The reaction mix is deposited on fresh peeled, chemically treated mica (APS [241] on MutSβ-DNA reaction and Ethanolamine [242] on MutSβ-MutLα-DNA reaction), blotted dry, rinsed with deionized water (Sigma-Aldrich), and dried with Nitrogen before transferring to the AFM for imaging. A summary of the experiment design is shown in Figure 4.4. Images are collected on several systems (MFP3D (Asylum Research), NanoScope III and IIIa (Digital Instruments)), at a scan speed of 10µm/s and scan resolution of 3.9µm/pixel. See our methods review paper [242] for detailed AFM deposition protocol. Image analysis is performed using Image Metrics (CHAPTER 3). A typical AFM image of MutSβ-DNA complex and the
structural properties we analyze are shown in Figure 4.5. A detailed description of the analysis using Image Metrics can be found in Section 3.5.3. Notably, the protein-DNA complexes are filtered for some types of analysis (Figure 4.6). A schematic of how the position analysis is performed is shown in Figure 4.7. It should be noted that our analysis measures DNA bend angles that protracts externally from the protein-DNA complex (Figure 4.5), which is indicative but does not actually capture the three-dimensional bend angles internally. The DREEM
DREEM images of MutSβ-DNA complex have been captured (Appendix Figure C.4), showing different external and internal bend angles. We will discuss more of the analysis in the results.
Figure 4.4 Experiment Design
The DNA substrates used are shown on the left pane. Different protein and nucleotide combinations (middle pane) are used to examine different stages (blue boxes) of the repair process. The reaction is timed for a set length of time
Figure 4.5 A Typical AFM Image of a Protein-DNA Complex and Its Analysis
Shown in the image is one DNA with two MutSβ complexes. Measured quantities are labeled and color coded on the image and explained in the side boxes. A. Stoichiometry. Stoichiometric relationships between protein complex and
DNA can be counted directly from distinctive individual complexes on the DNA. Protein stoichiometry within a complex can be measured by volume analysis [84]. B. Position. By measuring DNA profiles (sections along the DNA, see example in the inset), positions of protein complexes can be identified (arrows) to calculate specificity of
protein binding. C. DNA Bending. DNA bending is assessed through deflections of outgoing path from incoming path of DNA through a protein complex. It is an effective way to assess the internal conformations of the protein-
DNA complex. D. Protein-Protein Coordination. DNA-bound protein may interact (‘coordinate’) with another protein through short range (‘association’) or long range (‘looping’) interactions that may be significant in repair signaling. This interaction may be visualized as multi-protein complex or individual complexes that share borders
(‘neighboring’ complexes).
Figure 4.6 Selecting Particles for Stoichiometry and Position Analysis
(Left) Typically, our AFM image contains a mixture of proteins (red dots) and DNAs (blue squiggles). (Middle) To analyze the stoichiometry of protein complexes on a DNA (with at least one protein complex bound), free proteins and free DNAs are filtered (displayed in opaque colors in the figure). (Right) To measure the position distribution of a protein complex on DNA, the DNAs that contain more than one protein are filtered (i.e. only DNAs that are
Figure 4.7 Position Analysis
(A) Once the DNA height profile is plotted (top, adapted from Figure 4.5), the locations of the peaks can be mapped. In the middle, the schematic of the DNA substrate is shown with the slip-out location (marked in red)
matching one of the peaks’ location. A distribution of the peak locations can be plotted as a histogram after analyzing all the particles (bottom). The location of the slip-out will appear as a peak in the distribution. The
dashed line marks the location of the slip-out across the height profile, the DNA schematic, and the position distribution. For our analysis, we look at the position distribution on DNAs with a single protein bound. (B) For unblocked DNA, the protein can land on symmetric points (e.g. a distance of d) from either end of a DNA of length
2L, but only one location can be specific (purple) – other locations are non-specific (pink). Since we cannot distinguish the two DNA ends on unblocked DNA, only the short-arm length (location to the nearest end) of the protein’s location is measured, which ranges from 0 to L (half-length of the DNA). (C) The position distribution is a distribution plot of the short arm lengths. Because of the symmetric sites by measuring short arm length, the specific
binding events (purple) will be an overlay over the non-specific binding events (pink) in the plot (adapted from [150]). The areas that are masked by their respective colors, Asp and Ansp, represent the population of proteins that
specifically bind to the DNA and non-specifically bind to the DNA. The location of the peak frequency, Pmax,
represents the location of the slip-out (the binding site with the highest binding affinity). The location of the base- line frequency, Pmin, represents the average frequency of non-specific binding. The specificity of the specific site (i.e.
the slip-out) can be calculated as 𝑆 = 𝑁 × 𝐴𝑠𝑝
𝐴𝑛𝑠𝑝+ 𝑑 = 𝑁 × (
𝑃𝑎𝑣𝑔
𝑃𝑚𝑖𝑛− 1) + 1, where N is number of binding sites and
Pavg is the average occurrence probability of the whole distribution [150]. Accordingly, the larger the fraction of the specific binding population over the non-specific binding population (𝐴𝑠𝑝
𝐴𝑛𝑠𝑝), the higher the specificity of the