³²P-DNA:DNA and ³²P-RNA:RNA duplexes and the ³²P-RNA single strand substrates are shown, with the 5′ ³²P-label denoted by •. The principal site of RnhA incision of the RNA:DNA hybrid is indicated by a black arrowhead above the ³²P-labeled RNA strand. (B) Nucleic acid substrate specificity. Reaction mixtures (10 μl) containing 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl₂, either 20 nM (200 fmol) ³²P-RNA:DNA, ³²P-DNA:DNA or ³²P-RNA:RNA duplexes or 20 nM (200 fmol) ³²P-RNA single strand, and 8 nM (80 fmol) RnhA (where indicated by +) were incubated at 37°C for 20 min. 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. An alkaline hydrolysis ladder of the ³²P-labeled 24-mer RNA strand was analyzed in parallel in lane ^–OH. The radiolabeled RNAs were visualized by autoradiography. (C) Active site mutations. Reaction mixtures (10 μl) containing 25 mM Tris–HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl₂, 20 nM (200 fmol) ³²P-RNA:DNA hybrid duplex, and 8 nM (80 fmol) of wild-type RnhA, RnhA-E50Q or RnhA-D72N were incubated at 37°C for 20 min. RnhA was omitted from a control reaction in lane –. (D) Metal cofactor requirement. Reaction mixtures (10 μl) containing 25 mM Tris–HCl, pH 7.5, 50 mM NaCl, 20 nM ³²P-RNA:DNA hybrid duplex, 8 nM wild-type RnhA, and 5 mM of 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 –.