Characterization of binding-induced changes in dynamics suggests a model for sequence-nonspecific binding of ssDNA by replication protein A

Abstract

Single-stranded-DNA-binding proteins (SSBs) are required for numerous genetic processes ranging from DNA synthesis to the repair of DNA damage, each of which requires binding with high affinity to ssDNA of variable base composition. To gain insight into the mechanism of sequence-nonspecific binding of ssDNA, NMR chemical shift and (15)N relaxation experiments were performed on an isolated ssDNA-binding domain (RPA70A) from the human SSB replication protein A. The backbone (13)C, (15)N, and (1)H resonances of RPA70A were assigned for the free protein and the d-CTTCA complex. The binding-induced changes in backbone chemical shifts were used to map out the ssDNA-binding site. Comparison to results obtained for the complex with d-C(5) showed that the basic mode of binding is independent of the ssDNA sequence, but that there are differences in the binding surfaces. Amide nitrogen relaxation rates (R(1) and R(2)) and (1)H-(15)N NOE values were measured for RPA70A in the absence and presence of d-CTTCA. Analysis of the data using the Model-Free formalism and spectral density mapping approaches showed that the structural changes in the binding site are accompanied by some significant changes in flexibility of the primary DNA-binding loops on multiple timescales. On the basis of these results and comparisons to related proteins, we propose that the mechanism of sequence-nonspecific binding of ssDNA involves dynamic remodeling of the binding surface.

Fig. 1.

Comparison of OB-fold, ssDNA-binding motifs with and without oligonucleotides from three different OB-fold domains. (A,B) Superposition of the backbone of the A and B domains, respectively, from the crystal structure of RPA70AB in complex with the d-C₈-bound state (1JMC; Bochkarev et al. 1997) and in the absence of DNA (1FGU; Bochkareva et al. 2001). (C) The superposition of two subunits from the tetrameric Escherichia coli SSB X-ray structure bound to two molecules of d-C₂₈ (1EYG; Raghunathan et al. 2000) with the same subunits from the free protein (1KAW; Raghunathan et al. 1997). The L₁₂ DNA-binding loops and the L₄₅ loops are labeled in each structure. The figures were generated using MOLMOL (Koradi et al. 1996).

Fig. 2.

Binding of ssDNA induces changes in the structure and dynamics of RPA70A. (A) Changes in the chemical shifts of RPA70A induced by the binding of d-CTTCA. The weighted average of the chemical shift differences for the amide proton (¹H) and nitrogen (¹⁵N) (Evenäs et al. 2001) is given by Δδ_tot = , where ω_HN = 1.0, ω_N = 0.154, and Δδ_HN and Δδ_N are the chemical shift differences of the protein in the two states. The color coding represents residues belonging to three different groups; brown (δ_tot > 0.2 ppm), red (0.2 ppm > δ_tot > 0.1 ppm), and orange (0.1 > δ_tot > 0.07 ppm). Coordinates were extracted from the structure of RPA70AB in complex with d-C₈1 (Protein Data Bank code 1JMC). The figure was generated using MOLSCRIPT (Kraulis 1991). (B) Changes in the ¹⁵N order parameters of RPA70A induced by the binding of d-CTTCA. The radius of the tube is proportional to the value of J(0.87ω_H) calculated for the DNA-free protein. Residues are colored cyan if (S_bound² − S_free²) > 0.1. Residues that posses R_ex > 1.5 Hz in the DNA-bound state are colored red. The figure was created using MOLMOL (Koradi et al. 1996). (C,D) Comparison of RPA70A spectra obtained in the presence of two different ssDNA sequences. Regions from the amide backbone (¹⁵N–¹H HSQC) and the aliphatic side chain (¹³C–¹H CT-HSQC) spectra of RPA70A acquired in complex with d-CTTCA- (black contour lines) and d-C₅-bound protein (red contour lines). The spectra were acquired under conditions identical to those used for the relaxation experiments.

Fig. 3.

Backbone amide nitrogen (¹⁵N) relaxation rates (R₁ and R₂) and ¹H–¹⁵N NOE values for RPA70A in the absence and presence of d-CTTCA. The data were measured at 600 MHz and 298 K. The values for the free protein are represented by the gray squares and for the d-CTTCA complex by black diamonds. The average statistical error in the measurements is <5% for all relaxation parameters.

Fig. 4.

Effect of binding d-CTTCA on the internal dynamics of RPA70A. (A) Square of the generalized order parameter (S²) obtained from a Model-Free fit plotted as a function of residue number. The DNA-bound protein was fit to an isotropic tumbling molecule and the DNA-free state to an equilibrium between isotropic monomer and dimer. The fitting error is <3% for S² values of the free protein (black circles) and <1% for the complex with d-CTTCA (gray squares). Reduced spectral densities at (B) ¹H J(0.87)ω_H, (C) ¹⁵N J(ω_N), and (D) zero frequencies, calculated from the 600-MHz relation data in Figure 3 ▶. Note the large differences in the scales of the Y-axes in each panel. The black circles are data for free protein and gray squares are for the complex.