Fig. 3: Stepwise RNA cleavage by RNase H1 reveals a dominant rate-limiting step of hybrid unwinding.
From: RPA transforms RNase H1 to a bidirectional exoribonuclease for processive RNA–DNA hybrid cleavage
a, d, g Representative smFRET trajectories show a stepwise increase in E in the process of RNase H1 cleaving the 3′-RDH, GC-RDH, and AU-RDH substrates. The solid purple lines are the fitting results from an automated step-finding algorithm. The a.u. represents arbitrary units. b, e, h. The TDPs illustrate FRET transitions in the process of RNase H1 cleaving the 3′-RDH (n = 95), GC-RDH (n = 47), and AU-RDH substrates (n = 56), respectively. c, f, i. The dwell-time histograms are calculated from the first two pauses detected with the 3′-RDH (n = 70), GC-RDH (n = 165), and AU-RDH substrates (n = 107), respectively. Single exponential fittings to the histograms are shown in red. j The kinetic rates (k) obtained from the single exponential fitting are compared for the three substrates (n = 70 for 3′-RDH, n = 165 for GC-RDH, n = 107 for AU-RDH), respectively. Error bars represent SEM. k A representative gel showing that RNase H1 (15 nM) cleaves the FAM-labelled 53-bp RDH-FAM substrates (15 nM) at 0.07 mM Mg2+. The intensities of the cleavage products are compared with the 1-nt markers. Each experiment was repeated in triplicate. l A model of RNase H1 cyclically cleaving an RNA–DNA hybrid. Source data are provided as a Source Data file.
