Fig. 4: Spatial distribution, stoichiometry, and diffusive behavior of MCM in ATP after HSW.

a The fraction of ORC and MCM complexes that survive an in-situ HSW following incubation in ATPγS (left) or ATP (right). Percentages are the ratio of the total fluorescence of ORC (green) or MCM (red) before and after the HSW. Open circles are individual measurements, while the filled dots and error bars are the sample mean and S.D., respectively. In ATPγS (green), N = 6, whereas in ATP, N = 11. b (i–ii) The spatial distributions of the fluorescent foci, and (iii) the overall stoichiometry distribution. The gray bin labeled “other” accounts for all foci with ORC or MCM stoichiometries higher than 3. c Images and sample time traces that illustrate the motion of foci initially containing one (light red) or two (red) MCM. d Histograms of the diffusion constants of foci containing a single MCM (i) or two MCM (ii). Log-normal fits to the distributions of single (double) MCMs yield mean ± SEM of 0.006 ± 0.002 kbp² s⁻¹ (0.004 ± 0.001 kbp² s⁻¹), taking into account the error bars derived from bootstrapping the data set 100 times. e Summary plot of the diffusion constants derived from the data in d. f (i) Histogram of the diffusion constants of foci containing a single MCM (light red) or two MCM (red) imaged in high-salt buffer at an acquisition frequency of one frame every 120 s. The fitted diffusion coefficient was 0.0023 ± 0.0009 kbp² s⁻¹ (mean ± SEM). (ii) Histogram of the net displacements observed for the same MCM molecules as in (i). g (i) Histogram of the diffusion constants for foci containing dCas9-JF646 imaged in high-salt buffer at the same reduced acquisition frequency, with diffusion coefficient (3 ± 1) × 10⁻⁵ kbp² s⁻¹ (mean ± SEM). By one-way ANOVA, the distributions in f (i) and g (i) are statistically distinct (p < 0.0001). (ii) Histogram of the net displacements observed for the same dCas9 molecules as in (i).