Synergy between RecBCD subunits is essential for efficient DNA unwinding

Abstract

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.

Auxiliary ATP binding sites support DNA unwinding by RecBCD

“…If RecB and RecD have significantly different affinities towards nucleotides, one would expect to find that, at an intermediate ATP concentration, one subunit will be fully active while the second will not. To study the activity of the individual subunits we exploited a recently developed single-molecule optical tweezers assay 18 . Figure 2a shows a schematic representation of the experiment, where a DNA construct, consisting of a stem with a blunt end attached to two dsDNA “tracks”, is tethered between two beads trapped in separate optical traps.…”

“…To generate unwinding/translocation tracks of different lengths 18 , 600 and 4000 bp tracks were obtained using standard PCR reactions (Supplementary Table 11 , IDT) and nicked using Nt.BbvCI for the Biotin-terminated track and Nb.BbvCI for the Digoxigenin-terminated one (enzymes from New England Biolabs), resulting in complementary 29-nucleotides flanked with three nucleotides (5′-TGC-3′). For the symmetric geometry, the 600 biotin and digoxigenin tracks were mixed at equal molar ratios for DNA annealing, creating a ∼1200 bp fragment.…”

“…The RecC subunit “staples” the RecB and RecD subunits 5 , harbors the “pin” domain that has been proposed to split the duplex 5 , and recognizes the Chi sequence 6 , 7 . Previous studies have provided a wealth of knowledge on RecBCD’s biochemistry and structure 1 , 8 – 13 and revealed important features of the unwinding process, including its initiation, kinetic step size, and inter-subunit regulation 12 , 14 – 18 .…”

Three-dimensional tracking of tethered particles for probing nanometer-scale single-molecule dynamics using plasmonic microscope

“…This is because by far it is difficult to measure the spatial position and rotation of RecBCD on a single platform simultaneously. DNA length is often measured by magnetic and optical tweezers, 22-24 while RecBCD rotation is measured by a DNA origami-based method called ORBIT. 21 Since our tracking technique can probe the 3D coordinates of RecBCD in space, it is possible to obtain DNA length ( L ) and RecBCD rotation angle ( θ ) from the coordinates (Figure 3a).…”

A Tour de Force on the Double Helix: Exploiting DNA Mechanics To Study DNA-Based Molecular Machines

“…29,40 The well-characterized elasticity combined with its ease of construction makes DNA a popular choice as a molecular handle 41 for single-molecule studies of protein/RNA folding 42,43 and biomolecular motors. 44 More recently, researchers have utilized DNA origami technology to construct bundled DNA beams that are much more rigid than conventional dsDNA for ultra-high-resolution measurements. 45,46 ■ EXPLOIT DNA MECHANICS TO STUDY BIOLOGY DNA is under constant tension inside the cell: It is wrapped around histones, 47 unwound by helicases, 48 and twisted and untwisted by RNA polymerases 49 and topoisomerases, 50 to name a few examples.…”