Fig. 2: A single-molecule DNA curtain assay shows ATP hydrolysis-dependent H3–H4 loading onto DNA by Abo1.

a Schematic diagram of the single-tethered DNA curtain assay for histone deposition. In the presence of flow, DNA molecules are aligned and extended at the barrier, and can be visualized by TIRF microscopy. When the flow is stopped, DNA molecules recoil out of the evanescent field. b (Top) An image of DNA molecules (green) stained with YOYO-1. (Bottom) An image of Cy5-labeled H3–H4 (red) loaded by Abo1 in the presence of ATP. c Kymograph of a single DNA molecule in the yellow dashed box in b. When the flow is turned off, the Cy5 fluorescence signals disappear, ensuring that H3–H4 is specifically bound to DNA and not the surface. d, e Cy5-labeled H3–H4 loading onto DNA observed by the DNA curtain assay in the presence (w/ Abo1 + ATP) (d) or absence (e) of Abo1 (w/o Abo1 + ATP). In all images above, black bars left to image and arrows right to image indicate barrier position and flow direction, respectively. Scale bars represent 10 μm length unless indicated. f The ATP hydrolysis-dependence of Abo1 histone deposition activity as shown by DNA curtain assays with flow. g Quantification of histone H3–H4 deposition activity under different nucleotide conditions by measuring the fraction of DNA with bound H3–H4. Error bars represent the standard deviation (SD) of three experiments, and 100–200 molecules per experiment were analyzed for the quantifications.