Single-molecule kinetics reveal microscopic mechanism by which High-Mobility Group B proteins alter DNA flexibility

Abstract

Eukaryotic High-Mobility Group B (HMGB) proteins alter DNA elasticity while facilitating transcription, replication and DNA repair. We developed a new single-molecule method to probe non-specific DNA interactions for two HMGB homologs: the human HMGB2 box A domain and yeast Nhp6Ap, along with chimeric mutants replacing neutral N-terminal residues of the HMGB2 protein with cationic sequences from Nhp6Ap. Surprisingly, HMGB proteins constrain DNA winding, and this torsional constraint is released over short timescales. These measurements reveal the microscopic dissociation rates of HMGB from DNA. Separate microscopic and macroscopic (or local and non-local) unbinding rates have been previously proposed, but never independently observed. Microscopic dissociation rates for the chimeric mutants (~10 s(-1)) are higher than those observed for wild-type proteins (~0.1-1.0 s(-1)), reflecting their reduced ability to bend DNA through short-range interactions, despite their increased DNA-binding affinity. Therefore, transient local HMGB-DNA contacts dominate the DNA-bending mechanism used by these important architectural proteins to increase DNA flexibility.

Figure 1.

HMGB proteins studied in DNA–protein binding assays. (A) Box A of human HMGB2 bound to cisplatin-modified DNA (PDB: 1ctk) (15). Box B and the linker sequence of the tandem box structure are omitted for clarity. (B) Single-box domain of HMGB1 homologue Nhp6Ap from S. cerevisiae bound to a recognition sequence for the transcription factor SRY (PDB: 1j5n) (14). Dark spheres illustrate partial intercalators methionine in Nhp6Ap, and alanine in HMGB2 (box A), as well as the intercalating residue phenylalanine present in both. (C) Sequences of HMGB2 (box A), Nhp6Ap and two chimeras (termed M1 and M2) based on the box A motif (residues in blue), including cationic residues from the N-terminus of the Nhp6Ap protein (in red—the region of substituted residues is also highlighted) (5). Shaded boxes indicate helical regions, and intercalating residues are shown in black. M1 and M2 were purified with poly(His) tags as shown (in grey italics), which Nhp6Ap proteins lack. Box A of HMGB2 was studied with and without tags.

Figure 2.

Cycles of extension and release for DNA in an optical tweezers experiment. (A) DNA is tethered within a flow cell and extended between functionalized spheres. (B) DNA is extended in a solution containing HMGB protein. (C) Phage-λ DNA (48 500 bp in length) is extended (solid line) and overstretched at ∼60 pN. Release (dotted line) indicates that the process is reversible on the timescales of these experiments. Three sequential cycles of extension and recovery (shown in red, green and blue) illustrate the reproducibility of the data. (D) When DNA is exposed to a solution including 400 nM Nhp6Ap, three extension and release cycles (blue, green and red) reveal consistent changes in the persistence length, contour length and the overstretching force.

Figure 3.

Quantifying DNA–protein elasticity. (A) The extension data (solid symbols) of a typical single extension cycle for DNA (blue diamonds) and DNA in the presence of 400 nM Nhp6Ap (red circles). Vertical bars represent standard error of measurement when larger than symbols. Fits to the WLC model for bare DNA (blue line) provided values of P_ds = 48 ± 2 nm, B_ds = 0.340 ± 0.002 nm/bp and S_ds = 1400 ± 100 pN ( = 0.45). When the DNA was exposed to 400 nM Nhp6Ap (red line), these fitted values became P_ds = 6.7 ± 0.7 nm, B_ds = 0.380 ± 0.002 nm/bp and S_ds = 1240 ± 200 pN ( = 0.37). Residuals to these fits are shown on the right. (B) Fitted HMGB–DNA persistence length, P_ds(c), determined versus concentration of Nhp6Ap (red), constructs M1 (green) and M2 (yellow) and HMGB2 (box A) proteins with and without an N-terminal poly(His) tag (blue and cyan). Symbols represent averages of fitted parameters from three to six fitted data sets. Lines indicate fits to Equations 2 and 3, to determine the equilibrium constant, K_D, and the persistence length of DNA saturated with protein, P_L. (C) The HMGB–DNA contour length, B_ds(c), fit to Equations 2 and 4, to determine the contour length of DNA saturated with protein, B_L. The value of K_D is indistinct from the fits of Figure 3B. All fits assume a fixed binding site size (n = 7) and weak cooperativity (ω = 20), while ranges from 0.5 to 2.0. Fitted parameters for all proteins are summarized in Table 1.

Figure 4.

Quantifying DNA–protein stability. (A) The overstretching force for a DNA molecule (blue circles, triangles and diamonds) rises when that molecule is exposed to 400 nM Nhp6Ap (red circles, triangles and diamonds). The extension data of three contiguous cycles of extension (solid symbols) and release (open symbols) are plotted to illustrate the reproducibility of the data. Averaging extensions over the range of the graph (0.42–0.48 nm per base pair), and over each of the three cycles, the overstretching force is found to be 62.4 ± 0.4 pN in the absence and 70.2 ± 0.4 pN in the presence of 400 nM Nhp6Ap. (B) The observed overstretching force F_ov(c) fit to the site exclusion binding isotherm of Equations 3 and 6 to determine K_D and the overstretching force of DNA saturated with protein, . All fits assume a fixed binding site size (n = 7) and weak cooperativity (ω = 20), while ranges from 0.5 to 2.0. Fitted parameters are found in Table 2.

Figure 5.

Probing the dynamics of HMGB–DNA binding. (A) Cycles of extension and release (solid and dotted lines) for (A) 200 nM HMGB2 (box A) and (B) 200 nM M1 as a function of the rates shown (additional data can be found in Supplementary Figure S4). At pulling rates higher than the natural dissociation rate, DNA becomes torsionally constrained. Release to lower DNA extensions shows multiple plateaus, described in the text. (C) The average overstretching force as a function of the experimental pulling rate, v, for bare DNA (black) and DNA exposed to Nhp6Ap (red), constructs M1 (green) and M2 (yellow) and HMGB2 (box A) (blue) proteins. Data are averaged over active concentrations (c > K_D) as described in the text (and Supplementary Figure S5). Dotted lines are fits to Equation 8, giving the dissociation rate, k_off. Rates from these fits are shown in Table 2. (D) Fits to Equation 1 measure P_ds for bare DNA (black), 500 nM Nhp6Ap (red), 2500 nM M2 (yellow), 4000 nM M1 (green) and 200 nM HMGB2. Dotted lines mark values at the lowest pulling rate (100 nm/s). (E) The measured stiffness, S, of the HMGB–DNA complexes after overstretching, at low pulling rates (<1000 nm/s). (F) The fraction of DNA that is melted by force, f, decreases with increasing protein concentration, as dsDNA is progressively converted into a stable filament that cannot be overstretched at the observed forces.