Fig. 2: Spatial distribution of rapidly bound ORCJF646 on 10.4 kbp DNA containing an ARS1 (or mutated) origin (chromatinized or not).
a Tethered DNA molecules are introduced into the protein channel for a 5 s incubation with ORCJF646 and Cdc6 in the presence of ATP, followed by confocal scanning in the buffer channel at an acquisition frequency of one frame every 0.01 min (tf = 0.01 min). Created with BioRender.com. b (i–iv) Schematics of the origin regions, representative fluorescence scans over the full length of the DNA (red: ORCJF646; blue, H2AAF488; note that DNA molecules can be captured in opposite orientations), and spatial distributions of ORCJF646 acquired immediately after incubation in Ch4 and initial illumination in Ch3. Created with BioRender.com. Focusing on the latter: (i) Spatial distribution of ORCJF646 on DNA containing a non-chromatinized origin as deduced from the red diffraction-limited spots (Nfoci) collected from 106 distinct DNA molecules (NDNA-ORC). The dashed lines indicate the location of the NPSs, and the solid curve indicates the kernel density estimation of the data (PDF: probability density function). (ii) Spatial distribution of ORCJF646 on DNA containing a chromatinized origin as deduced from the red diffraction-limited spots (Nfoci) collected from 50 distinct DNA molecules (NDNA-ORC) analyzed and displayed as in (b-i). (iii) Spatial distribution of ORCJF646 on DNA containing a non-chromatinized mutated origin as deduced from the diffraction-limited red spots (Nfoci) collected from 70 distinct DNA molecules (NDNA-ORC) analyzed and displayed as in (b-i). (iv) Spatial distribution of ORCJF646 on DNA containing a chromatinized mutated origin as deduced from the red diffraction-limited spots (Nfoci) collected from 51 distinct DNA molecules (NDNA-ORC) analyzed and displayed as in (b-i). c ORCJF646 occupancy probability for the bins containing the (chromatinized or not) origin in (b-i)–(b-iv), as indicated by the corresponding color bar (ARS1: non-mutated origin; Mut ori: mutated origin; nuc: chromatinized). Nfoci-ARS1 = 145; Nfoci-ARS1+nuc = 67; Nfoci-Mut ori = 100; Nfoci-Mut ori+nuc = 70. Data are presented as mean values ± one-sigma Wilson confidence intervals. Statistical significance is determined by a two-sided binomial test (p valueARS/ARS1+nuc = 4.0 × 10−2; p valueARS1/Mut ori = 4.2 × 10−9; p valueARS1+nuc/Mut ori+nuc = 1.7 × 10−2; p-valueMut ori/Mut ori+nuc = 1.3 × 10−2): * p < 0.05, ****** p < 0.0000005. Source data are provided as a Source Data file.
