Studying protein-DNA interactions using atomic force microscopy

Abstract

Atomic force microscopy (AFM) has made significant contributions to the study of protein-DNA interactions by making it possible to topographically image biological samples. A single protein-DNA binding reaction imaged by AFM can reveal protein binding specificity and affinity, protein-induced DNA bending, and protein binding stoichiometry. Changes in DNA structure, complex conformation, and cooperativity, can also be analyzed. In this review we highlight some important examples in the literature and discuss the advantages and limitations of these measurements. We also discuss important advances in technology that will facilitate the progress of AFM in the future.

Keywords: Atomic force microscopy; Binding specificity; DNA bending; Protein-DNA interaction; Stoichiometry.

Figure 1. Binding position of mismatch repair proteins on DNA

A. AFM images (1 μm × 1 μm, 2 nm height scale) of wildtype (left) and F39A mutant (right) MutS binding to 738-bp duplex DNA containing a T-bulge. Arrows indicate protein-DNA complexes at different DNA sites: DNA ends (yellow), specific complexes at a T-bulge (red), and nonspecific complexes (pink). Adapted with permission from [28]. B. Distribution of different complexes of MutS, MutL, and MutH on DNA containing a single hemi-methylated d(GATC) site (dashed line). Left, scatter plot of observed complex size vs. relative position on un-looped DNA molecules. Volumes are expressed as the complex volume (V_C) scaled to a streptavidin label (V_SA). Points are colored according to density scalebar; open circles are points that were more enriched than >90% of species of the indicated volume by permutation analysis. Right, distribution of observed occupancy of all protein complexes along DNA (right). Adapted with permission from [29].

Figure 2. DNA bending measurements and Rad4-Rad23-induced DNA bend angle

A. and B. Schematics showing the end-to-end measurement and the tangent method, respectively, for quantifying DNA bending. C. Distributions of Rad4-Rad23-induced DNA bend angles at all internal (white, n = 335) and specific (blue, n = 189) binding sites in the 538-bp fluorescein-dT containing DNA. Gaussian fitting (red curve) is shown for specific binding events only. D. AFM image of Rad4-Rad23 bound to the 538-bp fluorescein-dT containing DNA. White arrows highlight representative binding events. E. Distribution and Gaussian fitting of intrinsic DNA bend angles of the 538-bp DNA duplex containing a fluorescein-dT at 30% from one end (n = 245). Inset: representative AFM image of a 538-bp DNA fragment. C–E. Adapted with permission from [54]. F. Co-crystal structure of Rad4-Rad23 bound to DNA containing a CPD (PDB: 2QSG).

Figure 3. Protein volume and stoichiometry of UV-DDB

A. Schematic showing one method of measurement of AFM volume of a protein particle. Image on right is a cross-section showing heights of image on left. The area, A_p, of a slice is taken at a certain height, h_p, above the background, H. The average height of the particle, h_avg, is determined (not shown). Volume = A_p (h_avg − H). Adapted with permission from [58]. B. Calibration curve relating the molecular weight of a complex to its measured AFM volume, mean ± SD of three separate determinations. The curve was generated using the following proteins in solution: (i) Pot1 monomer (65 kDa), (ii) PcrA monomer (86.4 kDa), (iii) UvrA monomer (105 kDa), (iv) Taq MutS dimer (181 kDa), (v) UvrA dimer (210 kDa), and (vi) Taq MutS tetramer (362 kDa). Linear fit to the data yields V(nm³)=1.471MW(kDa)−7.294 with R²=0.9886. C. Crystal structure of UV-DDB in complex with damaged DNA, showing dimer of dimers in surface representation (PDB: 4E5Z). D. AFM image of wildtype UV-DDB binding to UV-irradiated 517-bp duplex DNA. Pink and blue arrows indicate dimeric UV-DDB binding to one and two molecules of duplex DNA, respectively. E. Distributions and Gaussian fittings (dashed lines) of AFM volume of UV-DDB on one (gray bars, n = 339) and two strands (black bars, n = 79) of duplex DNA. F. AFM image of UV-DDB (K244E) bound to 517-bp duplex DNA as dimer of dimers. G. Distribution of AFM volume of UV-DDB (K244E) on DNA (n = 171). B, F, G. Adapted with permission from [21]. D, E. Adapted with permission from [70].