(A) Method to track DNA-loop dynamics through space and time. Upper: Schematic of the top view of a DNA-looping event segmented into three regions: region I (orange), region II (green), and the loop region (blue). Middle: Snapshot of DNA-looping event where DNA is labelled using Sytox Orange. Bottom: The integrated fluorescence intensity of the DNA generated by summing the intensity values along the perpendicular axis of the strand. The dashed red line represents a Gaussian fit to the data. Signal values above the fit’s offset define the looped region given in blue; signal values below this threshold correspond to the non-looped regions I and II, given in orange and in green. To convert the signal into DNA length, the integrated intensity of each region is divided by the total summed intensity of the DNA strand and multiplied by the total length of λ-phage DNA (48.5 kb). (B) Dynamics of DNA looping in H3-H4-depleted extract in metaphase and interphase. (Bi,iv left) DNA amount as a function of time computed for the looped region (blue) and non-looped regions I and II (green and orange). The dots represent experimental data and the solid lines represent exponential fits to the data. (Bi,iv right) The redistribution of DNA during the looping events shown in the left panel quantified as the change in DNA content in each region. (Bii,v) Change in the amount of DNA in the looped region and non-loop regions I and II (as in Bi,iv right) for the entire population of meta- and interphase looping events plotted as a function of the initial relative DNA extension of the corresponding molecules. Error bars correspond to standard deviations of data clustered by proximity. Points represent raw data. (Biii, vi) Analysis of loop extrusion symmetry shows predominantly non-symmetric extrusion (symmetry score ~1) in metaphase (Biii) and symmetric extrusion (symmetry score ~0) in interphase (Bvi). (C) Initial growth rates of DNA loop extrusion in metaphase (red) and interphase (blue) as a function of initial relative DNA extension. These rates were obtained from the slopes of the exponential fits to the loop data at time t = 0 for the subset of loop extrusion events that allowed for a fitting that converged within a tolerance (10−8relative change of the cost function), that corresponded to N = 21 out of 30 in interphase, and N = 24 out of 34 for metaphase. Error bars were obtained from error propagation of the uncertainties of the exponential fit parameters. (D) Box plots of the stall forces for DNA loop extrusion in metaphase and interphase obtained from the final relative extension of the DNA strand at the end of loop formation.