Fig. 5: Nucleosomes spatially constrain MCMJF646 at the origin. | Nature Communications

Fig. 5: Nucleosomes spatially constrain MCMJF646 at the origin.

From: A chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint

Fig. 5: Nucleosomes spatially constrain MCMJF646 at the origin.The alternative text for this image may have been generated using AI.

a ORC, Cdc6 and MCMJF646/Cdt-1 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; tf: frame acquisition rate. b Schematics of the origin regions, representative fluorescence scans over the full length of the DNA (red: MCMJF646; blue, H2AAF488; note that DNA molecules can be captured in opposite orientations), and spatial distributions of MCMJF646 acquired immediately after introduction into the flow cell. (i) MCMJF646 spatial distribution on non-chromatinized DNA. 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) MCMJF646 spatial distribution on chromatinized DNA. (iii) MCMJF646 spatial distribution on non-chromatinized mutated DNA. (iv) MCMJF646 spatial distribution on chromatinized mutated DNA. c MCMJF646 occupancy probability for the bins containing the (chromatinized or not) origin for the datasets in (b-i)–(b-iv), as indicated by the corresponding color bar (ARS1: non-mutated origin; Mut ori: mutated origin; nuc: chromatinized). Nfoci-ARS1 = 50; Nfoci-ARS1+nuc = 97; Nfoci-Mut ori = 115; Nfoci-Mut ori+nuc = 96. 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   =  1.3 × 10−5; p valueARS1/Mut ori   =  7.3 × 10−2; p valueARS1+nuc/Mut ori+nuc   =  1.7 × 10−14; p valueMut ori/Mut ori+nuc  = 3.4 × 10−1): n.s. not significant, **** p < 0.00005 and ****** p < 0.0000005. d ORC, Cdc6, and MCMJF646/Cdt-1 are incubated with DNA molecules as in (a) but at an acquisition frequency of one frame per minute (tf = 1 min). e Histograms of the relative displacements between 2 min intervals (Δt = 2 min) for (i) dCas9JF646, (ii) for MCMJF646 on DNA containing a non-chromatinized origin or (iii) chromatinized origin. Nfoci, number of molecules; NΔx, number of relative displacements; σ, standard deviation. Source data are provided as a Source Data file. Created with BioRender.com.

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