Motor step size and ATP coupling efficiency of the dsDNA translocase EcoR124I

The Type I restriction-modification enzyme EcoR124I is an archetypical helicase-based dsDNA translocase that moves unidirectionally along the 3′–5′ strand of intact duplex DNA. Using a combination of ensemble and single-molecule measurements, we provide estimates of two physicochemical constants that are fundamental to a full description of motor protein activity—the ATP coupling efficiency (the number of ATP consumed per base pair) and the step size (the number of base pairs transported per motor step). Our data indicate that EcoR124I makes small steps along the DNA of 1 bp in length with 1 ATP consumed per step, but with some uncoupling of the ATPase and translocase cycles occurring so that the average number of ATP consumed per base pair slightly exceeds unity. Our observations form a framework for understanding energy coupling in a great many other motors that translocate along dsDNA rather than ssDNA

Impediment of E. coli UvrD by DNA-destabilizing force reveals a strained-inchworm mechanism of DNA unwinding

Escherichia coli UvrD is a non-ring-shaped model helicase, displaying a 3′–5′ polarity in DNA unwinding. Using a transverse magnetic tweezer and DNA hairpins, we measured the unwinding kinetics of UvrD at various DNA-destabilizing forces. The multiform patterns of unwinding bursts and the distributions of the off-times favour the mechanism that UvrD unwinds DNA as a dimer. The two subunits of the dimer coordinate to unwind DNA processively. They can jointly switch strands and translocate backwards on the other strand to allow slow (∼40 bp/s) rewinding, or unbind simultaneously to allow quick rehybridization. Partial dissociation of the dimer results in pauses in the middle of the unwinding or increases the translocation rate from ∼40 to ∼150 nt/s in the middle of the rewinding. Moreover, the unwinding rate was surprisingly found to decrease from ∼45 to ∼10 bp/s when the force is increased from 2 to 12 pN. The results lead to a strained-inchworm mechanism in which a conformational change that bends and tenses the ssDNA is required to activate the dimer