Fig. 5: Pre-separation of the hybrids unleashes the 5′-to-3′ exoribonuclease activity of RNase H1.
From: RPA transforms RNase H1 to a bidirectional exoribonuclease for processive RNA–DNA hybrid cleavage
a A schematic showing the experimental design of the smFRET assay with the forked RDH template. RNase H1 degrading the 3′ end of the ssRNA detaches the substrate from the surface, resulting in the simultaneous disappearance of both fluorophore signals. b A representative smFRET trajectory showing fluorophore signals’ simultaneous disappearance before the increase in E. The a.u. represents arbitrary units. c The percentage of the smFRET trajectories with the forked and blunt RDH substrates showing the simultaneous disappearance of both fluorophore signals before E reaches 0.9. n = 241 and 179. d A representative gel showing the cleavage products of RNase H1 (10 nM) on the FAM-labelled blunt RDH and forked RDH substrates (15 nM) at 0.05 mM Mg2+. The 16-nt cleavage product is highlighted with the red dotted rectangle. Each experiment was repeated in triplicate. e A representative kymograph showing the bidirectional RNA-Cy3 (brown) degradation by RNase H1 under 25 pN. f The percentage of the RNase H1-mediated bidirectional RNA–DNA hybrid degradation event under 15, 20, and 25 pN. n = 9, 13, and 17. Source data are provided as a Source Data file.
