(A) Geometry of the single-molecule unzipping assay. Dashed arrows denote directions of trap movement (20 nm/s) during unzipping (red arrow) or rezipping (black arrow). Two DNA handles connect to the template DNA, which consists of two tandem NPS repeats and an end hairpin. Diagram illustrates nucleosome unzipping, with the second NPS replaced with a pre-assembled mononucleosome. For simplicity, linkers and restriction sites flanking the NPS are not shown. (B, C) Unzipping (red) and rezipping (black) traces of bare NPS DNA (B) and a single WT human nucleosome (C). The presence of the nucleosome on the second NPS causes characteristic high force (20–40 pN) transitions that correspond to disruption of histone-DNA contacts. The unzipped basepairs (bp) are normalized to the beginning of the second NPS. The nucleosome rezipping trace matches that of bare NPS DNA, indicating complete histone removal during unzipping. (D) Representative unzipping traces of tetrasome (cyan), WT (red), H2A.Z (blue), and uH2B (green) nucleosomes. For clarity, only the region after entering the second NPS (corresponding to the boxed region in (C)) is shown, with the unzipped bp normalized to the beginning of the second NPS. The three dashed lines are entry, dyad, and exit of the second NPS, respectively. Rezipping traces, identical to those of B and C, are not shown. (E) Topography maps are plotted as force-weighted residence time (RT) histograms of the unzipping fork along bare NPS DNA, tetrasome and different types of nucleosomes during unzipping at constant trap separation speed of 20 nm/s. The gray histograms with colored stripes (excluding Bare NPS DNA and WT Nucleosome) are residual plots found by subtracting the WT nucleosome RTs. Unzipped bp are normalized to the beginning of the second NPS core. Major peaks are highlighted with gray dashed lines, with the peak positions (in bp) labeled above the peaks. (Left to right: 17, 22, 26, 31, 35, 41, 52, 61, 69, 109, 112, 122 bp). n = 34, 41, 34, 39, 35, 10, respectively for NPS DNA, hWT, H2A.Z, M3_M7, uH2B nucleosome and tetrasome. See also Figure 1—figure supplement 1 for representative unzipping traces and analysis.