Fig. 1: Design and characterization of a labeled chromatinized origin within 10.4 kbp DNA.
a Structural depictions of the yeast nucleosome (PDB:1ID3), including in blue the mutated residue on the H2A histones for adding fluorescent labels. b (left panel) Schematic of the flow cell used in the single-molecule experiments. (right upper panel) Schematic of the 10.4 kilobase pair (kbp) DNA held in an optical trap that contains the ARS1 origin of replication flanked by two nucleosome positioning sites (NPSs). The DNA is chromatinized via salt gradient dialysis prior to its introduction into the single-molecule flow cell (Supplementary Fig. 1.2). Created with BioRender.com. (right lower panel) Confocal scan showing that signal from H2AAF488 is detected as a single diffraction-limited spot localized at the NPSs. c (left panel) Spatial distribution of H2AAF488on DNA described in panel (b), acquired immediately after introduction into the flow cell and deduced from the blue diffraction-limited spots (Nfoci) collected from 248 distinct DNA molecules (NDNA). Dashed lines indicate the location of the NPSs, and the solid curve indicates the kernel density estimation of the data (PDF: probability density function). (right panel) Stoichiometry distribution of H2AAF488 in the bin containing the chromatinized origin. Data are presented as mean values ± one-sigma Wilson confidence intervals. Filled white circles indicated at left designate the fitted values based on the model described in the Methods and in Supplementary Fig. 1.3. Data in both panels derives from four chromatinized samples (Supplementary Fig. 1.4). Source data are provided as a Source Data file.
