Fig. 4: Spatial distribution of stably bound ORC^JF646 on 10.4 kbp DNA containing an ARS1 (or mutated) origin (chromatinized or not).

a ORC^JF646 and Cdc6 are incubated with DNA molecules for 30 min with ATP. Then, the DNA-protein complex is flushed into the single-molecule flow cell, tethered, and transferred to the buffer channel for imaging; t_f: frame acquisition rate. Created with BioRender.com. b (i-iv) Schematics of the origin regions, representative fluorescence scans over the full length of the DNA (red: ORC^JF646; blue, H2A^AF488; note that DNA molecules can be captured in opposite orientations), and spatial distributions of ORC^JF646 immediately after introduction into the flow cell. (i) ORC^JF646 spatial distribution on non-chromatinized DNA, deduced from red diffraction-limited spots (N_foci) using 35 DNA molecules (N_DNA-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) ORC^JF646 spatial distribution on chromatinized DNA, deduced from red diffraction-limited spots (N_foci) using 43 DNA molecules (N_DNA-ORC). (iii) ORC^JF646 spatial distribution on non-chromatinized mutated DNA, deduced from red diffraction-limited spots (N_foci) using 10 DNA molecules (N_DNA-ORC). (iv) ORC^JF646 spatial distribution on chromatinized mutated DNA, deduced from red diffraction-limited spots (N_foci) using 2 DNA molecules (N_DNA-ORC, note that the total number of DNA molecules analyzed with and without ORC^JF646 is 47, see Supplementary Fig. 4.3). 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). Created with BioRender.com (c) ORC^JF646 occupancy probability for the bins containing the origin in (b-i)–(b-iv), as indicated by the corresponding color bar (ARS1: non-mutated origin; Mut ori: mutated origin; nuc: chromatinized). N_foci-ARS1 = 38; N_{foci-ARS1+nuc} = 43; N_{foci-Mut ori} = 10; N_{foci-Mut ori+nuc} = 2. Data are presented as mean values ± one-sigma Wilson confidence intervals. Statistical significance is determined by a two-sided binomial test (p value_ARS/ARS1+nuc  = 2.6 × 10⁻¹; p value_{ARS1/Mut ori}  = 3.4 × 10⁻¹⁴; p value_{ARS1+nuc/Mut ori+nuc}  = 0.0; p value_{Mut ori/Mut ori+nuc}  = 1.0). n.s. not significant, ****** p < 0.0000005. Source data are provided as a Source Data file.