Figure 4

DNA bend angle analyses at DNA lesion sites. (a) Schematic representation of DNA bend angle measurements on a DNA substrate with a lesion incorporated at 50% of the DNA length. The DNA bend angle Θ is obtained as 180°—Φ. (b) DNA fragments in AFM images are selected by thresholding. (c) Selected DNA fragments are skeletonized by tracing the DNA backbone with short (2 nm) rigid segment lines. The contour length of the DNA fragments is automatically determined as the length of the trace (skeleton) line. The 50% position of each DNA is located automatically (blue circle). (d) The angle Φ between tangent lines to points at a user defined distance (here: 8 nm) from the DNA center is automatically measured for each DNA, and the corresponding DNA bend angles Θ are returned by the software. (e) The measured DNA bend angles, here for undamaged DNA, are plotted and a (single or multiple) Gaussian fit to the resulting distribution reveals the conformational state(s) in the system as the center(s) of the Gaussian peak(s). Broad Gaussian widths indicate high flexibility of a conformation. In addition, a shift in bend angles for a DNA lesion substrate compared to undamaged DNA can be indicative of either static distortions, altered DNA bendability, or a release of DNA helix constraints by the lesion. Scale bars: (a) 50 nm, (d) 500 nm.