Fig. 2: ATP and tension dependencies of real-time DNA unwinding kinetics.

a Schematic of optical trapping assays. A DNA fork-like construct is tethered between two functionalized micron-sized beads. One bead is hold in the optical trap (red cone), while the other is immobilized on a micropipette. In the presence of ATP, DNA unwinding is recorded as a change in tether extension (Δx) under constant mechanical tension (F). The blue arrow indicates the 5′ to 3′ translocation of the helicase along the hairpin. b The DNA construct consists of a 559 bp DNA hairpin (sequence in Supplementary Methods) with a ∼2.6-kb long dsDNA handle labelled with digoxigenin (red dots) and a 5′-poly(dT)₃₅ labeled with biotin (blue dot). The single stranded poly(dT)₃₅ tail serves as a helicase loading site (Methods). c Representative traces of the WT Twinkle (blue), ΔZBD (orange) and ΔC-tail (magenta) variants (F = 6 pN). DNA unwinding (increase in bp) is often followed by rewinding (decrease in bp). Raw data (grey background) was smoothed using a low-pass 10 Hz filter. d–g ATP dependencies of the average unwinding processivity (d), unwinding rate (e), pause-free velocity (f), and pause occupancy (g) for the WT helicase (blue), and ΔC-tail (magenta) variant. The dotted line in (f) represents the Michaelis-Menten fit for the pause-free velocity of WT Twinkle as a function of ATP concentration (R² = 0.89). Data in (d, g) were recorded at F = 6 pN, the minimal tension enabling consistent activity detection across all ATP concentrations (WT, N = 46 independent activities; Δ-Ctail, N = 36). h–k Tension dependencies of the average unwinding processivity (h), unwinding rate (i), pause-free velocity (j) and pause occupancy (k) for WT Twinkle (blue, N = 45) and the ΔZBD variant (orange, N = 32). For all panels, data points represent to the average of multiple independent measurements, and error bars indicate the s.e. For this figure (d–k) source data are provided as a Source Data file.