Srs2/PARI DNA helicase mediates abscission inhibition in response to chromatin bridges in yeast and human cells

  1. Monica Dam
  2. Nicola Brownlow
  3. Audrey Furst
  4. Coralie Spiegelhalter
  5. Manuel Mendoza

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Abstract

The coordination of chromosome segregation with cytokinesis is crucial for maintaining genomic stability. Chromatin bridges, arising from DNA replication stress or catenated chromosomes, can interfere with this process, leading to genomic instability if not properly managed. Here, we uncover that the budding yeast DNA helicase Srs2 and its human homolog PARI delay the timing of abscission events in the presence of chromatin bridges. We demonstrate that Srs2 is essential for delaying abscission in yeast cells with chromatin bridges, and preventing their damage by cytokinesis. In human cells, PARI similarly plays a key role in delaying abscission events, such as midbody severing and actin patch disassembly during cytokinesis, in response to chromatin bridges caused by topoisomerase II inhibition. Our results also show that PARI functions within the Aurora B-mediated abscission checkpoint pathway. These findings reveal an evolutionarily conserved role of the Srs2/PARI DNA helicase in maintaining genomic integrity by modulating abscission timing in response to chromatin bridge formation.

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    Reply to the reviewers

    Response to Reviewers

    We thank the three reviewers for their insightful and constructive comments, which have helped improve the manuscript. Our replies to each comment are provided below.

    Reviewer #1

    __Evidence, reproducibility and clarity __

    The abscission checkpoint, also known as NoCut, is a genome protection mechanism that remains poorly understood. This pathway is conserved from yeast to humans and protects the genome against chromosome bridges, a dangerous missegregation event that can have catastrophic consequences on genome stability. Dam et al now report the role of Srs2, a DNA helicase, as a key factor in the abscission checkpoint. The authors establish Srs2 as bona fide factor in this pathway by showing its involvement in abscission delays when chromatin bridges are induced. Importantly, yeast defective for Srs2 show increased levels of DNA damage when the frequency of chromatin bridges is increased. The authors also provide genetic evidence supporting a model whereby the interaction of SrS2 with PCNA s required for abscission regulation. In the second part of the manuscript, the authors study the human homologue of SRS2, PARI, in abscission regulation. The manuscript provides convincing evidence that PARI is also required for abscission delays in the presence of chromatin bridges. Critically, this role is specific for chromosome missegregation as abscission delays in response to nucleoporin depletion remain intact in PARI-depleted cells. Thus there is a conserved requirement for these DNA helicases in the abscission checkpoint.

    Overall, these are important advances in our understanding of the abscission checkpoint. The data is high quality and convincing in general. However, the impact of PARI depletion on genome stability needs to be further demonstrated to support key claims in the manuscript. Specifically:*

    Disruptions of the abscission checkpoint in human cells result in bi-nucleation or increased levels of DNA damage. In this context, the authors need to show that PARI-depleted cells with increased frequency of chromatin bridges exhibit increased levels of bi-nucleation, DNA damage or both.

    We thank the reviewer for its positive assessment of our work. While our data establish that Srs2 inhibits abscission to prevent DNA damage in yeast, we agree with the reviewer that we have not tested the consequences of PARI loss on DNA damage or cytokinesis failure in HeLa cells. We will address this in the revised version of our study.

    Significance

    The abscission checkpoint, remains poorly understood. There is evidence in the literature that disruptions in this pathway increase susceptibility to cancer. The identification of the Srs2/PARI helicases as key components in this pathway is a considerable step forward in this field.

    Reviewer #2

    __Evidence, reproducibility and clarity __

    The Aurora B-mediated abscission checkpoint ("NoCut" in yeast) prevents tetraploidization or chromatin breakage in the presence of chromatin bridges in cytokinesis and the mechanisms of its activation are a matter of active investigation. In the present study, Dam et al propose that the conserved Srs2/PARI DNA helicase is required for the activation of the abscission checkpoint in response to chromatin bridges generated by DNA replication stress or topoisomerase inhibition. This is a timely and very interesting topic and the potential identification of a novel regulatory protein that activates the abscission checkpoint would be important. However, in my opinion, some Figures are of relatively low quality and need improving, there are apparent discrepancies between data and important control experiments are missing, which preclude the reader from fully evaluating the conclusions of this study. Some direct evidence of the role of Srs2/PARI on DNA bridges is also required. Also, it would be nice to investigate mechanistic details of the potential Srs2/PARI functions in the abscission checkpoint, and how it fits with other recently published signaling pathways that activate the abscission checkpoint in cytokinesis.

    Specific comments:

    1. The DNA channel (Ht2B-mCherry) in Figure 1A is of very low quality to be able to verify the authors interpretations of when the individual chromatin bridges are resolved (probably broken). For example, in the WT movie, they claim that the bridge is intact in frames 10 min and 14 min (yellow arrow) and that the bridge is resolved at 16 min (asterisk); however, I'm not convinced this is the case, because I can only see a very small portion of the bridge already at the 10 min and 14 min time-points. In my opinion, this bridge could have been broken much earlier, probably at 10 min. Also, WT +HU, is this bridge really intact at 10 min and at 14 min? In Srs2Δ + HU, the bridge appears broken to me much earlier, perhaps at 30 min. There is a distinct possibility that the authors could not calculate the resolution times accurately from these movies (please also see my next comment, #2). The authors could perhaps use a more sensitive bridge marker such as GFP-BAF.

    To clarify our approach, chromosome segregation was considered complete only when bridges were no longer detectable, while discontinuous or faint bridges were still classified as unresolved, as stretched DNA may result in weak nucleosome signals. This definition aligns with the bridge resolution times reported in Figure 1B-E. To improve clarity, we have revised the Results section to specify our classification criteria, and added all frames from the time-lapse movies in Figure 1A as a new figure (Supplementary Figure S1).

    In Figure 1B, they conclude that Srs2Δ cells treated with HU exhibit increased time from anaphase onset to bridge resolution compared with WT or Srs2Δ cells. This result appears at odds with data from Fig. 2C showing that Srs2Δ+HU finish abscission at similar times to WT or Srs2Δ cells as judged by plasma membrane morphology. (final cut). Given that the final cut of the plasma membrane should cause chromatin bridges to break, if Srs2 is required for an abscission delay in response to HU-induced chromatin bridges, I would expect Srs2Δ + HU cells to exhibit accelerated plasma membrane cut and also faster chromatin bridge resolution compared with controls. This discrepancy could at least in part be caused by the relatively low quality of movies used for the calculations in Fig. 1.

    This is a perceptive point. To clarify, we analyzed the timing of chromosome segregation, membrane ingression at the abscission site, and abscission relative to anaphase onset, as shown in the new Supplementary Figure S2. In HU-treated cells (both WT and srs2∆), bridge resolution and membrane ingression occur around the same time (~10 minutes after anaphase onset), with srs2∆ cells exhibiting slightly earlier membrane contraction. This suggests that bridges resolve during cytokinesis (see also our reply to the next comment) but does not distinguish whether they break prematurely or resolve normally. Our key finding is that membrane abscission is delayed in HU-treated cells in an Srs2-dependent manner, raising the question of whether this delay is important to prevent bridge breakage. This hypothesis is tested and supported by Figure 2D, where delaying cytokinesis (via cyk3∆) reveals the protective role of Srs2.

    Fig. 2 shows faster abscission times (membrane cut) in Srs2Δ+HU cells compared with WT+HU. The authors interpret this data as evidence for a role of Srs2 in abscission delay in response to HU-induced chromatin bridges (page 7 and elsewhere). However, there is no direct evidence that the cells analyzed in Fig.2 exhibited DNA bridges in cytokinesis. One could argue that HU-induced DNA replication stress caused DNA lesions at the nuclear chromatin, which affected completion of cytokinesis in the absence or presence of Srs2. What proportion of HU-treated cells in cytokinesis exhibit DNA bridges? Judging from Fig. 1D this could be as low as 0-20%. The authors should analyze HU-treated cells that clearly exhibit DNA bridges, either by live-cell imaging or in fixed cells experiments. As it stands and together with my previous comments #1 and 2, I'm not convinced this data fully supports a role for Srs2 in the abscission delay in response to HU-induced DNA bridges.

    We appreciate the reviewer's concern. The presence of chromatin bridges in HU-treated cells during cytokinesis (membrane ingression) is documented in the new Supplementary Figure S2, as noted in our response to the previous comment. Additionally, our previous study (Amaral 2016, PMID: 27111841, Figure 1D) demonstrated that under the same HU treatment conditions used here, >90% of wild-type cells exhibit chromatin bridges during cytokinesis. This strongly supports the conclusion that the effects observed in Figure 2 are linked to the presence of DNA bridges.

    In Fig. 2D, there is no evidence to support that Mre11 foci are caused by bridge breakage, and not by replication-stress induced DNA lesions at the main nucleus (no DNA bridge is evident, also see comment #3).

    The use of the cyk3 mutant in Figure 2D specifically addresses this concern. If Mre11 foci resulted from replication stress-induced lesions in the main nucleus, delaying cytokinesis should have no impact on damage levels. However, we observe that delaying cytokinesis via the cyk3 mutation significantly reduces Mre11 foci, strongly suggesting that these foci arise from chromatin bridge breakage rather than replication stress, and that delaying cytokinesis provides extra time to solve the chromosome segregation problem. This conclusion is further supported by previous studies showing that cyk3∆ delays cytokinesis (Amaral 2016, PMID: 27111841, Figure 2C; Onishi et al. 2013, PMID: 23878277). We have clarified this point in the revised text.

    Figure 3: the authors use a top2-4 mutant strain to generate DNA bridges from catenated DNA and investigate the potential role of Srs2 in the abscission delay. However, no DNA bridges are obvious in the cells shown in Fig. 3. What proportion of top2-4 mutant cells in cytokinesis exhibit DNA bridges? Does this explain the striking difference in the percentage of cells that haven't completed abscission after 30-60 min in WT+HU vs Top2-4 cells? Please also see my previous comments above.

    The *top2-4 *mutant is well-characterized, and under the conditions used here, 100% of cells exhibit DNA bridges during cytokinesis (see for example Amaral et al., 2016, Figure 3A). We have clarified this point in the revised text. Notably, previous work has shown that top2-4-induced bridges are thicker and more persistent than those caused by HU-induced replication stress. This difference might contribute to the more severe abscission defect observed in top2-4 cells, though we have not directly tested this.

    The authors propose that association of Srs2 with PCNA is required for complete inhibition of abscission in top2-4 mutant cells with chromatin bridges. Assuming a role for Srs2 in abscission timing in cytokinesis with chromatin bridges is fully proven, it is essential that the authors also investigate the localization of Srs2 and PCNA on chromatin bridges, using GFP-tagged proteins or appropriate antibodies in fixed and/or living cells. This would suggest a direct role of these proteins on chromatin bridges and considerably strengthen the authors hypothesis. Alternatively, Srs2 and PCNA may indirectly affect abscission timing through their well-established roles at nuclear chromatin.

    The perturbations used in Figure 4 have been previously shown to disrupt Srs2-PCNA and PCNA-chromatin interactions (Armstrong et al., 2012; Ayyagari et al., 1995; Johnson et al., 2016; Kubota et al., 2013), as referenced in our manuscript. Given this well-established evidence, we believe additional imaging experiments would be redundant. Moreover, we do not claim that Srs2 or PCNA must specifically localize to chromatin bridges for NoCut function. Instead, our data demonstrate their genetic requirement for abscission inhibition in the presence of bridges. Whether these proteins localize exclusively on bridges or more broadly on chromatin remains unresolved, a point we explicitly discuss in the manuscript.

    In Fig. 4D, the authors show an abscission delay in elg1Δ mutant cells in the presence of dicentric bridges compared with cytokinesis without bridges and interpret this as evidence that artificially retaining PCNA on dicentric chromatin bridges is sufficient to inhibit abscission. It is important that the authors demonstrate that PCNA localizes to dicentric bridges in elg1Δ mutant, but not in ELG1 control, cells, e.g., by immunofluorescence, to support their claim and their proposed model.

    As noted in our previous response, the association of PCNA with chromatin throughout the cell cycle and its regulation by Elg1 have been extensively characterized in prior studies. Given this established evidence, additional imaging experiments would be redundant.

    We also clarify that we do not claim that PCNA is specifically retained on chromatin bridges in elg1Δ mutants. Rather, our model is based on the overall retention of PCNA on chromatin in elg1Δ cells, as demonstrated in published studies.

    Notably, elg1Δ mutants without dicentric bridges retain PCNA on chromatin but do not exhibit delayed abscission. However, only elg1Δ mutants with chromatin bridges inhibit abscission, indicating that PCNA retention alone is not sufficient—it is the presence of a bridge with retained PCNA that is critical. This distinction has been clarified in the revised manuscript.

    In Fig. 5, the authors claim that HeLa cells treated with the Top2 inhibitor ICRF193 exhibit delayed midbody resolution compared with controls and that depletion of PARI by siRNA accelerates abscission in ICRF-treated cells. They interpret this as evidence for a role of PARI in the abscission delay in response to ICRF-induced chromatin bridges. However, no bridges are visible at any time-frame in cells in Fig. 5B raising the possibility that the observed time-differences are due to some effect of ICRF in cytokinesis without bridges. I'm also not convinced that in Fig. 5B the midbodies in NT/ICRF/230 min, siPARI/DMSO/110 min and siPARI/ICRF/150 min were resolved as indicated by the authors, as I can definitely see both midbody arms very clearly in these photos. The p-values are also just below the p

    We acknowledge that the chromatin bridges in Figure 5B are challenging to visualize and may appear discontinuous. This is not due to poor image quality but likely reflects the low chromatin density of these structures. To clarify this, we now include magnified and contrast-enhanced images to better highlight the bridges, and quantification in Fig. 5C. Additionally, in the revised manuscript, we will provide new images using GFP-BAF, which directly binds DNA, to more clearly demonstrate the presence of chromatin bridges in ICRF-treated cells. These data will confirm that most cytokinetic cells in ICRF-treated conditions exhibit bridges.

    Regarding the midbodies shown in Figure 5B, the presence of one or both arms intact does not indicate unresolved abscission but rather that the midbody has been severed, a distinction we explicitly describe in the manuscript.

    Concerning the statistical analysis, we note that the p-value threshold of 0.05 is a widely accepted convention for statistical significance, and we have applied it appropriately in our analysis.

    Finally, regarding the EM images in Figure 5C, these are single-section images, which do not allow us to determine definitively whether the bridges are physically broken when they appear discontinuous. It is possible that portions of the bridge extend outside the sectioned image. Regardless, we do not claim that these bridges are intact or broken. Rather, our key conclusion is that their presence at the abscission site in ICRF-treated cells is not affected by PARI knockdown, supporting our model.

    In Fig. 6, the authors examine actin patches in PARI-depleted and control cells as a marker of abscission. Although a role for PARI in actin patch formation would be very interesting, I'm not sure how it fits with the present story. The actin inside the intercellular canal described by Bai et al (removal of which correlates with abscission) appears very different to the accumulations of actin at the base of the intercellular canal described by Sreigemann et al and by Dandoulaki et al. I can definitely see actin patches (similar to the ones in Steigemann et al) in Fig. 6 NT/ICRF, but I can't see any at the other treatments (I disagree with the arrows). Incidentally, I can see a DNA bridge only in NT/ICRF, but not in the other treatments.

    We have revised our description of this figure for greater clarity. In control cells, actin accumulates at the cleavage furrow during anaphase and gradually disperses (clears) as cytokinesis progresses. We do not see patches in untreated cells, and we have updated the y-axis label in Figure 5B from “% of cells with actin patches” to “% of cells with actin clearance” to better reflect our observations.

    Actin patches were observed only in ICRF-193-treated cells and were often associated with chromatin bridges. Cells that successfully disassembled these actin patches were classified as having completed actin clearance. Our data indicate that PARI depletion increases the fraction of cells that clear chromatin from the division plane, facilitating actin patch disassembly.

    The actin patches observed in our study closely resemble those reported by Steigemann et al., and notably, we used the same cell line as in that study. Regarding Bai et al., they used both phalloidin and actin-GFP. For example, Figure 5C in Bai et al., shows examples of both actin patches near chromatin bridges, which resemble those in our study, and filamentous actin structures within the intercellular canal, which appear distinct.

    Finally, a bridge fragment lacking actin patches is visible in PARI knockdown cells treated with ICRF, and we have now highlighted this in the revised figure.

    1. Midbody resolutions are clearer in Fig. 7, perhaps with the exception of siPARI/DMSO. However, no DNA bridges are visible, raising again the possibility that the authors investigate effects in cytokinesis without DNA bridges.

    See our response to point 8: while bridges are difficult to visualize, our analysis confirms that ICRF treatment induces bridges that persist during cytokinesis.

    Can the authors investigate whether the helicase activity of PARI is required for the abscission checkpoint, by depletion-reconstitution experiments with a helicase-mutant protein?

    PARI lacks detectable Walker motifs and associated ATPase activity, suggesting PARI lacks helicase activity (Moldovan et al., 2012). Therefore, we have not pursued depletion-reconstitution experiments with a helicase-mutant protein.

    The authors should investigate localization of PARI to the midbody/ DNA bridge in cytokinesis with chromatin bridges. Recent reports have proposed that a Top2-MRN-ATM-Chk2 pathway activates the Aurora B-dependent abscission checkpoint in human cells (PMIDs: 37638884, 33355621). The authors should examine localization of Aurora B and some of the above proteins in control and PARI-deficient cells to establish if/how PARI fits in the above pathway.

    As noted in our manuscript, we attempted to visualize PARI at midbodies and DNA bridges but were unable to detect any signal. This could be due to either its absence in these regions or its low concentration, making detection challenging.

    We agree that investigating the Top2-MRN-ATM-Chk2 pathway in this context is important. We will examine the localization of key pathway components, including Aurora B, in control and PARI-deficient cells, and include the results in the revised manuscript.

    1. The authors use ICRF to generate chromatin bridges. If ICRF is continuously present in their assays, one would expect it to inhibit Top2 and impair the abscission checkpoint (PMIDs: 37638884, 33355621). How do the authors reconcile this with their proposed model?

    This is an important point. Studies from the Zachos lab have shown that Topoisomerase IIα-DNA covalent complexes (Top2ccs) accumulate near the midbody in cells with chromatin bridges and play a key role in initiating abscission checkpoint signaling by recruiting MRN, ATM, and Aurora B. Supporting this model, ICRF-193 treatment does not alter midbody disassembly timing in HeLa cells, as shown in Petsalaki et al., 2023 (Figure S4D).

    However, our results indicate that ICRF-193-treated HeLa cells exhibit delayed midbody severing, suggesting that at least some aspects of abscission checkpoint signaling remain active under these conditions. One possible explanation for this discrepancy is the difference in ICRF-193 concentration: our study uses a low dose (250 nM) versus 10 µM in the Zachos group study. We favor the hypothesis that this lower dose preserves sufficient Top2 activity to support some level of checkpoint signaling while still effectively generating chromatin bridges.

    Additional comments:

    Page 8: "Although SIM-defective Srs2 has a lower affinity to SUMOylated PCNA, it can still interact with PCNA". The authors should test this experimentally or provide appropriate references supporting this claim.

    We have clarified our statement and provided the reference: Although SIM-defective Srs2 has a lower affinity to SUMOylated PCNA, it can still interact with non-SUMOylated PCNA (Armstrong et al. 2012).

    1. Page 6: "Deletion of SRS2 further increased the fraction of anaphase cells with RPA foci, rising to approximately 30% in the absence of HU..."; however, this rise was not statistically significant as indicated in Fig. 1C.

    Thank you for noting this - we have removed this statement.

    Fig. 1C, D: SDs are missing. Fig. 1E: please show the p-values.

    These data in Figures 1C-D represent percentages from cells pooled from two independent experiments with similar results. P-values were calculated using Dunn’s multiple comparison test. Standard deviations are not applicable in this case. We have included the p-values for Figure 1E.

    Fig. 2D: please show SDs and individual values.

    These data represent percentages from cells pooled from independent experiments with similar results. P-values were calculated using Fisher’s exact test. Standard deviations and individual values are not applicable in this case.

    1. Why do the authors show the spindle pole body in their movies?

    We do this to infer the time of anaphase onset; see our response to points 1-3 and Fig. S2.

    Fig. 4A: WT and top2-4 cells have the same symbol in the graph.

    We have changed the symbols.

    Significance

    Strengths: potentially novel regulator of the abscission checkpoint. Timely and interesting topic of broad scientific interest.

    Limitations: problems with quality of some data and withy the interpretation. Also, more mechanistic evidence is required to significantly advance our knowledge in the field.

    Reviewer #3

    Evidence, reproducibility and clarity:

    Summary: Building on the specific connection between DNA bridges that bear marks of replication stress and the NoCut checkpoint (Amaral 2016, 2017), which prevents completion of cytokinesis, Dam et al. test the helicase Srs2/PARI for a role in this checkpoint pathway. The authors have produced a thorough study investigating the role of this helicase in both yeast and mammalian cells in the presence of DNA bridges. The manuscript includes clear evidence that Srs2 is important to resolve chromatin bridges, remove replication protein A (RPA) from chromatin, and delay cytokinesis under replication stress. Further, the authors show that loss of Srs2 under replication stress increases DNA damage, marked by elevated MRE11 foci in a manner dependent on cytokinesis (i.e., dependent on Cyk3). Srs2 deletion also partially abrogates the abscission delay seen upon topo-II inactivation. They further report that Srs2 must interact with PCNA to delay abscission in S. cerevisiae. While chromatin bridges formed when a dicentric chromosome is present escape detection by the NoCut checkpoint, inactivation of Elg1, which unloads PCNA and associated factors following DNA replication, results in delayed abscission. In HeLa cells, the Srs2 ortholog PARI is shown to similarly help promote abscission delay in the presence of DNA bridges following topoisomerase inhibition, as loss of PARI through siRNA knockdown prevents this abscission delay. Mechanistically, when PARI levels are reduced in HeLa cells, actin patches that function to stabilize the midbody and protect DNA bridges do not form/persist robustly as in cells with intact PARI. Consistent with a specific role in sensing the presence of a DNA bridge, depletion of PARI did not impact abscission checkpoint activity in response to depletion of the NPC component, Nup153. Finally, the authors show that PARI depletion reduced time to abscission to the same extent as treatment with an Aurora B inhibitor, and PARI depletion in conjunction with Aurora B inhibition did not reduce abscission timing further than singular treatments, suggesting that PARI works within the Aurora B-mediated NoCut signaling cascade.

    Major comments: The manuscript is well written and, in general, the conclusions are thoroughly supported, but there are a few recommendations for addition or revision.

    The first of these is for a more thorough introduction of helicases potentially involved in cytokinesis and more clear rationale for why the focus is on Srs2.

    We appreciate the reviewer’s suggestion and have expanded the introduction to better contextualize helicases in cytokinesis and clarify our focus on Srs2.

    Figure 1 E lacks statistical analysis. In addition, the text referring to 1E leads to confusion because the distinction between "RPA foci during anaphase" and "RPA coated chromatin bridges" is not made clear. The authors should clarify that the data presented in 1E shows quantification of cells with RPA foci during anaphase, not RPA coated chromatin bridges, and use consistent wording between the text and figure/figure legend. Further, how cells with RPA foci were identified, and what is classified as an RPA focus from images should be described in the methods.

    We appreciate the reviewer’s feedback. In the revised manuscript, we have included statistical analysis for Figure 1E and clarified the distinction between "RPA foci during anaphase" and "RPA-coated chromatin bridges" to ensure consistency. Additionally, we have updated the Methods section to specify how cells with RPA foci were identified and what criteria were used to classify RPA foci based on the imaging data.

    In some cases, it is unclear whether DNA bridge formation is prevented vs aberrantly broken. For example, under Top2 inactivation, does the absence of Srs2 prevent bridge formation or promote their breakage along with premature midbody abscission? Confirming the frequency of chromatin bridge formation would address this and, further, monitoring RPA persistence would validate whether RPA clearance from bridges is consistently correlated with Srs2 activity (an interesting observation from Figure 1 that is not followed up on). Similarly, other conditions that appear to interfere with abscission delay (e.g., disrupting Srs2-PCNA interaction) should be monitored for whether the formation of DNA bridges has been altered.

    We agree this is important and will address it in a full revision. We will quantify chromatin bridge formation under Top2 inactivation to determine whether Srs2 mutations affect bridge frequency or stability. Additionally, we will monitor RPA persistence in top2 cells to assess whether RPA clearance correlates with Srs2 activity. While we find it unlikely that bridge formation is prevented by srs2 mutations, as Top2 is essential for decatenation, our experiments will directly test this possibility.

    In Figure 4A, the data show that the PIP-box is required for timely abscission. Imaging data from yeast strains with the PIP-box deletion alone should be included, rather than only showing the deletion in combination with the SIM deletion.

    We agree with the reviewer’s suggestion, and will include imaging data from yeast strains with the PIP-box deletion alone in the revised manuscript.

    While the authors state that PARI and PCNA were not detectable at bridges in mammalian cells, it would be worth examining whether RPA is persistent on DNA bridges in mammalian cells depleted of PARI to understand how closely this pathway resembles the features found in yeast.

    Here too, we agree with the reviewer’s suggestion, and will include imaging data from HeLa cells visualizing RPA in the revised manuscript.

    In Figure 6, the authors should describe in the methods how cells with actin patches were identified and quantified and explain what criteria must be met to be identified as an actin patch. Actin patches were described as "disassembling more quickly" in PARI-depleted cells, but the images look as if actin patches are not forming properly in these cells. The images are crisp and clear, but a change in wording may be necessary to accurately describe the data.

    Thank you for pointing this out. We agree that the wording was confusing (see our reply to reviewer 2, comment 9) and have revised our description of this figure for greater clarity. In control cells, actin accumulates at the cleavage furrow during anaphase and gradually disperses (clears) as cytokinesis progresses. We do not see patches in untreated cells, and we have updated the y-axis label in Figure 5B from “% of cells with actin patches” to “% of cells with actin clearance” to better reflect our observations. Actin patches were observed only in ICRF-193-treated cells and were often associated with chromatin bridges. Cells that successfully disassembled these actin patches were classified as having completed actin clearance. Our data indicate that PARI depletion increases the fraction of cells that clear chromatin from the division plane, facilitating actin patch disassembly.

    Minor suggestions to improve the manuscript are:

    Include a diagram that shows hallmarks of cell division and what is being tracked in particular assays (e.g., DNA bridge duration vs time to abscission).

    Thank you for this suggestion, which we have implemented in Figure S2A.

    In the elegant CLEM experiments presented in Figure 5, organelle labels could be added to orient the readers.

    We added organelle labels to CLEM images.

    The data in supplemental Figure 2 should be moved to Figure 5. The fact that there are similar levels of chromatin bridges is vital information and stresses that the defect lies in detection and response to the bridge as opposed to formation of bridges when PARI is depleted.

    We agree, and have moved Figure S2 to Figure 5 (now Figure 5C).

    Significance

    The link between DNA bridges and NoCut/abscission checkpoint signaling is a fundamental aspect of cell cycle regulation. This manuscript makes a significant contribution to our understanding of this pathway by introducing a novel role for the helicase Srs2/PARI in execution of an abscission delay in the presence of DNA bridges. This is an important contribution as there is sparse information about cellular factors that mediate detection and response to DNA bridges, which is vital to protecting genome integrity. Although, as the authors themselves state, "the molecular mechanisms by which Srs2 and PARI function in NoCut remain unclear," this study, with some revisions, merits publication as it reveals a conserved role for a factor in this important response pathway and provides new insights into why certain DNA bridges (i.e., bridges formed by dicentric chromosomes) are not recognized by the NoCut pathway.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary: Building on the specific connection between DNA bridges that bear marks of replication stress and the NoCut checkpoint (Amaral 2016, 2017), which prevents completion of cytokinesis, Dam et al. test the helicase Srs2/PARI for a role in this checkpoint pathway. The authors have produced a thorough study investigating the role of this helicase in both yeast and mammalian cells in the presence of DNA bridges. The manuscript includes clear evidence that Srs2 is important to resolve chromatin bridges, remove replication protein A (RPA) from chromatin, and delay cytokinesis under replication stress. Further, the authors show that loss of Srs2 under replication stress increases DNA damage, marked by elevated MRE11 foci in a manner dependent on cytokinesis (i.e., dependent on Cyk3). Srs2 deletion also partially abrogates the abscission delay seen upon topo-II inactivation. They further report that Srs2 must interact with PCNA to delay abscission in S. cerevisiae. While chromatin bridges formed when a dicentric chromosome is present escape detection by the NoCut checkpoint, inactivation of Elg1, which unloads PCNA and associated factors following DNA replication, results in delayed abscission. In HeLa cells, the Srs2 ortholog PARI is shown to similarly help promote abscission delay in the presence of DNA bridges following topoisomerase inhibition, as loss of PARI through siRNA knockdown prevents this abscission delay. Mechanistically, when PARI levels are reduced in HeLa cells, actin patches that function to stabilize the midbody and protect DNA bridges do not form/persist robustly as in cells with intact PARI. Consistent with a specific role in sensing the presence of a DNA bridge, depletion of PARI did not impact abscission checkpoint activity in response to depletion of the NPC component, Nup153. Finally, the authors show that PARI depletion reduced time to abscission to the same extent as treatment with an Aurora B inhibitor, and PARI depletion in conjunction with Aurora B inhibition did not reduce abscission timing further than singular treatments, suggesting that PARI works within the Aurora B-mediated NoCut signaling cascade.

    Major comments: The manuscript is well written and, in general, the conclusions are thoroughly supported, but there are a few recommendations for addition or revision. The first of these is for a more thorough introduction of helicases potentially involved in cytokinesis and more clear rationale for why the focus is on Srs2.

    Figure 1 E lacks statistical analysis. In addition, the text referring to 1E leads to confusion because the distinction between "RPA foci during anaphase" and "RPA coated chromatin bridges" is not made clear. The authors should clarify that the data presented in 1E shows quantification of cells with RPA foci during anaphase, not RPA coated chromatin bridges, and use consistent wording between the text and figure/figure legend. Further, how cells with RPA foci were identified, and what is classified as an RPA focus from images should be described in the methods.

    In some cases, it is unclear whether DNA bridge formation is prevented vs aberrantly broken. For example, under Top2 inactivation, does the absence of Srs2 prevent bridge formation or promote their breakage along with premature midbody abscission? Confirming the frequency of chromatin bridge formation would address this and, further, monitoring RPA persistence would validate whether RPA clearance from bridges is consistently correlated with Srs2 activity (an interesting observation from Figure 1 that is not followed up on). Similarly, other conditions that appear to interfere with abscission delay (e.g., disrupting Srs2-PCNA interaction) should be monitored for whether the formation of DNA bridges has been altered.

    In Figure 4A, the data show that the PIP-box is required for timely abscission. Imaging data from yeast strains with the PIP-box deletion alone should be included, rather than only showing the deletion in combination with the SIM deletion.

    While the authors state that PARI and PCNA were not detectable at bridges in mammalian cells, it would be worth examining whether RPA is persistent on DNA bridges in mammalian cells depleted of PARI to understand how closely this pathway resembles the features found in yeast.

    In Figure 6, the authors should describe in the methods how cells with actin patches were identified and quantified and explain what criteria must be met to be identified as an actin patch. Actin patches were described as "disassembling more quickly" in PARI-depleted cells, but the images look as if actin patches are not forming properly in these cells. The images are crisp and clear, but a change in wording may be necessary to accurately describe the data.

    Minor suggestions to improve the manuscript are:

    Include a diagram that shows hallmarks of cell division and what is being tracked in particular assays (e.g., DNA bridge duration vs time to abscission).

    In the elegant CLEM experiments presented in Figure 5, organelle labels could be added to orient the readers.

    The data in supplemental Figure 2 should be moved to Figure 5. The fact that there are similar levels of chromatin bridges is vital information and stresses that the defect lies in detection and response to the bridge as opposed to formation of bridges when PARI is depleted.

    Significance

    The link between DNA bridges and NoCut/abscission checkpoint signaling is a fundamental aspect of cell cycle regulation. This manuscript makes a significant contribution to our understanding of this pathway by introducing a novel role for the helicase Srs2/PARI in execution of an abscission delay in the presence of DNA bridges. This is an important contribution as there is sparse information about cellular factors that mediate detection and response to DNA bridges, which is vital to protecting genome integrity. Although, as the authors themselves state, "the molecular mechanisms by which Srs2 and PARI function in NoCut remain unclear," this study, with some revisions, merits publication as it reveals a conserved role for a factor in this important response pathway and provides new insights into why certain DNA bridges (i.e., bridges formed by dicentric chromosomes) are not recognized by the NoCut pathway.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    The Aurora B-mediated abscission checkpoint ("NoCut" in yeast) prevents tetraploidization or chromatin breakage in the presence of chromatin bridges in cytokinesis and the mechanisms of its activation are a matter of active investigation. In the present study, Dam et al propose that the conserved Srs2/PARI DNA helicase is required for the activation of the abscission checkpoint in response to chromatin bridges generated by DNA replication stress or topoisomerase inhibition. This is a timely and very interesting topic and the potential identification of a novel regulatory protein that activates the abscission checkpoint would be important. However, in my opinion, some Figures are of relatively low quality and need improving, there are apparent discrepancies between data and important control experiments are missing, which preclude the reader from fully evaluating the conclusions of this study. Some direct evidence of the role of Srs2/PARI on DNA bridges is also required. Also, it would be nice to investigate mechanistic details of the potential Srs2/PARI functions in the abscission checkpoint, and how it fits with other recently published signaling pathways that activate the abscission checkpoint in cytokinesis.

    Specific comments:

    1. The DNA channel (Ht2B-mCherry) in Figure 1A is of very low quality to be able to verify the authors interpretations of when the individual chromatin bridges are resolved (probably broken). For example, in the WT movie, they claim that the bridge is intact in frames 10 min and 14 min (yellow arrow) and that the bridge is resolved at 16 min (asterisk); however, I'm not convinced this is the case, because I can only see a very small portion of the bridge already at the 10 min and 14 min time-points. In my opinion, this bridge could have been broken much earlier, probably at 10 min. Also, WT +HU, is this bridge really intact at 10 min and at 14 min? In Srs2Δ + HU, the bridge appears broken to me much earlier, perhaps at 30 min. There is a distinct possibility that the authors could not calculate the resolution times accurately from these movies (please also see my next comment, #2). The authors could perhaps use a more sensitive bridge marker such as GFP-BAF.
    2. In Figure 1B, they conclude that Srs2Δ cells treated with HU exhibit increased time from anaphase onset to bridge resolution compared with WT or Srs2Δ cells. This result appears at odds with data from Fig. 2C showing that Srs2Δ+HU finish abscission at similar times to WT or Srs2Δ cells as judged by plasma membrane morphology. (final cut). Given that the final cut of the plasma membrane should cause chromatin bridges to break, if Srs2 is required for an abscission delay in response to HU-induced chromatin bridges, I would expect Srs2Δ + HU cells to exhibit accelerated plasma membrane cut and also faster chromatin bridge resolution compared with controls. This discrepancy could at least in part be caused by the relatively low quality of movies used for the calculations in Fig. 1.
    3. Fig. 2 shows faster abscission times (membrane cut) in Srs2Δ+HU cells compared with WT+HU. The authors interpret this data as evidence for a role of Srs2 in abscission delay in response to HU-induced chromatin bridges (page 7 and elsewhere). However, there is no direct evidence that the cells analyzed in Fig.2 exhibited DNA bridges in cytokinesis. One could argue that HU-induced DNA replication stress caused DNA lesions at the nuclear chromatin, which affected completion of cytokinesis in the absence or presence of Srs2. What proportion of HU-treated cells in cytokinesis exhibit DNA bridges? Judging from Fig. 1D this could be as low as 0-20%. The authors should analyze HU-treated cells that clearly exhibit DNA bridges, either by live-cell imaging or in fixed cells experiments. As it stands and together with my previous comments #1 and 2, I'm not convinced this data fully supports a role for Srs2 in the abscission delay in response to HU-induced DNA bridges.
    4. In Fig. 2D, there is no evidence to support that Mre11 foci are caused by bridge breakage, and not by replication-stress induced DNA lesions at the main nucleus (no DNA bridge is evident, also see comment #3).
    5. Figure 3: the authors use a top2-4 mutant strain to generate DNA bridges from catenated DNA and investigate the potential role of Srs2 in the abscission delay. However, no DNA bridges are obvious in the cells shown in Fig. 3. What proportion of top2-4 mutant cells in cytokinesis exhibit DNA bridges? Does this explain the striking difference in the percentage of cells that haven't completed abscission after 30-60 min in WT+HU vs Top2-4 cells? Please also see my previous comments above.
    6. The authors propose that association of Srs2 with PCNA is required for complete inhibition of abscission in top2-4 mutant cells with chromatin bridges. Assuming a role for Srs2 in abscission timing in cytokinesis with chromatin bridges is fully proven, it is essential that the authors also investigate the localization of Srs2 and PCNA on chromatin bridges, using GFP-tagged proteins or appropriate antibodies in fixed and/or living cells. This would suggest a direct role of these proteins on chromatin bridges and considerably strengthen the authors hypothesis. Alternatively, Srs2 and PCNA may indirectly affect abscission timing through their well-established roles at nuclear chromatin.
    7. In Fig. 4D, the authors show an abscission delay in elg1Δ mutant cells in the presence of dicentric bridges compared with cytokinesis without bridges and interpret this as evidence that artificially retaining PCNA on dicentric chromatin bridges is sufficient to inhibit abscission. It is important that the authors demonstrate that PCNA localizes to dicentric bridges in elg1Δ mutant, but not in ELG1 control, cells, e.g., by immunofluorescence, to support their claim and their proposed model.
    8. In Fig. 5, the authors claim that HeLa cells treated with the Top2 inhibitor ICRF193 exhibit delayed midbody resolution compared with controls and that depletion of PARI by siRNA accelerates abscission in ICRF-treated cells. They interpret this as evidence for a role of PARI in the abscission delay in response to ICRF-induced chromatin bridges. However, no bridges are visible at any time-frame in cells in Fig. 5B raising the possibility that the observed time-differences are due to some effect of ICRF in cytokinesis without bridges. I'm also not convinced that in Fig. 5B the midbodies in NT/ICRF/230 min, siPARI/DMSO/110 min and siPARI/ICRF/150 min were resolved as indicated by the authors, as I can definitely see both midbody arms very clearly in these photos. The p-values are also just below the p<0.05 threshold, which could in part be due to the quality of the movies quantified. Also, in Fig. 5C, the authors show evidence of DNA at the midbody in ICRF-treated cells by CLEM; however, this DNA appears broken before abscission in both cases and could not have been derived from premature abscission.
    9. In Fig. 6, the authors examine actin patches in PARI-depleted and control cells as a marker of abscission. Although a role for PARI in actin patch formation would be very interesting, I'm not sure how it fits with the present story. The actin inside the intercellular canal described by Bai et al (removal of which correlates with abscission) appears very different to the accumulations of actin at the base of the intercellular canal described by Sreigemann et al and by Dandoulaki et al. I can definitely see actin patches (similar to the ones in Steigemann et al) in Fig. 6 NT/ICRF, but I can't see any at the other treatments (I disagree with the arrows). Incidentally, I can see a DNA bridge only in NT/ICRF, but not in the other treatments.
    10. Midbody resolutions are clearer in Fig. 7, perhaps with the exception of siPARI/DMSO. However, no DNA bridges are visible, raising again the possibility that the authors investigate effects in cytokinesis without DNA bridges.
    11. Can the authors investigate whether the helicase activity of PARI is required for the abscission checkpoint, by depletion-reconstitution experiments with a helicase-mutant protein?
    12. The authors should investigate localization of PARI to the midbody/ DNA bridge in cytokinesis with chromatin bridges. Recent reports have proposed that a Top2-MRN-ATM-Chk2 pathway activates the Aurora B-dependent abscission checkpoint in human cells (PMIDs: 37638884, 33355621). The authors should examine localization of Aurora B and some of the above proteins in control and PARI-deficient cells to establish if/how PARI fits in the above pathway.
    13. The authors use ICRF to generate chromatin bridges. If ICRF is continuously present in their assays, one would expect it to inhibit Top2 and impair the abscission checkpoint (PMIDs: 37638884, 33355621). How do the authors reconcile this with their proposed model?

    Additional comments:

    1. Page 8: "Although SIM-defective Srs2 has a lower affinity to SUMOylated PCNA, it can still interact with PCNA". The authors should test this experimentally or provide appropriate references supporting this claim.
    2. Page 6: "Deletion of SRS2 further increased the fraction of anaphase cells with RPA foci, rising to approximately 30% in the absence of HU..."; however, this rise was not statistically significant as indicated in Fig. 1C.
    3. Fig. 1C, D: SDs are missing. Fig. 1E: please show the p-values.
    4. Fig. 2D: please show SDs and individual values.
    5. Why do the authors show the spindle pole body in their movies?
    6. Fig. 4A: WT and top2-4 cells have the same symbol in the graph.

    Significance

    Strengths: potentially novel regulator of the abscission checkpoint. Timely and interesting topic of broad scientific interest.

    Limitations: problems with quality of some data and withy the interpretation. Also, more mechanistic evidence is required to significantly advance our knowledge in the field.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    The abscission checkpoint, also known as NoCut, is a genome protection mechanism that remains poorly understood. This pathway is conserved from yeast to humans and protects the genome against chromosome bridges, a dangerous missegregation event that can have catastrophic consequences on genome stability. Dam et al now report the role of Srs2, a DNA helicase, as a key factor in the abscission checkpoint. The authors establish Srs2 as bona fide factor in this pathway by showing its involvement in abscission delays when chromatin bridges are induced. Importantly, yeast defective for Srs2 show increased levels of DNA damage when the frequency of chromatin bridges is increased. The authors also provide genetic evidence supporting a model whereby the interaction of SrS2 with PCNA s required for abscission regulation. In the second part of the manuscript, the authors study the human homologue of SRS2, PARI, in abscission regulation. The manuscript provides convincing evidence that PARI is also required for abscission delays in the presence of chromatin bridges. Critically, this role is specific for chromosome missegregation as abscission delays in response to nucleoporin depletion remain intact in PARI-depleted cells. Thus there is a conserved requirement for these DNA helicases in the abscission checkpoint. Overall, these are important advances in our understanding of the abscission checkpoint. The data is high quality and convincing in general. However, the impact of PARI depletion on genome stability needs to be further demonstrated to support key claims in the manuscript. Specifically: Disruptions of the abscission checkpoint in human cells result in bi-nucleation or increased levels of DNA damage. In this context, the authors need to show that PARI-depleted cells with increased frequency of chromatin bridges exhibit increased levels of bi-nucleation, DNA damage or both.

    Significance

    The abscission checkpoint, remains poorly understood. There is evidence in the literature that disruptions in this pathway increase susceptibility to cancer. The identification of the Srs2/PARI helicases as key components in this pathway is a considerable step forward in this field.

  5. Version published to 10.1101/2024.11.13.623343v2 on bioRxiv
  6. Version published to 10.1101/2024.11.13.623343v1 on bioRxiv