FIGURE 3.

Single-turnover kinetics of Twinkle helicase activity on replication fork structures. Twinkle hexamer (3.2 nm) was pre-incubated with the indicated radiolabeled DNA substrate 1–6 (2.5 nm) prior to simultaneous addition of ATP and 100-fold excess of dT₂₀₀, followed by incubation at specified time points as described under “Experimental Procedures” for single-turnover kinetic assays. Reaction products were resolved on native 12% polymacrylamide gels and analyzed. A, Twinkle (3.2 nm hexamer) unwinding kinetics on forked duplex substrate with single-stranded 5′ and 3′ arms (substrate 1) (filled circle) or 5′ flap substrate (substrate 2) (open circle). Inset, 3′ flap substrate (substrate 3) (filled triangle) or synthetic replication fork with duplex leading and lagging strand arms (substrate 4) (open triangle). B, Twinkle (6.4 nm hexamer) unwinding kinetics on 5′ single strand tailed duplex (substrate 5) (filled square) or 3′ single strand tailed duplex (substrate 6) (open square). Average values of at least three independent experiments with standard deviations indicated by error bars are shown.