(A) Electrophoretogram of a single-turnover unwinding experiment. Preincubation of fluorescein-labeled forked duplex DNA (30 nM, gc36) with RecQ-dH (100 nM) was followed by rapid mixing with ATP (3 mM) plus excess ssDNA trap strand (3 µM) (final post-mixing concentrations). Reactions were stopped by the addition of EDTA (40 mM) and loading dye at different time points (0–150 s, cf. panel B) using a quenched-flow instrument or by manual mixing. Amounts of DNA species (forked duplex and ssDNA, depicted by cartoons) labeled with fluorescein (asterisk) were detected by a fluorescence imager. “– “denotes a 150 s control reaction in which ATP was absent. (B) Single-turnover unwinding kinetics of forked DNA substrates with GC contents of 36% (light gray), 48% (gray) and 79% (black) of RecQ-dH and RecQWT. Error bars represent SEM calculated from three experiments. Solid lines show fits based on: the n-step model at n = 5 for both helicase constructs (see scheme on panel C); simultaneous model (Equation 1 and panel C) at n = 4 for RecQ-dH and n = 5 for RecQWT; and delayed release model (Equation. and panel C) at n = 4 for RecQ-dH and n = 5 for RecQWT. (C) Common scheme for the modified n-step and derived simultaneous melting and delayed release models. In the models, unwinding starts from the ssDNA-dsDNA junction. Of all DNA-RecQ complexes (D.R), only a fraction (fP, D.Rp, lower row) unwinds the dsDNA segment in a single run, consisting of n consecutive irreversible kinetic steps (k(1)…k(n)). In the n-step model, the rate constant of each unwinding kinetic step is identical (k(1) = k(n)). In contrast to this, in the simultaneous melting and delayed release models, the rate constant of each unwinding kinetic step depends on the base pair energy of the dsDNA segment to be unwound (segment length according to kinetic step size), as described in Equation 1 and 2, respectively. A fraction of DNA bound helicase molecules (fN, D.RN, upper row) is unable to successfully unwind DNA due to the limited processivity of the enzyme and/or more complex unwinding patterns. After dissociating from the DNA substrate, these enzyme molecules can rebind to the substrate at rate constant krebind and start a new unwinding run (D.R). (D–E) Determined χv2 values from fitting the (D) n-step model for RecQ-dH (filled circles) and RecQWT (open circles) or (E) fitting the simultaneous melting (green) and delayed release models (red) for the indicated helicase construct. Other determined parameters are listed in Supplementary file 1 Table S2.