Twinkle helicase activity on immobile D-loop DNA substrates.
A–D, indicated concentrations of Twinkle helicase were incubated with the specified immobile D-loop (substrates 7–9) or bubble (substrate 10) DNA substrates (0.5 nm) at 37 °C for 30 min as described under “Experimental Procedures.” Reaction products were resolved on non-denaturing 12% polyacrylamide gels. Representative gel images from at least three independent experiments are shown. E, quantitative analysis of percent DNA substrate unwound from A to D. Immobile D-loop with single-stranded 5′ tail (substrate 7), filled circle; immobile D-loop with single-stranded 3′ tail (substrate 8), open circle; immobile D-loop with flush invading strand (substrate 9), filled triangle; bubble DNA substrate (substrate 10), open triangle. Average values of at least three independent experiments with standard deviations indicated by error bars are shown.