The Heterogeneity of DNA Unwinding by RecBCD Molecules Reflects Individual Conformations Stabilized by Ligand Binding: A Manifestation of the Ergodic Hypothesis

Abstract

Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular properties and activities, and reveal interesting variations between individual molecules. The application of these techniques to the RecBCD helicase of Escherichia coli has put to rest some long-standing debates and given some mechanistic insights about its functions. Importantly, previous single-molecule analysis showed that the DNA unwinding rates of individual enzyme molecules vary significantly. However, the origin of this heterogeneity was unknown. Here we investigated the question by defining the components of the distribution, and the behavior of individual molecules in the distribution. The distribution of DNA unwinding rates is not unimodal: one third of the molecules unwound DNA significantly slower than the majority of the population. Although any individual RecBCD molecule unwound DNA at a constant rate, we discovered that transiently pausing a single enzyme-DNA complex, by depleting Mg2+-ATP, changed the rates of some enzyme molecules. The proportion of molecules that changed rate increased exponentially with the duration of the pausing, with a half-time of 4 sec, suggesting a conformational change. We suggest that substrate binding locks the enzyme in one of a broad range of conformations that affect the rate-limiting translocation behavior of RecBCD, resulting the molecule-to-molecule variation in the helicase activity.