Purified GFP-dRNH1 is catalytically inactive and specific for RNA–DNA hybrids in vitro. (A) Schematic illustration of GFP-dRNH1. HBD, hybrid binding domain; HC, hybrid catalytic domain; His, His tag; LR, linker region. (B) Purified GFP-RNaseH1, both GFP-wtRNH1 and GFP-dRNH1, were resolved on a 4–12% Bis-Tris SDS-PAGE gel and stained with Coomassie G-250. Mw, molecular weight in kDa. (C) Products from enzymatic reactions following incubations of oligonucleotide substrates with GFP-wtRNH1 or GFP-dRNH1 were resolved on a polyacrylamide gel and stained with SYBR Gold. Bp, DNA size in base pairs. (D) RNA–DNA hybrids 60 bp in length were labeled with ³²P, and 1 nM of labeled substrate was incubated with increasing concentrations of GFP (left), GFP-dRNH1 (middle), or S9.6 antibody (right; 1, 10, 20, 40 nM). The resulting complexes were resolved on a native polyacrylamide gel. Unbound RNA–DNA hybrids are indicated at the bottom of the gel. (E) dsRNA 60 bp in length was labeled with ³²P, and 1 nM of substrate was incubated with increasing concentrations of GFP (left), GFP-dRNH1 (middle), or S9.6 antibody (right; 1, 10, 20, 40, 80 nM). The resulting complexes were resolved on a native polyacrylamide gel. Unbound dsRNA is indicated at the bottom of the gel.