(A) TIRF-smFRET time traces of DF-7,1mismatch(1nt)-Flap alone and in the presence of FEN1. (B) Surface-immobilized confocal-smFRET time traces of NonEQ DF-6,1Flap in the presence of FEN1-R47A at 5 ms temporal resolution. (C) The effect of blocking 5’flap threading on DNA bending by FEN1. Schematic showing the strategy used to block 5’flap threading into the cap-helical gateway by introducing NeutrAvidin/biotin linkage at the 5’end of the 5’flap of DF-30,1 (termed of DF-30,1blocked-dsDNA) prior to the addition of FEN1 (upper panel). Surface-immobilized confocal-smFRET time traces of DF-30,1blocked-dsDNA alone and in the presence of FEN1 at 5 ms temporal resolution (lower panel). The substrate was immobilized by surface-coated-NeutrAvidin via the biotin group on the 5’flap. (D) A bar chart comparing final bent FRET states of DF-30,1trapped-dsNDA, DF-30,1blocked-dsDNA, SF-30,0trapped-dsDNA and SF-30,0blocked-dsDNA using burst confocal-smFRET histograms from freely diffusing substrates acquired at sub-ms temporal resolution. FEN1 concentrations were 5000 nM for SF-30,0internal-blocked, 1000 nM for DF-30,1internal-blocked,1000 nM for SF-30,0internal-trapped and 200 nM for DF-30,1internal-trapped. To trap a threaded 5’flap, FEN1 was first pre-incubated with the substrate before NeutrAvidin was added to bind the biotin on the 5’flap (as shown in the schematic in the upper panel). The FRET value in each condition represents the average of N = 3 and the uncertainty corresponds to the standard error of their fits. (E) TIRF-smFRET time traces of DF6,2Flap alone and in the presence of FEN1.