Extended Data Fig. 4: RecBCD unwinding rate and pausing characteristics at solution pH 6.
From: Rotation tracking of genome-processing enzymes using DNA origami rotors
a, Average unwinding rate as a function of ATP concentrations fit to a Michaelis–Menten dependence with vmax = 290 ± 10 bp s−1, KM = 130 ± 10 μM. Data are mean ± s.e.m. (n = 47, 94, 80, 37 and 110 (from left to right) trajectories from at least 3 independent biological replicates for each condition). b, ATP concentration dependence of the pause frequency. Pause frequency was determined as the average number of pauses per second for each single-molecule trajectory. Data are mean ± s.e.m. (n = 47, 94, 80, 37 and 110 (from left to right) trajectories, from at least 3 independent biological replicates for each condition). c, Median duration of pauses during forward unwinding at various concentrations of ATP. The error bars are the s.d. of the median derived from resampling (n = 76, 62, 27 and 18 (from left to right) events, from at least 3 independent biological replicates for each condition). d, Mean backtracking distance at various concentrations of ATP. Data are mean ± s.e.m. (n = 20, 22, 16 and 10 (from left to right) events, from at least 3 independent biological replicates for each condition). e, Median recovery pause duration after a backtracking event at various concentrations of ATP. The error bars are the s.d. of the median derived from resampling. The P values for the differences between the 25-μM-ATP data point and the 50-, 75- and 300-μM-ATP data points are 0.05, 0.004 and 0.09, respectively, derived from two-sided Kolmogorov–Smirnov test of the distributions of the pause durations (n = 20, 22, 16 and 10 events for the 25, 50, 75, and 300 μM data points, respectively). Note that not all trajectories contain a backtracking event. Data were acquired at 10% glycerol, solution pH 6, 500 Hz. f, Schematic of a kinetic model of DNA unwinding induced by RecBCD. During DNA unwinding, pausing occurs frequently and some pauses lead to enzyme backtracking; the enzyme can exit the backtracking state and resume DNA unwinding through a recovery pause intermediate, which is distinct from the initial pause state.
