Glyoxal binds to force melted DNA. (a) An extension/relaxation cycle of DNA is shown as a solid/dotted line in black. After stretching DNA in the presence of 500 nM of glyoxal (blue solid line), the DNA is held at the extension marked by the blue arrow for 30 minutes. Upon relaxation (blue dotted line), the strands fail to anneal, as the exposed nucleotides have been modified by glyoxal. A subsequent extension/relaxation cycle (green lines) confirms that the modification is permanent. (b) Relaxation data for DNA stretched to the lengths denoted by the arrows and corresponding to the lengths of approximately ¼, ½ and ¾ of the overstretching transition (in blue, green and red, respectively). The data is fit to a superposition of the WLC and FJC models as described in the text, for data where b_ss > b_ds. The fraction of ssDNA stabilized by glyoxal is 0.22 ± 0.03, 0.33 ± 0.01 and 0.44 ± 0.05 respectively. Figures are adapted from Shokri, et al.65

HMG proteins stabilize DNA melting. (a) Increasing concentrations of HMGB1(box A+B) raise the force necessary to melt DNA. Extension/relaxation data are shown as solid/dotted lines. To emphasize this change, the graph is split; data to the right of the dotted line is expanded on the force axis shown to the right. (b) Increasing concentrations of HMGB2(box A) reveal little change in the melting force until much higher concentrations are added to solution. (c) The averaged midpoint of the melting force for a range of protein concentrations for HMGB1(box A+B) (green) and HMGB2(box A) (blue). Error bars are determined from standard deviations of at least four measured melting curves. Fits to the data yield an equilibrium association constant for HMGB1(box A+B) (Kn = 7.2 ± 1.7 × 10⁸ M^-1) and for HMGB2(box A) (Kn = 0.28 ± 0.10 × 10⁸ M^-1). Figures adapted from McCauley, et al.91

Constant force experiments measure the intercalation rate of a threading intercalator. (a) A pair of covalently linked [Ru(phen)₂dppz]²⁺ molecules yield Δ,Δ-[μ-bidppz(phen)_zRu₂]⁴⁺ (shortened to ΔΔ-P). These complexes initially bind into the major groove, as shown by a predicted model in blue. Over several hours, the complex threads and intercalates into the double helix, resulting in a structure modeled in red. (b) A series of constant force experiments reveal an increase in extension over time as intercalation occurs. The fits shown are to a single exponential dependence on time. (c) Fitted time constants as a function of pulling force. An exponential fit to this data gives a length change due to threading of 0.239 ± 0.025 nm, or the number of base pairs that must melt for each intercalation event must be 1.08 ± 0.11. Extrapolating to zero force gives a reaction time constant of 2.0 ± 0.5 hours. These figures adapted from Paramanathan et al.127

Pol III α polymerase domains bind to single- and double-stranded DNA. (a) A sequence of extension and relaxation cycles (solid and dotted lines) for DNA in 100 nM full length α. The initial extension (blue) shows little change from bare DNA (black), though relaxation shows that the force-melted DNA has failed to reanneal. Subsequent cycles (green, yellow and red) show some protein unbinding and subsequent DNA reannealing, but binding is complete at the final cycle. An increase in the melting force indicates stabilization of dsDNA. The figure is split about the dotted line; data to the right are expanded onto the scale shown. (b) Extension and relaxation cycles (blue, green and red) of N-terminal construct α1-917 show complete annealing upon relaxation and no protein binding to ssDNA. Stabilization of dsDNA is evident, indicating that a dsDNA binding site is contained within the construct. (c) Three data cycles (blue, green, and red) for the N-terminal construct α1-835 show no detectable binding to either ssDNA or dsDNA. Comparing the data from panel (b) and (c) indicates a dsDNA binding site within the residues 836-917. Thus the (HhH)₂ motif is predominantly responsible for dsDNA binding. These figures are adapted from McCauley, et al.64

Finding the equilibrium melting force for proteins that bind to ssDNA and dsDNA. (a) Structure of the fragment of T4 gene 32 protein (gp32) known as *III, which excludes the N-terminal residues 1-20 (present in the fragment *I) and C-terminal residues 254-301 present in the full structure.152 The major lobe, containing an OB-fold, is shown in pink, while the minor, zinc – coordinating lobe is in green. A C-terminal binding flap is shown in red. DNA binds into the region between the two lobes (PDB code: 1GPC). (b) Structure of the fragment of the T7 gene 2.5 protein (gp2.5), which lacks 26 residues at the C-terminus that are believed to participate in the formation of the dimer (shown in blue and violet).151 Each 206 residue protein consists of a β-barrel (OB-fold, in cyan) and an α-helix (dark blue), while the long tail coordinates a calcium atom (yellow). DNA binds into the structure above center, interacting with structurally conserved aromatic residues that surround the basic core of the OB-fold (PDB code: 1JE5). (c) Extension (solid lines) and relaxation (dotted lines) for DNA (black) and DNA in the presence of 200 nM of T4gp32 (*I). The non-equilibrium melting force varies strongly with the pulling rate (250 nm/s in blue, 100 nm/s in green, and 25 nm/s in red). (d) The equilibrium melting force for DNA (black), T4gp32 (*I) in 100 mM Na⁺ (red) and T7gp2.5-Δ26C in 25 mM Na⁺ (blue) are found by holding a constant extension at the midpoint of the melting transition. The lightly shaded curves were collected by pausing during relaxation. Figures (c) and (d) adapted from Pant, et al.45 and Shokri, et al.48

Force-extension experiments manipulate dsDNA as a function of constant force or extension. In this diagram of constant extension experiments, force-extension data are shown as solid symbols followed by relaxation data as open symbols. (a) DNA is tethered between two beads and (b) as the extension is increased, the tension rises (filled blue circles). (c) & (d) At a critical force, base pairing and stacking are disrupted as dsDNA is melted by force and converted into two parallel ssDNA strands. (e) If relaxed, these strands anneal and revert to dsDNA, though some hysteresis is evident (open blue circles). (f) Force-melted DNA may be stretched further until the oppositely labeled strands separate (green, yellow and red). Polymeric models that describe dsDNA (blue line) and ssDNA (green line) are shown and described in the text. All data is collected in 10 mM Hepes buffer, pH 7.5 with 100 mM Na⁺, unless noted.

HMG proteins alter the flexibility of dsDNA. (a) Non-specific binding into the minor groove bends the backbone ∼ 111° in this crystal structure of HMG-D from D. melanogaster,89 which shows a box motif typical of the HMGB family (PDB code: 1QRV). (b) Four sets of extension data for dsDNA (blue circles) and dsDNA in the presence of 3 nM of HMGB1(box A+B) (green circles). The average of these data sets may be fit to the WLC model of Equation 1 (solid blue and green lines). The addition of protein reduces the persistence length from 46 ± 2 nm to 22 ± 2 nm. The contour length and bulk stiffness show less definite changes from b_ds = 0.339 ± 0.001 nm/bp and K_ds = 1200 ± 50 pN to b_ds = 0.339 ± 0.003 nm/bp and K_ds = 939 ± 100 pN for dsDNA with 3 nM HMGB1(box A+B). Fits apply only to data below 45 pN. (c) Fitted persistence lengths as a function of concentration for HMGB2(box A) (blue) and HMGB1(box A+B) (green). Uncertainties are determined from the fits to averaged data sets as shown above. The open circles along the left hand axis are the persistence lengths measured in the absence of any protein, shown as a reference. Fits to the concentration are described in the text and the data determine the equilibrium binding constant per ligand for HMGB1(box A+B) to be Kn = 5.9 ± 1.6 × 10⁸ M^-1 and Kn = 0.15 ± 0.06 × 10⁸ M^-1 for HMGB2(box A). Figures (b) and (c) adapted from McCauley, et al.91

Intercalating ligands characterized by the change in the length of DNA. (a) Averages of the force-extension data for DNA in the presence of varying amounts of the intercalating ligand [Ru(phen)₂dppz]²⁺. Each curve is the average of at least three different extension cycles. DNA lengthens as ligand is added. As the applied force increases the length change increases for a given concentration, indicating that higher forces enable greater binding. (b) Data points are determined from the fractional elongation of DNA as a function of ligand concentration shown in panel a. Solid lines are fits to the data as described in the text. Parameters obtained from these fits are plotted versus force, including the (c) equilibrium association binding constant and (d) binding site size. Higher forces favor binding and reduce the binding site size, which decreases from 2.9 ± 0.1 to 2.6 ± 0.1 base pairs. Fitting the equilibrium association constant to exponential force dependence gives a binding constant in the absence of force of 9.0 ± 1.0 × 10⁵ M^-1. Figures from Mihailovic, et al112 and Vladescu, et al.115

Structure of Pol III catalytic α polymerase subunit from E. coli. (a) Linear structure of α with structural domains indicated from (b) crystal structure of α amino acids 1-917.132 The polymerase core includes the palm, thumb and fingers domains that assume the classical right-hand fold. Residue numbers marking the boundaries of these regions are from the crystal structure (PDB code: 2HNH). (c) Homology model of the C-terminus predicts an OB-fold domain (not present in the crystal structure) for residues 978-1078. ssDNA is shown interacting with the residue F1031.152,162,163 (d) dsDNA is predicted to bind nonspecifically with the residues 833-889, which contains the dual helix-hairpin-helix motif, noted as (HhH)₂ within the sequence map.135,136 Figures (c) and (d) are from McCauley, et al.64

Equilibrium binding to DNA for various constructs of α. (a) Binding to dsDNA is characterized by the change in the observed melting force. Data was collected for full length α (blue), the N-terminal constructs α1-917 (green) and α1-835 (yellow), and the C-terminal construct α917-1160 (red). Error bars are the standard error of four extension cycles. Fits to the binding isotherm described in the text give the equilibrium association constant for full length α as K_ds = 28 ± 7 × 10⁶ M^-1, while the N-terminal constructs α1-917 and α1-835 measured K_ds = 3.5 ± 0.9 × 10⁶ M^-1 and K_ds = 0.4 ± 0.1 × 10⁶ M^-1, respectively. The C-terminal fragment α917-1160 showed little binding and an upper limit was set of K_ds < 0.03 × 10⁶ M^-1. (b) ssDNA binding was quantified by fits to the WLC and FJC models as described in the text. Data was collected for full length α (blue), the N-terminal constructs α1-917 (green) and α1-835 (yellow) and the C-terminal construct α917-1160 (red). Error bars are determined from uncertainties of the fits. Equilibrium association binding constants for full length α showed K_ss = 22 ± 6 × 10⁶ M^-1 and α917-1160, which contains the putative OB-fold region, gave K_ss = 10 ± 3 × 10⁶ M^-1. N-terminal constructs showed little binding to ssDNA. α1-917 and α1-835 were limited to K_ss < 0.1 × 10⁶ M^-1 and K_ss < 0.01 × 10⁶ M^-1, respectively. Figures are from McCauley, et al.64

Determining equilibrium binding to ssDNA and dsDNA for various salt concentrations. (a) Measured equilibrium melting force as a function of protein concentration in 100 mM, 75 mM and 50 mM Na⁺ (data points in green, yellow and red, respectively for T4 gp32 (*I) and violet, blue and cyan for T7 gp 2.5 - Δ26C). Fits shown are to Equation 11. (b) Fitted values of the equilibrium association binding constant (including cooperativity) for T7 gp2.5 (blue) and T4 gp32 (red) to ssDNA. Uncertainties are on the order of the symbols shown. (c) The melting force dependence upon the pulling rate. Selected data shows 50 nM (red), 100 nM (yellow) and 200 nM (green) of gp32 (*I) in 100 mM Na⁺, and 230 nM (cyan), 300 nM (blue) and 460 nM (violet) of gp2.5 – Δ26C in 50 mM Na⁺. DNA in the absence of protein is shown in black. Dotted lines represent fits to Equation 12. (d) Equilibrium association binding constant to dsDNA for T4 gp32 (red) and T7 gp2.5 (blue). Uncertainties are on the order of the symbols shown. Linear fits are shown as solid lines for wild type proteins and dotted lines for truncated fragments. Figures adapted from Pant, et al.44 and Shokri, et al.48