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

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 molecules1,2,3. 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 behaviour4,5,6. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably6,7,8, 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 Mg2+-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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Figure 1: Unwinding of DNA by individual RecBCD molecules is heterogeneous, with a fixed rate for the duration of DNA translocation.
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.

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Acknowledgements

We are grateful to members of the laboratory for their comments on this work. S.C.K. was supported by the National Institutes of Health (GM-62653 and GM-64745).

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B.L., R.J.B. and S.C.K. conceived the general ideas, designed the experiments and interpreted the data. B.L. performed experiments. B.L. and S.C.K. analysed the data and wrote the manuscript. R.J.B. passed away on July 3, 2010; this work is dedicated to his collegiality and contributions.

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Correspondence to Stephen C. Kowalczykowski.

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Liu, B., Baskin, R. & Kowalczykowski, S. DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate. Nature 500, 482–485 (2013). https://doi.org/10.1038/nature12333

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