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. 2023 Mar 20;51(7):3307–3326. doi: 10.1093/nar/gkad186

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Observations of Rep at stalled replisomes. (A) Duration of a pause is decreased at increasing concentrations of Rep. In the presence of 0.25 nM dCas9 only, mean pause duration is determined from fitting a Gaussian distribution function (140 ± 60 s (S.E.M.), n = 25 pauses). In the presence and absence of dCas9-cgRNA1 and Rep-AF647, mean pause duration is determined by fitting single-exponential decay functions to the data (No dCas9-cgRNA1, No Rep-AF647, 5 ± 2 s, n = 57 pauses; dCas9-cgRNA1 and 5 nM Rep-AF647, 80 ± 40 s, n = 22 pauses; dCas9-cgRNA1 and 10 nM Rep-AF647, 20 ± 7 s, n = 92 pauses; dCas9-cgRNA1 and 20 nM Rep-AF647, 20 ± 9 s, n = 80 pauses). Pause durations of titrated Rep conditions represent pauses where a Rep-AF647 intensity was observed above a threshold. (B) Example traces of the number of Rep-AF647 present during a pause as a function of time. Two distinct types are observed; Rep-AF647 intensity above the threshold is reached at t > 0 (top), and Rep-AF647 intensity above the threshold is reached at t = 0. (C) The probability of observing the two Rep-AF647 activities during a pause for each concentration of Rep. Error bars indicate the margin of error for each concentration. (D) Distribution of the wait time (t_w) for a Rep-AF647 molecule to associate to the replication fork in t> 0 events. Mean wait time for each concentration of Rep-AF647 was determined from fitting a single-exponential decay function to the data: 5 nM Rep-AF647, 30 ± 40 s (n= 18 pauses); 10 nM Rep-AF647, 9 ± 3 s (n= 76 pauses); 20 nM Rep-AF647, 3 ± 1 s (n= 78 pauses). (E) Histogram showing distributions of the number of Rep-AF647 molecules that associated first during a pause at t_w reveal predominantly monomeric stoichiometry at all concentrations used. (F) Pause resolve time (t_R) for t= 0 (squares) and t> 0 (circles) events. The mean pause resolve times were determined by fitting a single-exponential decay function to the data of each concentration: 5 nM Rep-AF647, (t= 0) no fit converged (n = 4 pauses), and (t> 0) 50 ± 60 s (S.E.M.); 10 nM Rep-AF647, (t= 0) 12 ± 2 s (n = 31 pauses), and (t> 0) 13 ± 4 s; 20 nM Rep-AF647, (t= 0) 17 ± 7 s (n = 25 pauses), and (t> 0) 20 ± 8 s. Comparison of distributions was conducted by Kruskal-Wallis test for multiple comparisons with Dunn's procedure, where *, ** and **** denote statistical significance with p ≤0.05, 0.01 and 0.0001, respectively. Absence of markers or ns denotes no significance difference (p > 0.05).