Journal: bioRxiv
Article Title: Replisome passage through the cohesin ring
doi: 10.1101/2024.10.30.621121
Figure Lengend Snippet: a, Schematic and example kymograph of the DNA replication assay. The DNA template, lagging strand replication product, as well as the globular-coiled leading strand product are indicated. Intensity fluctuations of the latter are likely due to diffusive motion in and out of the TIRF excitation volume. Replication rates in the presence and absence of TCM are shown. Each data point represents a DNA replication segment. Black lines and error bars represent the weighted mean and standard error (* p < 0.0001, unpaired t-test). n is the total number of segments. b, Schematics and representative kymograph of the events during replisome passage through the cohesin ring. First, the replisome approaches cohesin. On encounter, a stretch force transiently builds up (distributed SYTOX Orange intensity increase). Following passage, progressive SYTOX Orange intensity increase results from the two newly synthesised leading and laggings strands. c, The outcomes of replisome-cohesin encounters were aggregated from 4 biological replicates (Replication), 2 replicates (+ Chl1) and 2 replicates (immobilised cohesin), n is the total number of observed encounters. d, Example kymograph of cohesin encounters by replisomes including LD655-labelled CMG. Only cohesin was imaged until 10 min, then excitation was changed to image CMG and DNA. Forces generated in the run-up to jumps were calculated from the DNA stretch factor and jump size. The force is assumed to be negligible if no jump was observed. n is the total number of passage events. d, Comparison of jump sizes and stalling times during CMG-, CMG with cohesion establishment factor-, and replisome-cohesin encounters. Black lines and error bars represent means and standard errors (* p < 0.0001 jump size, p = 0.0038 stalling time, unpaired t-tests).
Article Snippet: All experiments were performed on a Nikon Eclipse Ti2-E inverted TIRF microscope equipped with a SR HP Apo TIRF 100x/1.49 oil immersion objective and a LU-NV-D laser bed.
Techniques: Generated, Comparison
