Figure 8.

MBD2sc exchanges between neighboring mCpG sites demonstrated by NMR and the DNA tightrope assay.A, overlay of ¹H-¹⁵ N HSQC spectra of select amide resonances corresponding to the MBD2 DNA-binding domain (V22, I23, G27, K43) upon binding to DNA with either the first (orange), second (blue), or both (black) CpG sites methylated. With both sites methylated, we observe an averaging of the peak indicating rapid exchange on the order of ≤1 ms. B, schematic drawing of the ligated mCpG-mini DNA substrate used for the DNA tightrope assay. C, representative fluorescence image (top) and kymograph (bottom) of MBD2sc on the mCpG-mini DNA tightrope. Arrows at the bottom pointing to pausing events. D, histogram of the distance between transient pausing events on mCpG-mini DNA tightropes (N = 83). MBD, methyl-CpG–binding domain; CpG, cytosine-guanosine dinucleotide; mCpG, methylated CpG.