Extended Data Fig. 8: RecBCD unwinding kinetics measured using ensemble stopped-flow assay.

a, Design of the DNA substrate for the stopped-flow experiments, as described in Extended Data Fig. 5a, b, except that either a 3′ or a 5′ ssDNA overhang was added as indicated (dashed lines), to prepare the respective substrates with overhangs. b, Stopped-flow fluorescence measurements for blunt-end substrates and substrates with 6-nt 3′ or 10-nt 5′ overhangs, showing slower kinetics for the substrate with the 5′ overhang. c, Stopped-flow fluorescence measurements for substrates with 10-nt 5′ overhang or 10-nt 5′ overhang with G-C pairs in the initial 5 base pairs converted to A-T. d, Comparison of predicted ensemble time course (red line) based on the initiation and unwinding rates derived from single-molecule data (collected at a solution pH 8) to measured stopped-flow time courses (coloured symbols) on the blunt-end substrate at several pH values. To simulate the ensemble time course without fit parameters, we modelled initiation as a single exponential process using our measured initiation rates from the single-molecule data, and unwinding as a series of 1-bp unwinding steps with each molecule in the simulation having an unwinding rate drawn from our experimentally measured distribution of unwinding rates from the single-molecule data. As expected, owing to the fact that the silica coverslip surface is charged (leading to local pH shift of about 2 units; see Supplementary Discussion), the predicted curve from the initiation and unwinding rates measured by single-molecule experiments at solution pH 8 (surface pH 6) matches the stopped-flow data measured at pH 6. e, Comparison of stopped-flow data (red symbols) for blunt-end substrates at pH 6 to the time courses predicted from both initiation and unwinding rates (red) or from unwinding rates alone (black line), derived from single-molecule data obtained at a solution pH 8 (surface pH 6). Thus, the inclusion of the initiation phase in the simulation is required to match the experimental results. f, g, Comparison of stopped-flow data at pH 6 (coloured symbols) shown in b, c to predicted ensemble time courses (lines with matched colour) using initiation and unwinding rates derived from the single-molecule data at a solution pH 8 (surface pH 6), as described for d. All stopped-flow experiments were conducted at 50 μM ATP, 10% glycerol, pH 6 (except for d, for which the pH is indicated in the legend) and are representative of at least 3 individual experiments for each condition. Plots with the same colours are duplicates of the same dataset placed in different panels for the purposes of comparison.