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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 3. — Visualization of stalled replication rescue by Rep at the single-molecule level. (A) Schematic representation of single-molecule stalled rolling-circle replication assay. The dCas9-cgRNA1-Atto647 complex is pre-incubated with the rolling-circle DNA template, and further incubated with DnaBC before immobilization to the flow cell. The addition of the replisome components results in the initiation of replication until the dCas9-cgRNA1-Atto647 roadblock has been encountered. Stalled replication is imaged by visualizing the Sytox orange-stained DNA (gray) and dCas9-cgRNA1-Atto647 complex (magenta). (B–F) Example montages and (G) mean DNA product length of (B) rolling-circle DNA replication in the absence of protein roadblocks and Rep proteins (89 ± 5 kbp; n= 81; replication efficiency of 4 ± 1% (S.E.M.)). (C) Stalled DNA replication by dCas9-cgRNA1-Atto647 complex in the presence of all replisome components (2.0 ± 0.1 kbp; n= 80; no replicating products observed). (D) stalled replication rescue by Rep WT following removal of the dCas9-cgRNA1-Atto647 complex (65 ± 4 kbp; n= 75; 2 ± 1%). (E) Stalled replication in the presence of Rep K28A (2 ± 1 kbp; n= 80; no replicating products observed). (F) stalled replication in the presence of Rep ΔC33 (2 ± 1 kbp; n= 80; no replicating products observed). (G) Total DNA product length after 3 min, where bars represent the reported mean ± S.E.M, as listed for (B–F). Comparison of distributions was conducted using one-way ANOVA with Tukey's multiple comparisons post-hoc test, where *** denotes statistical significance with p ≤ 0.001 and absence of markers indicates no significant difference (p > 0.05).