FIGURE 5.

Twinkle helicase promotes branch migration of mobile three-way junction DNA structure preferentially in the 5′ to 3′ direction. A and B, indicated concentrations of Twinkle helicase hexamer were incubated with the specified DNA substrate (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. C, quantitative analysis of percent branch migration product in the 5′ to 3′ direction (substrate 11) (A, filled circle) or 3′ to 5′ direction (substrate 13) (B, open circle). Average values of at least three independent experiments with standard deviations indicated by error bars are shown. D, Twinkle-catalyzed branch migration is dependent on hydrolyzable ATP. Reaction mixtures containing 5 nm Twinkle and the 5′ to 3′ mobile three-way junction DNA substrate (substrate 11) (0.5 nm, A) were incubated and analyzed as described above. E, indicated concentrations of Twinkle helicase hexamer were incubated with a forked duplex DNA substrate (substrate 12) (0.5 nm) at 37 °C for 30 min as described under “Experimental Procedures.” The third oligonucleotide added to the reaction mixture requires helicase-catalyzed unwinding of the duplex region to anneal to one of the two unwound strands. Reaction products were resolved on non-denaturing 12% polyacrylamide gels. Representative gel images from at least three independent experiments are shown. F, quantitative analysis of percent helicase product. Average values of at least three independent experiments with standard deviations indicated by error bars are shown.