Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA

Abstract

B-DNA becomes unstable under superhelical stress and is able to adopt a wide range of alternative conformations including strand-separated DNA and Z-DNA. Localized sequence-dependent structural transitions are important for the regulation of biological processes such as DNA replication and transcription. To directly probe the effect of sequence on structural transitions driven by torque, we have measured the torsional response of a panel of DNA sequences using single molecule assays that employ nanosphere rotational probes to achieve high torque resolution. The responses of Z-forming d(pGpC)(n) sequences match our predictions based on a theoretical treatment of cooperative transitions in helical polymers. "Bubble" templates containing 50-100 bp mismatch regions show cooperative structural transitions similar to B-DNA, although less torque is required to disrupt strand-strand interactions. Our mechanical measurements, including direct characterization of the torsional rigidity of strand-separated DNA, establish a framework for quantitative predictions of the complex torsional response of arbitrary sequences in their biological context.

Fig. 1.

Feedback enhanced rotor bead tracking assay and torsional response of control DNA. (A) Experimental setup for dynamic torque measurements. The DNA molecule is attached to the cover slip surface by a single digoxigenin:α-digoxigenin interaction (light blue), which acts as a swivel. The magnetic bead (MB, orange) is attached via multiple fluorescein:α-fluorescein interactions (dark blue), producing a torsional constraint. The fluorescent rotor bead (RB, green) is attached to the side of the DNA molecule via biotin:neutravidin interactions. A specific “sequence of interest” (SOI, red) is placed between the MB and the RB attachment of the DNA molecule. The angular position ϕ of the MB is controlled by rotating the magnets, and the angle ψ of the RB is measured using videomicroscopy. The twist θ in the DNA is obtained as θ = ϕ - ψ with the equilibrium twist defined as θ = 0. Feedback control of MB angle can be used to impose a twist clamp. (B) Torque-twist diagram for 4.6-kb control DNA tethers. Unless stated otherwise, all such curves are obtained from four independent twist clamp experiments, and unwinding curves are superimposable with rewinding. Twist was ramped at 0.05 turns/s, torque was obtained from rotor angular velocities averaged over a 3-s window, and data were subsequently binned along the twist axis and averaged (red line). The postbuckling torque τ_B, critical torque for the B-L transition τ_L, and torsional spring constant (where l is the length of the DNA and P_t,eff is the effective twist persistence length) can be extracted from the torque-twist plot. DNA twist and rotor angular velocities were obtained from raw traces (insert) showing the cumulative angles of the magnets (orange trace) and RB (green trace) as a function of time. (C) Torque-twist plot showing complete conversion of B-DNA to L-DNA under negative torque for an N base pair long control DNA held under 3.2 pN tension. A faster twist ramp and proportionately larger bin sizes have been used for data in the plateau region. By saturating the transition, the extrapolated change in helicity per base pair Δθ_0,L can be obtained. From the slope κ_L the twist persistence length of L-DNA can be calculated. (D) L-DNA helicity and elasticity vary with tension. Δθ_0,L and effective twist persistence length P_t,eff,L of the L state are displayed ± SEM, based on linear fits to the data spanning −15 and -20 pNnm. Averaged traces are each based on three independent measurements.

Fig. 2.

Torsional response of GC repeats: Z-DNA formation. (A) Templates containing 50 bp (yellow, Z50) or 22 bp (green, Z22) GC repeat SOIs were assayed in PBS under 1.6 pN. Negative control DNA (black line) under identical conditions serves as a reference. Individual unwinding curves (fine lines) were variable and differed from the consistent behavior of rewinding curves (averaged, bold lines). Fits to a cooperative transition model (see SI Text) are displayed (inset) along with the corresponding experimental data (violet, Z50; red, Z22). (B) Overlay of individual unwinding (dark blue) and rewinding (light blue) experiments for Z50 under low salt conditions (Upper), showing reversibility. Under high salt conditions (Lower) the B-Z coexistence plateau occurs at lower absolute torques, and unwinding (bold dark red) and rewinding (fine light red) traces show hysteretic behavior. (C) The response of 22 bp GC repeats is affected by the inclusion of atomic spacers (Z22sp) and by increases in ionic strength (HS). Comparison of unwinding (fine lines) with averaged rewinding curves (bold lines) shows reduced hysteresis in Z22sp. (D) The torsional response of the DNA handles has been subtracted from averaged rewinding curves in order to show the inferred response of the SOI alone, plotted as torque vs. twist/bp.

Fig. 3.

Torsional responses of mismatch bubbles. (A) Torque-twist curve for d(pT)₁₀₀•d(pT)₁₀₀ SOI (T100) and negative control DNA (gray tones) in PBS, together with fits to the cooperative transition model (light blue and green lines). (B) Response of 100 bp (dark green, Bub100) and 50 bp (light green, Bub50) d(pGpApCpT)_n•d(pGpApCpT)_n SOIs assayed in PBS under 10 pN of tension. Red and yellow lines show transition model fits. Characteristic curves show a plateau at a critical torque τ_crit, a posttransition torsional spring constant κ_post, and a change in helicity Δθ₀ for the transition. (C) The response of Bub100 is insensitive to increased ionic strength. Torque-twist curves are shown for Bub100 in PBS (green) and in high ionic strength (red line) alongside control DNA (light green and orange, respectively) under 10 pN of tension. Transition model fits are shown in black.

Fig. 4.

A model for cooperative structural transitions in homogeneous sequences under torque. (A) Fits are shown for Z50, Z22, Bub100, and T100, demonstrating the range of behaviors explained by the model described in SI Text. (B) Visualization of the effect of cooperativity in our model, shown by a series of calculated torque-twist curves, using all the parameters from Z50 except for the domain wall penalty J, which was varied from 0.5 to 12 kcal/mol.