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Summary

Jia et al. designed biophysical assays to investigate the torsional dynamics of the T7 replisome to show how DNA replication is highly regulated by torsion. They demonstrate that the buildup of torsional stress during replication can lead to replisome stalling, which, if prolonged, significantly impairs the ability to effectively restart replication. The study also highlights the role of the helicase CTD in the exchange of DNAP during a stall, which facilitates the reactivation of the replisome.

Major

1. It is unclear whether the experiments were performed on chromatin or naked DNA strands. If the latter, it is worth mentioning that this is a key limitation of doing these assays ex vivo. (Page 1)

2. Please include information on the length of the DNA you are working with for this analysis. (Page 3)

3. Please provide estimates of both the spatial and temporal resolution available for the system you are using. This can help clarify your results and conclusions.  (Page 3)

4. It would be more helpful if the authors elaborated on their decision to generate the ∆CTD helicase, explain how such modification impacts binding, and describe how its functional properties differ from the wild type. (Page 6)

5. The conclusion drawn on page 6, ‘The synergistic cooperation does not rely on any known specific interactions…’ cannot be fully drawn. While it is clear the CTD does not impact this, it is unknown if there are other interactions. Please clarify this statement. (Page 6)

6. Providing quantitative values associated with claims in the text would strengthen your arguments. For example, on page 4, you state, “As torque increases (mimicking a decrease in topoisomerase activity), the replisome’s pause-free velocity decreases with a concurrent increase in pausing, leading to a significantly lower velocity including pauses.” There are additional times on page 5, first paragraph, page 5, second paragraph, page 7 last paragraph, etc. where the paper could use this.  (Page 5, 7)

7. The findings in Figure 4 would benefit from a clear explanation of what constitutes a “no restart” case, as all results shown were from the “restart” case only. (Page 8)

8. On page 9, provide the number describing what ‘short’ DNA substrate is. (Page 9)

9. The discussion of replisome velocity and frequency of pausing is not well-supported by the results (Fig. 1E). While replisome velocity is shown, it is unclear how the “concurrent increase in pausing” was determined, and there is no data given to support this statement in the main paper. Please clarify how pauses in replication start and stop with the introduction of torsion. (Page 15)

10. Please provide The rationale for presenting a Gaussian curve instead of indicating a standard deviation in Figure 2D should be addressed, as this choice may seem unnecessary to some readers. (Page 17)

11. It is unclear how the results of the fork regression assay (Fig. 3) provide evidence that the ∆CTD replisome regresses using the chicken-foot mechanism. While it is reasonable to assume that regression of the ∆CTD replisome uses the chicken-foot mechanism, as it lacks exonuclease activity, there is insufficient evidence to rule out the possibility of exonuclease activity. Additional condition of Exo- DNAP +  WT helicase is recommended to test this mechanism in Figure 3A. (Page 19)

12. There is no distinction between the energetics of left-handed (L) and right-handed (R) rotations in Figure 3B. The presence or absence of the exonuclease does not guarantee equivalent torque, which should be clarified in the discussion. (Page 19)

13. Discuss additional enzyme roles in-vivo in the discussion section and how this impacts your conclusions.

Minor

1. There is unclear word choice in the sentence “However, even the native full complement of topoisomerases is insufficient to fully relieve torsional stress across the genome.” (Page 2). Does this just mean that although the cells’ natural (native) defense against torsional stress is to utilize (complement with) topoisomerases, this alone is not enough to fully relieve the stress? If so, we suggest replacing the underlined portion with “topoisomerase activity.”

2. The sentence “Subsequently, with the cylinder angle held constant, the replisome rotates DNA, converting the (-) supercoiling to (+) supercoiling which then hinders replication, generating increasing (+) torsion as the replisome proceeds” should be revised. The correct ordering should be 1, Replisome rotation; 2, converting (-) to (+); 3, generate torsion as (+) supercoils build up; 4, the supercoils hinder replication. Now the phrasing implies replication is hindered before the (+) torsion builds up. (Page 3)

3. The term “plectoneme” should be defined to ensure that all readers, regardless of their background, can understand its significance in the context of the study. (Page 5)

4. The term “exo-replisome” is confusing word choice and should be renamed for clarity, perhaps to “-Exo replisome.” (Page 7)

5. To enhance understanding, the manuscript should provide a more thorough explanation of the number of turns for each condition discussed . (Page 8)

6. The last sentence of page 9 is a run-on sentence. Please revise. (Page 9)

7. The phrase “This mimics” on page 10 lacks a clear antecedent and should be revised to improve clarity. (Page 10)

8. Abbreviations in the discussion (SSB, CMG) should be defined for clarity. (Page 11, 13)

9. The difference between (+) and (-) rotation of the cylinder in Figure 1B should be clarified to enhance understanding. Directionalities like counterclockwise and clockwise should be labeled if shown in the diagram. (Page 15)

10. It is unclear how the circles in the graph contribute to Figure 1D. Consider using opacity on the arrow to indicate movement in the circle. Also, completing a rotation with an additional panel and then defining that as “one turn” would improve the connection to the y-axis. (Page 15)

11. The distinction between the dashed lines that indicate the unwinding period and the "no restart" region in Figures 4b-d should be clarified to avoid confusion. (Page 20)

12. There is a missing dashed line in Figure 4c that should be included for completeness. (Page 20)

Competing interests

The authors declare that they have no competing interests.