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. 2013 Aug 22;500(7463):482-5.
doi: 10.1038/nature12333. Epub 2013 Jul 14.

DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate

Bian Liu  1 , Ronald J Baskin, Stephen C Kowalczykowski
Affiliations

DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate

Bian Liu et al. Nature. .

Abstract

Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme-DNA complex by depleting Mg(2+)-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity, but in fact reflects one microstate from the equilibrium ensemble that a single molecule manifests during an individual processive translocation event.

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Figures

Figure 1
Figure 1. Unwinding of DNA by individual RecBCD molecules is heterogeneous, with a fixed rate for the duration of DNA translocation
a, Visualization of a RecBCD unwinding an individual DNA molecule: (top) experimental scheme; (bottom) sequential images. b, Time courses for unwinding DNA (lacking a χ sequence) by different RecBCD molecules: black: absence of RecBCD; colors: individual RecBCD enzymes. Errors are shown as standard error of the fit. c, Distribution of unwinding rates for wild-type RecBCD and motor-mutants fit to the sum of two Gaussian functions and a single Gaussian, respectively. The distribution of the motor mutants was summed to represent equal numbers of each protein. Errors are the s.d.
Figure 2
Figure 2. The DNA unwinding rate of single enzymes is stochastically changed to a velocity within the original distribution, after transient depletion of Mg2+-ATP
a, DNA unwinding by three representative RecBCD enzymes. The gray block indicates the pause duration. Errors are shown as standard error of the fit. b, The rates before and after pausing (N = 173). Error bars represent the standard error of the fit. c, Distribution of rates before (blue) and after (red) pausing for molecules with an initial rate of 1450 to 1550 bp/s (blue box, panel b; N = 36); before pausing, the selected bin had a mean velocity of 1493 ±27 bp/s (SD); after pausing and redistribution, the mean velocity was 1245 ±453 bp/s (SD) (median = 1411 bp/s). d, Proportion of molecules that changed rates after pausing plotted versus pause duration and fitted to an exponential curve; error bars are expected bounds assuming a binomial distribution of switching events. e, Scatter plot of the relative rate changes after two pauses (N = 34).
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