The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA-binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear. Here, we present a novel optical tweezers assay that allows monitoring the activity of RecBCD's individual subunits, when they are part of an intact full complex. We show that RecBCD and its subunits are able to generate forces up to 25-40 pN without a significant effect on their velocity. Moreover, the isolated RecD translocates fast but is a weak helicase with limited processivity. Experiments at a broad range of [ATP] and forces suggest that RecD unwinds DNA as a Brownian ratchet, rectified by ATP binding, and that the presence of the other subunits shifts the ratchet equilibrium towards the post-translocation state.
Keywords: E. coli; RecBCD; biochemistry; chemical biology; helicase; molecular biophysics; optical tweezers; single molecule biophysics; structural biology.
© 2019, Zananiri et al.