The nucleolar protein GNL3 prevents resection of stalled replication forks

  1. Rana Lebdy
  2. Marine Canut
  3. Julie Patouillard
  4. Jean‐Charles Cadoret
  5. Anne Letessier
  6. Josiane Ammar
  7. Jihane Basbous
  8. Serge Urbach
  9. Benoit Miotto
  10. Angelos Constantinou
  11. Raghida Abou Merhi
  12. Cyril Ribeyre

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Abstract

Faithful DNA replication requires specific proteins that protect replication forks and so prevent the formation of DNA lesions that may damage the genome. Identification of new proteins involved in this process is essential to understand how DNA lesions accumulate in cancer cells and how they tolerate them. Here, we show that human GNL3/nucleostemin, a GTP‐binding protein localized mostly in the nucleolus and highly expressed in cancer cells, prevents nuclease‐dependent resection of nascent DNA in response to replication stress. We demonstrate that inhibiting origin firing reduces resection. This suggests that the heightened replication origin activation observed upon GNL3 depletion largely drives the observed DNA resection probably due to the exhaustion of the available RPA pool. We show that GNL3 and DNA replication initiation factor ORC2 interact in the nucleolus and that the concentration of GNL3 in the nucleolus is required to limit DNA resection. We propose that the control of origin firing by GNL3 through the sequestration of ORC2 in the nucleolus is critical to prevent nascent DNA resection in response to replication stress.

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  1. Version published to 10.15252/embr.202357585
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    Reply to the reviewers

    Reply to reviewers.

    We deeply thank the reviewers for the time spent on evaluating our manuscript as well as providing comments and suggestions to improve our study.

    __Reviewer #1 (Evidence, reproducibility and clarity (Required)): __

    *In this manuscript Lebdy et al. describe a new role of GNL3 in DNA replication. They show that GNL3 controls replication fork stability in response to replication stress and they propose this is due to the regulation of ORC2 and the licensing of origins of replication. Their data suggest that GNL3 regulates the sub nuclear localization of ORC2 to limit the number of licensed origins of replication and to prevent resection of DNA at stalled forks in the presence of replication stress.

    While many of the points of the manuscript are proven and well supported by the results, there are some experiments that could improve the quality and impact of the manuscript. The main issue is that the connection between the role of GNL3 in controlling ORC2, the firing of new origins and the protection of replication forks is not clearly established. At the moment the model relies on mainly correlative data. In order to further substantiate the model, we propose to address some of the following issues:*

    1. *The authors indicate that RPA and RAD51 accumulation at stalled forks is not affected by GNL3 depletion. These data should be included and other proteins should be analysed. In addition, the role of helicases could be explored through the depletion of the main helicases involved in the remodelling of the forks. * Response: As asked by the reviewer we will add the fractionation experiments that show that the level of RAD51 and RPA on chromatin is not affected by GNL3 depletion. So far, the other proteins we checked (RIF1 and BRCA1), both involved in nascent strand protection, did not show clear differences. Therefore, we concluded that depletion of GNL3 does not seem to affect the recruitment of major proteins required for protection of nascent DNA. Of course, we cannot exclude that other proteins may be affected by GNL3 depletion, but testing all the possible candidates would be time consuming with a very low chance of success. In addition, fractionation experiments are possibly not quantitative enough to uncover small differences and may be not that informative. Thus it remains possible that RPA exhaustion may be the cause of resection in absence of GNL3 as suggested by the work conducted in Lukas’ lab (Toledo et al. 2013. https://pubmed.ncbi.nlm.nih.gov/24267891/). To test this hypothesis, we will analyze if resection in absence of GNL3 is still occurring in a well-characterized cell line that overexpress the three RPA subunits that we obtained from Lukas’ lab.

    To our knowledge not many helicases have been shown to be involved in remodeling of stalled forks. The best example is RECQ1, however we feel that testing RECQ1 involvement in resection upon GNL3 depletion will complicate our story without adding much regarding the mechanism. We hope the reviewer understands our concern.

    • The proposed model implies that GNL3 depletion leads to increased origin licensing. FThe authors should address if the primary effect of GNL3 depletion is on origin firing by using CDC7 inhibition in the absence of stress (Rodríguez-Acebes et al., JBC 2018). *

    __Response: __This is an excellent point raised by the reviewer. To test if the primary effect of GNL3 depletion in on origin firing we will test if the defect in replication fork progression is dependent on CDC7 using DNA fibers experiments and CDC7 inhibitor.

    • A way to prove that origin firing mediates the effect of GNL3 on fork protection would be to reduce the number of available origins. The depletion of MCM complexes has been shown to limit the number of back-up origins that are licensed and leads to sensitivity to replication stress (Ibarra et al., PNAS 2008). If GNL3 depletion results in increased number of origins, this effect should be prevented by the partial depletion of MCM complexes. *

    __Response: __This is also an excellent point. We will test if MCM depletion decreases resection upon GNL3 depletion and treatment with HU. In addition, we will integrate in the manuscript experiments that we have done recently that show that treatment with roscovitine, a CDK inhibitor that impairs origin firing, decreases the level of resection observed in absence of GNL3. We think this experiment strengthens the results obtained with CDC7 inhibitors.

    *Alternatively, the authors could try to modulate the depletion of GNL3. Origin licensing takes place in the G1 phase and thus the depletion of GNL3 by siRNA could affect the following S phase. Using an inducible degron for GNL3 depletion would allow to deplete GNL3 in G1 or S phase specifically. If the model is correct, the removal of GNL3 in S phase should not affect fork protection but removing GNL3 in the previous G2/M phase should reduce the number of licensed origins and lead to impaired fork protection. *

    __Response: __This is obviously a good point given the fact that GNL3 deletion is not viable (see responses to reviewer 2). We tried to develop an auxin induced degron of GNL3, but we could not obtain homozygous clones, meaning that our clones had always an untagged GNL3 allele. Since GNL3 is essential its tagging may impair its function, explaining why we could not obtain homozygous clones. However, we are planning to optimize the design using other degrons system (for instance Halo-tag) to address the role of GNL3 specifically during S-phase. But we think this is above the scope of the present study.

    *In addition to the connection GNL3-origin firing-fork protection, it is unclear how the lack of GNL3 in the nucleolus and the change in the sub nuclear localization of ORC2 controls origin firing and resection. The strong interaction observed between GNL3-dB and ORC2, and the subsequent change in ORC2 localization does not explain how origin licensing can be affected. In this sense, the authors could address: *

    1. *Does the depletion of GNL3 and the expression of GNL3-dB affect the formation of the ORC complex, its subnuclear localization or its binding to chromatin? The authors have not explored if the interaction of GNL3 with ORC2 is established in the context of the ORC complex. An IF showing NOP1 with PLA data from GNL3-dB and ORC2 is needed to analyse how the expression of increasing amounts of GNL3-dB affects ORC2. * __Response: __We tested if GNL3 depletion impacts ORC2 and ORC1 recruitment on chromatin, but we could not observe significant differences. No clear differences were observed upon GNL3-dB expression either. One reason for this may be due to the excess of ORC complex on the chromatin, in addition chromatin fractionation is likely not sensitive enough to observe small differences. We think that quantitative ChIP-seq of ORC2 or other ORC subunits upon GNL3 depletion is required to visualize such differences, but this is above the scope of the study, and this constitutes the following of this project. We also tried to look at subnuclear localization of ORC2 using immunofluorescence, but the signal was not specific enough to observe differences. We think that the increased interaction (PLA) of ORC2 with GNL3-dB (Figure 5E) demonstrates a change in ORC2 subnuclear localization. To confirm this, we will perform the excellent experiment proposed by the reviewer to test if increasing level of GNL3-dB affects its interaction with ORC2 using PLA.

    We do not think that the interaction between ORC2 and GNL3 is established in the context of the ORC complex since only ORC2 (and not the other ORC) was significantly enriched in the GNL3 Bio-ID experiment. The full list of proteins from the Bio-ID experiment (Figure 4A) will be provided in the revised version. Therefore, we think that either GNL3 regulates ORC2 subnuclear localization that in turns impact the ORC complex or GNL3 regulates ORC2-specific functions. More and more evidences show that ORC2 plays roles possibly independently of the ORC complex (see Huang et al. 2016 https://doi.org/10.1016/j.celrep.2016.02.091 or Richards et al. 2022 https://doi.org/10.1016/j.celrep.2022.111590 for instance). Future work should uncover how these ORC2 functions may regulate origins activity.

    *In order to confirm if the mislocalization of ORC2 by the expression of GNL3-dB increases origin firing and mediates the effects on fork protection the authors could check DNA resection levels inhibiting CDC7 in high GNL3-dB conditions. Also, the levels of MCM2, phosphor-MCM2, CDC45, have not been analysed upon expression of GNL3-dB. *

    __Response: __This is a good point; we will test if the resection observed upon expression of GNL3-dB is dependent on origin firing using CDC7 inhibitor. We have not measured the level of the cited proteins but instead we performed DNA combing to measure Global Instant Fork Density. We now show that expression of GNL3-WT suppresses the increased origin firing observed upon GNL3 depletion, in contrast expression of GNL3-dB does not suppress it. This important result indicates that origin firing is increased upon GNL3-dB expression, providing a link between aberrant localization and increased firing. These data will be part of the revised version of the manuscript.

    *The data in the paper suggest that GNL3 may affect the role of ORC2 in centromeres. Since depletion of GNL3 leads to increased levels of gH2AX, it would be interesting to address if this damage is due to incomplete replication in centromeres by analysing the co-localization of gH2AX and centromeric markers both in unstressed conditions and upon the induction of replication stress. *

    __Response: __This is indeed and interesting comment, however since it has been previously shown that gH2AX signal is rather strong upon GNL3 depletion (see Lin et al. 2013. https://pubmed.ncbi.nlm.nih.gov/24610951/ ; Meng et al. 2013. https://pubmed.ncbi.nlm.nih.gov/23798389/) we do not think that co-localization experiments with CENP-A for instance will be informative given the high number of gH2AX foci.

    *Minor points: *

    1. *In the initial esiRNA screen the basal levels of gH2AX should also be shown. * Response: Our negative control is the transfection of an esiRNAs that targets EGFP (a gene that is not expressed in the tested cell line). This esiRNAs is ranked at the end of the list and therefore constitutes the basal level of gH2AX signal. In any case it is well-established that GNL3 depletion increases gH2AX signal (see Lin et al. 2013. https://pubmed.ncbi.nlm.nih.gov/24610951/ ; Meng et al. 2013. https://pubmed.ncbi.nlm.nih.gov/23798389/).

    *Figure EV1B: I think the rank needs another RS mark to see better the effect of each esiRNA on DNA lesions (high variability in all the conditions showed). *

    __Response: __We understand this issue, but we cannot repeat this set of experiments for technical reasons (reagents and cost mainly). Anyway, we believe that the role of GNL3 is response to replication stress is extensively addressed by other experiments of this manuscript.

    *Figure 1C and Figure EV1D/E: the quantification of the pCHK1/CHK1 levels could be included to show that there are no changes in phosphorylation upon GNL3 depletion. *

    Response: it is a good point; we will put quantification in the revised version.

    *In the first section of the results, at the end Figure 4B is incorrectly called for. *

    __Response: __Thanks for the comment, we will modify accordingly.

    The levels of GLN3 expression in 293 cells should be already included in section GNL3 interacts with ORC2.

    __Response: __We will add a figure that shows the level of expression in 293 cells.

    The full MS data needs to be included for both GNL3 and ORC2.

    __Response: __This will be integrated in the revised version.

    Figure 4B should be improved, since there is a faint band in the IgG mouse control.

    __Response: __it is true that the figure is not perfect, but we believed that our Bio-ID and PLA experiments fully demonstrate the interaction between GNL3 and ORC2.

    __Reviewer #1 (Significance (Required)): __

    *The work is nicely written, the figures are well presented and the experiments have the necessary controls. It provides relevant information to understand how replication stress is controlled and linked to replication fork protection through origin firing. These results are relevant to the field, linking GNL3 to origin firing and with potential to help understand the role of GNL3 in cancer. They provide new information and can give rise to new studies in the future. Many of the conclusions of the manuscript are well supported. Additional support for some of the main claims would strengthen the results and also increase the impact providing a bigger conceptual advance by performing some of the suggested experiments. *

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

    *This manuscript explores the role of GNL3/nucleostemin in DNA replication and specifically in the response of DNA replication to DNA damage. GNL3 is a predominantly nucleolar protein, previously characterised as a GTP-binding protein and shown to be necessary for effective recruitment of the RAD51 recombinase to DNA breaks. The entry point for this report is a mini screen, based on proteins identified previously by the authors to associate with replication forks by iPOND, for factors that increase gamma-H2Ax (an indicator of DNA damage) after treatment with the Top1 inhibitor camptothecin (CPT). In this mini-screen GNL3 emerged as the top hit.

    The authors put forward the hypothesis that GNL3 is able to sequester the replication licensing factor ORC2 in the nucleolus and that failure of this mechanism leads to excessive origin firing and DNA resection following CPT treatment.*

    • The model put forward is interesting, but currently rather confusing. However, for the reasons upon which I expand below, I do not believe that the data provide a compelling mechanistic explanation for the effects that are reported and I am left not being certain about some of the links that are made between the various parts of the study, even though individual observations appear to be of good quality. *

    *Specific points: *

    *The knockdown of GNL3 is very incomplete. In this regard, the complementation experiments are welcome and important. However, is it an essential protein? Can it be simply deleted with CRISPR-Cas9?

    *__Response: __There are obviously variations between experiments but overall, the depletion of GNL3 using siRNA seems good in our opinion. Deletion of GNL3/nucleostemin leads to embryonic lethality in mouse (Beekman et al. 2006. https://pubmed.ncbi.nlm.nih.gov/17000755/ ; Zhu et al. 2006. https://pubmed.ncbi.nlm.nih.gov/17000763/). ES cells deleted for GNL3 can be obtain but do not proliferate probably because of their inability to enter in S-phase (Beekman et al. 2006. https://pubmed.ncbi.nlm.nih.gov/17000755/). We wanted to test if it was the case in our cellular model and we tried to delete it using CRISPR-Cas9. We managed to obtain few clones deleted for GNL3, but they grow really poorly prevented us to do experiments. To bypass this, and as suggested by the reviewer 1, we tried to make an auxin-induced degron of GNL3. Unfortunately, we did not manage to obtain homozygous clones, only heterozygous. One possibility could be that the tagging induced a partial loss of function of GNL3, and since GNL3 is essential, it may explain why we did not obtain homozygous clones. We may also want to use alternative degron systems such as Halo-Tag, but we believe this is out of the scope of the study.

    __ __*Global instant fork density is not quite the same as actually measuring origin firing. Ideally, it would be good to see some more direct evidence of addition origin firing e.g. by EdU-seq (Macheret & Halazonetis Nature 2018) but this would be quite a significant additional undertaking. However, given the authors have performed DNA combing with DNA counterstain, they should be able to provide accurate measurements of origin density and inter-origin distance. *

    __Response: __As indicated by the reviewer EdU-seq would need a lot of development since we are not using this approach in our team. In addition, this method can detect replication origins only if performed in the beginning of S-phase, meaning that only the early firing origins will be detected and not the others. GIFD measurement is actually directly linked with origin firing since it is counting the forks to duplicate the genome. The measurements of IODs have at least two main limitations: (1) there is a bias for short IODs due to the length of analyzed fibers and (2) it focuses only on origins within a cluster not globally. Overall, we believe that GIFD is the method of choice to measures origins firing. In addition, these experiments have been done by the lab of Etienne Schwob (see acknowledgments), a leader in the field.

    *'Replication stress' is induced with CPT. This term is frequently used to describe events that lead to helicase-polymerase uncoupling (e.g. O'Connor Mol Cell 2015) but that is not the case with CPT, which causes fork collapse and breaks. Are similar effects seen with e.g. UV or cisplatin? Additionally, a clear statement of the authors definition of replication stress would be welcome. *

    __Response: __We will better define the term ‘replication stress’ in the revised version of the manuscript. It should be understood, in our case, that any impediment that leads to replication fork stalling and measurable by DNA combing or Chk1 phosphorylation. We have not performed experiments using UV and cisplatin.

    *It is really not clear how the authors explain the link between potential changes in origin firing and resection. i.e. What is the relationship between global origin firing and resection at a particular fork, presumably broken by encounter with a CPT-arrested TOP1 complex. What is the link mechanistically? This link needs elaborating experimentally or clearly explaining based on prior literature. *

    • *__Response: __Most of our results on resection has been performed with hydroxyurea, but it is true that we saw resection in absence of GNL3 in response to CPT. Treatment with HU or CPT reduces fork speed and activates additional replication origins (see Ge et al. 2007 https://pubmed.ncbi.nlm.nih.gov/18079179/ for HU or Hayakawa et al. 2021 https://pubmed.ncbi.nlm.nih.gov/34818230/ for CPT ). When GNL3 is depleted, more forks are active, meaning more targets for HU and CPT. In addition, it is likely that the firing of additional origins in response to HU and CPT is stronger in absence of GNL3. Because of this we believe that factors required to protect stalled forks may be exhausted explaining why resection is observed. This is inspired by the work of Lukas’ lab (Toledo et al. 2013 https://pubmed.ncbi.nlm.nih.gov/24267891/) and is described in the figure 6. One obvious candidate that may be exhausted is RPA, to test this we will check if resection upon GNL3 depletion and treatment with HU is still occurring in cell lines provided by Lukas’ lab that overexpress RPA complex (described in Toledo et al.). We will explain our model more carefully in the revised version.

    *Related to this, I remain unconvinced that the experiments in Figure 3 show that the effects of ATRi and Wee1i on origin firing and on resection are contingent on each other. I do not believe that the authors have adequately supported the statement (end of pg 9) 'We conclude that the enhanced resection observed upon GNL3 depletion is a consequence of increased origin firing.' The link between origin firing and resection needs really needs further substantiation and / or explanation.

    *__Response: __Our rational was the following. Inhibition of ATR or WEE1 increase replication origin firing, a situation that may be like the one observed for GNL3 depletion. In Toledo et al, they show that inhibition of WEE1 or ATR induces exhaustion of RPA. This exhaustion is reduced in presence of CDC7 inhibitor, roscovitine (a CDK inhibitor that inhibits origin firing) or depletion of CDC45, indicating that this is due to excessive origin activation. In our case we show that the resection observed upon WEE1 or ATR inhibition is reduced upon treatment with CDC7 inhibitor. We conclude that excessive replication origin firing induces DNA resection. Since we observed the same thing upon GNL3 depletion (but not upon BRCA1 depletion) we conclude that excessive origin firing favors DNA resection likely through exhaustion of RPA. As indicated above we will test this hypothesis by overexpressing RPA. In addition, we now show that treatment with roscovitine decreases resection upon GNL3 depletion (this will be part of the revised manuscript), an experiment that we believe confirms that excessive replication origins firing is responsible for resection upon GNL3 depletion. As suggested by reviewer 1, we will also test if depletion of MCM also reduces resection observed in absence of GNL3.

    *It is not clear whether the binding of ORC2 to GNL3 also sequesters other components of the origin recognition complex? Does loss of the ability of GNL3 to bind ORC2 actually lead to more ORC bound to chromatin? How does GNL3 contribute to regulation of origin firing under normal conditions? Is it a quantitatively significant sink for ORC2 and what regulates ORC2 release? *

    Response: The results of GNL3 Bio-ID were extremely clear, we could not significantly detect any other ORC subunits than ORC2 (these data were not present in the manuscript but will be added in the revised version), therefore we believe that GNL3 may sequester/regulate only ORC2. We tried to see if GNL3 depletion was changing the binding of ORC1 and ORC2 to the chromatin, but we could not see any difference, one possibility may be that small differences are not detectable by chromatin fractionation. We believe that ChIP-seq or ORC2 or other ORC subunits in absence of GNL3 is required but this it out of the scope of the study. GNL3 may regulates the stability of the ORC complex on chromatin via ORC2 but GNL3 may also regulates other ORC2 functions, at centromeres for instance. It has been shown indeed that ORC2 plays roles possibly independently of the ORC complex (see Huang et al. 2016 https://doi.org/10.1016/j.celrep.2016.02.091 or Richards et al. 2022 https://doi.org/10.1016/j.celrep.2022.111590 for instance). How exactly this is affecting origin firing is still mysterious. This is something we are planning to address in the future.

    We do not know if it is a quantitatively sink for ORC2 or how this is regulated, however we believe that the ability of GNL3 to accumulate in the nucleolus may sequester ORC2. Consistent with this, we show that a mutant of GNL3 (GNL3-dB) that diffuses in the nucleoplasm interacts more with ORC2 in the nucleoplasm suggesting a release. As suggested by reviewer 1 we will now test if the interaction between ORC2 and GNL3-dB is dependent on the level of expression of GNL3-dB. In addition, we now show that expression of GNL3-dB increases replication origin firing like GNL3 depletion (data that will be added in the revised version), suggesting that regulation of ORC2 is the major cause of increased firing upon GNL3 depletion.

    *Minor points: *

    *All blots should include size markers *

    __Response: __We will add them

    *Some use of language is not sufficiently precise. For instance: ** - the meaning of 'DNA lesions' at the end of the first paragraph of the introduction needs to be more explicit. *

    • the approach to measurement of these 'lesions' (monitoring gamma-H2Ax) needs to be spelled out explicitly, e.g. line 4 of the last paragraph of the introduction. *
    • 'we observed that the interaction between GNL3-dB and ORC2 was stronger' ... I do not see how number of foci indicates necessarily the strength of an interaction. *
    • in many places throughout 'replication origins firing' should be 'replication origin firing' (or 'firing of replication origins'). *

    __Response: __We will correct these language mistakes.

    __Reviewer #2 (Significance (Required)): __

    The model put forward here has the potential to shed light on an important facet of the cellular response to DNA damage, namely the control of origin firing in response to replication stress that will certainly be of interest to the DNA repair / replication community and possibly more widely. The roles of GNL3 are poorly understood and this study could improve this state of affairs. However, the gaps in the mechanism outlined above and somewhat confusing conclusions do limit the ability of the paper to achieve this at present.

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

    *In this study, Lebdy et al propose a new mechanism to regulate the resection of nascent DNA at stalled replication forks. The central element of this mechanism is nucleolar protein GNL3, whose downregulation with siRNA stimulates DNA resection in the presence of stress induced by HU (Figure 1). Resection depends on the activity of nucleases MRE11 and CtIP, and can be rescued by reintroducing exogenous GNL3 protein in the cells (Figure 1G). GNL3 downregulation decreases fork speed and increases origin activity, without any strong effect on replication timing (Figure 2). Inhibition of Dbf4-dependent kinase CDC7 (a known origin-activating factor) also restricts fork resection (Figure 3). GNL3 interacts with ORC2, one of the subunits of the origin recognition complex, preferentially in nucleolar structures (Figure 4). A mutant version of GNL3 (GNL3-dB) that is not sufficiently retained in the nucleoli fails to prevent fork resection as the WT protein (Figure 5). In the final model, the authors propose that GNL3 controls the levels of origin activity (and indirectly, stalled fork resection) by maintaining a fraction of ORC2 in the nucleoli (Figure 6). *

    This model is interesting and provocative, but it also relies on a significant degree of speculation. The authors are not trying to "oversell" their observations, because the Discussion section entertains different interpretations and possibilities, and the model itself contains several interrogative statements (e.g. "ORC2-dependent?"; "exhaustion of factors?").

    • While the article is honest about its own limitations, the major concern remains about its highly speculative nature. I have some questions and suggestions for the authors to consider that could contribute to test (and hopefully support) their model. *
    1. *If GNL3 downregulation induces an excess of licensed origins and mild replicative stress resulting in some G2/M accumulation (Figure 2), what is the consequence of longer-term GNL3 ablation? Do the cells adapt, or do they accumulate signs of chromosomal instability? (micronuclei, chromosome breaks and fusions, etc) * __Response: __This is an important point also raised by Reviewer 2: deletion of GNL3 leads to embryonic lethality in mouse and ES cells deleted for GNL3 do not proliferate and fail to enter into S-phase. Consistent with this, the clones deleted for GNL3 that we obtained using CRISPR-Cas9 grow poorly, thus preventing us to do experiments. To our knowledge micronuclei and chromosome breaks have never been analyzed upon transient depletion of GNL3 using siRNA. However, it is well established that depletion of GNL3 induces phosphorylation of H2A.X) and the formation of ATR, RPA32 and 53BP1 foci due to S-phase arrest (Lin et al. 2013. https://pubmed.ncbi.nlm.nih.gov/24610951/ ; Meng et al. 2013. https://pubmed.ncbi.nlm.nih.gov/23798389/). DNA lesions have also been visualized by comet assay (Lin et al. 2019. https://pubmed.ncbi.nlm.nih.gov/30692636/). Consistent with this we observed a weak increased of DNA double-strand breaks upon GNL3 depletion using pulse-field gel electrophoresis as well as mitotic DNA synthesis (MiDAS). We can integrate this data in the revised version of the manuscript if required. To sum up, it is clear that GNL3 depletion is inducing problems during S-phase that may lead to possible genomic rearrangements.
    • The model relies on the link between origin activity and stalled fork resection that is almost exclusively based on the results obtained with CDC7i (Figure 3). But CDC7 has other targets besides pre-RC components at the origins, such as Exo1 (from the Weinreich lab, cited in the study), MERIT40 and PDS5B (from the Jallepalli lab, also cited). The effect of CDC7i could be exerted through these factors, which are linked to fork stability and DNA resection. The loss of BRCA1 (Figure 3F) could somehow entail the loss of control over these factors. Could the authors check the possible participation of these proteins?*

    __Response: __It is true that CDC7 has other targets than pre-RC components. We therefore decided to inhibit origin firing using roscovitine, a broad CDK inhibitor, a strategy previously used in Lukas lab (Toledo et al. 2013. https://pubmed.ncbi.nlm.nih.gov/24267891/). We observed that treatment with roscovitine decreased significantly resection observed upon GNL3 depletion, confirming the link between origin activity and stalled fork resection. This will be integrated in the revised version of the manuscript. As asked by Reviewer 1, we will also perform depletion of MCM to strength our model.

    Exo1 is indeed a target of CDC7 as shown by the Weinreich lab (Sasi et al. 2018. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6111017/) however the authors do not formally demonstrate that Exo1 phosphorylation is required for its activity. We observed that depletion of Exo1 significantly reduced resection upon GNL3 depletion (data that will be added in the revised version), indicating that the effect of CDC7 inhibitor could be exerted via the control of Exo1. This is why our BRCA1 control is important, it is well stablished that Exo1 is required for nascent strand degradation upon BRCA1 depletion (Lemaçon et al. 2017. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643552/) but CDC7 inhibition has no effect on resection upon BRCA1 depletion suggesting that resection by Exo1 may not be regulated by CDC7 in our context.

    As stated by the reviewer MERIT40 and PDS5B are targets of DDK kinases (Jones et al. 2021 https://doi-org.insb.bib.cnrs.fr/10.1016/j.molcel.2021.01.004) and seem to be required for protection of nascent DNA and in response to HU. However, little is known about the role(s) of these proteins and we think that adding them will complicate message. We hope the reviewer understands this.

    The model also relies on the fact that GNL3-dB mutant (not retained in the nucleoli) is not sufficient to counteract fork resection induced by HU (Figure 5G). The authors should test directly whether GNL3-dB induces extra origin activation, using their available DNA fibers-based technique.

    __Response: __This is an excellent point. We have now GIFD (Global Instant Fork Density) data that shows that the number of active forks is increased upon dB GNL3-dB expression. It demonstrates that when GNL3 is no longer retained in the nucleolus more origins are active. These data will be integrated in the revised version of the manuscript, and we believe further support the regulation of ORC2 by GNL3.

    *Finally, the model implies an exquisite regulation of the amount of ORC2 protein, which could influence the number of active origins and the extent of fork resection in case of stress. In this scenario, one could predict that ORC2 ectopic expression would have similar, or even stronger effects, than GNL3 downregulation. Is this the case? *

    __Response: __We completely agree with this prediction. However, we are afraid that overexpression of ORC2 may have indirect effects due to the many described functions of ORC2, therefore it may be difficult to interpret the data. We will give a try anyway.

    *Even if the connection between origins and fork resection could be firmly established, the molecular link between them remains enigmatic. The authors hint (as "data not shown") that it is neither mediated by RPA nor RAD51. Unfortunately, the reader is left without a clear hypothesis about this point. *

    __Response: __We will add data that show that RPA and RAD51 recruitment is not affected by GNL3 depletion. However, the sensitivity of chromatin fractionation approach may be too weak to detect low differences. Based on the work of Lukas Lab (Toledo et al. 2013 https://pubmed.ncbi.nlm.nih.gov/24267891/) one possible mechanism may be exhaustion of the pool of RPA. This may link the excessive activation of origins observed upon GNL3 depletion and resection. To test this, we will check if resection upon GNL3 depletion and treatment with HU is still occurring in cell lines that overexpress RPA complex (described in Toledo et al.) that we obtained from Lukas’ lab.

    __ __ **Referees cross-commenting**

    __ __In addition to each reviewer's more specific comments, the three reviews share a main criticism: the lack of mechanistic information about the proposed link between origin activity and resection of nascent DNA at stalled forks.

    __Reviewer #3 (Significance (Required)): __

    In principle, this study would appeal to the readership interested in fundamental mechanisms of DNA replication and the cellular responses to replicative stress.

    For the reasons outlined in the previous section, I believe that in its current version the study is not strong enough to provide a new paradigm about origins being regulated by partial ORC2 sequestering at nucleoli. The other potentially interesting advance is the connection between frequency of origin activity and the extent of nascent DNA resection at stalled forks, but the molecular link between both remains unknown.

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    Referee #3

    Evidence, reproducibility and clarity

    In this study, Lebdy et al propose a new mechanism to regulate the resection of nascent DNA at stalled replication forks. The central element of this mechanism is nucleolar protein GNL3, whose downregulation with siRNA stimulates DNA resection in the presence of stress induced by HU (Figure 1). Resection depends on the activity of nucleases MRE11 and CtIP, and can be rescued by reintroducing exogenous GNL3 protein in the cells (Figure 1G). GNL3 downregulation decreases fork speed and increases origin activity, without any strong effect on replication timing (Figure 2). Inhibition of Dbf4-dependent kinase CDC7 (a known origin-activating factor) also restricts fork resection (Figure 3). GNL3 interacts with ORC2, one of the subunits of the origin recognition complex, preferentially in nucleolar structures (Figure 4). A mutant version of GNL3 (GNL3-dB) that is not sufficiently retained in the nucleoli fails to prevent fork resection as the WT protein (Figure 5). In the final model, the authors propose that GNL3 controls the levels of origin activity (and indirectly, stalled fork resection) by maintaining a fraction of ORC2 in the nucleoli (Figure 6).

    This model is interesting and provocative, but it also relies on a significant degree of speculation. The authors are not trying to "oversell" their observations, because the Discussion section entertains different interpretations and possibilities, and the model itself contains several interrogative statements (e.g. "ORC2-dependent?"; "exhaustion of factors?").

    While the article is honest about its own limitations, the major concern remains about its highly speculative nature. I have some questions and suggestions for the authors to consider that could contribute to test (and hopefully support) their model.

    1. If GNL3 downregulation induces an excess of licensed origins and mild replicative stress resulting in some G2/M accumulation (Figure 2), what is the consequence of longer-term GNL3 ablation? Do the cells adapt, or do they accumulate signs of chromosomal instability? (micronuclei, chromosome breaks and fusions, etc)
    2. The model relies on the link between origin activity and stalled fork resection that is almost exclusively based on the results obtained with CDC7i (Figure 3). But CDC7 has other targets besides pre-RC components at the origins, such as Exo1 (from the Weinreich lab, cited in the study), MERIT40 and PDS5B (from the Jallepalli lab, also cited). The effect of CDC7i could be exerted through these factors, which are linked to fork stability and DNA resection. The loss of BRCA1 (Figure 3F) could somehow entail the loss of control over these factors. Could the authors check the possible participation of these proteins?
    3. The model also relies on the fact that GNL3-dB mutant (not retained in the nucleoli) is not sufficient to counteract fork resection induced by HU (Figure 5G). The authors should test directly whether GNL3-dB induces extra origin activation, using their available DNA fibers-based technique.
    4. Finally, the model implies an exquisite regulation of the amount of ORC2 protein, which could influence the number of active origins and the extent of fork resection in case of stress. In this scenario, one could predict that ORC2 ectopic expression would have similar, or even stronger effects, than GNL3 downregulation. Is this the case?
    5. Even if the connection between origins and fork resection could be firmly established, the molecular link between them remains enigmatic. The authors hint (as "data not shown") that it is neither mediated by RPA nor RAD51. Unfortunately, the reader is left without a clear hypothesis about this point.

    Referees cross-commenting

    In addition to each reviewer's more specific comments, the three reviews share a main criticism: the lack of mechanistic information about the proposed link between origin activity and resection of nascent DNA at stalled forks.

    Significance

    In principle, this study would appeal to the readership interested in fundamental mechanisms of DNA replication and the cellular responses to replicative stress.

    For the reasons outlined in the previous section, I believe that in its current version the study is not strong enough to provide a new paradigm about origins being regulated by partial ORC2 sequestering at nucleoli. The other potentially interesting advance is the connection between frequency of origin activity and the extent of nascent DNA resection at stalled forks, but the molecular link between both remains unknown.

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript explores the role of GNL3/nucleostemin in DNA replication and specifically in the response of DNA replication to DNA damage. GNL3 is a predominantly nucleolar protein, previously characterised as a GTP-binding protein and shown to be necessary for effective recruitment of the RAD51 recombinase to DNA breaks. The entry point for this report is a mini screen, based on proteins identified previously by the authors to associate with replication forks by iPOND, for factors that increase gamma-H2Ax (an indicator of DNA damage) after treatment with the Top1 inhibitor camptothecin (CPT). In this mini-screen GNL3 emerged as the top hit.

    The authors put forward the hypothesis that GNL3 is able to sequester the replication licensing factor ORC2 in the nucleolus and that failure of this mechanism leads to excessive origin firing and DNA resection following CPT treatment.

    The model put forward is interesting, but currently rather confusing. However, for the reasons upon which I expand below, I do not believe that the data provide a compelling mechanistic explanation for the effects that are reported and I am left not being certain about some of the links that are made between the various parts of the study, even though individual observations appear to be of good quality.

    Specific points:

    The knockdown of GNL3 is very incomplete. In this regard, the complementation experiments are welcome and important. However, is it an essential protein? Can it be simply deleted with CRISPR-Cas9?

    Global instant fork density is not quite the same as actually measuring origin firing. Ideally, it would be good to see some more direct evidence of addition origin firing e.g. by EdU-seq (Macheret & Halazonetis Nature 2018) but this would be quite a significant additional undertaking. However, given the authors have performed DNA combing with DNA counterstain, they should be able to provide accurate measurements of origin density and inter-origin distance.

    'Replication stress' is induced with CPT. This term is frequently used to describe events that lead to helicase-polymerase uncoupling (e.g. O'Connor Mol Cell 2015) but that is not the case with CPT, which causes fork collapse and breaks. Are similar effects seen with e.g. UV or cisplatin? Additionally, a clear statement of the authors definition of replication stress would be welcome.

    It is really not clear how the authors explain the link between potential changes in origin firing and resection. i.e. What is the relationship between global origin firing and resection at a particular fork, presumably broken by encounter with a CPT-arrested TOP1 complex. What is the link mechanistically? This link needs elaborating experimentally or clearly explaining based on prior literature.

    Related to this, I remain unconvinced that the experiments in Figure 3 show that the effects of ATRi and Wee1i on origin firing and on resection are contingent on each other. I do not believe that the authors have adequately supported the statement (end of pg 9) 'We conclude that the enhanced resection observed upon GNL3 depletion is a consequence of increased origin firing.' The link between origin firing and resection needs really needs further substantiation and / or explanation.

    It is not clear whether the binding of ORC2 to GNL3 also sequesters other components of the origin recognition complex? Does loss of the ability of GNL3 to bind ORC2 actually lead to more ORC bound to chromatin? How does GNL3 contribute to regulation of origin firing under normal conditions? Is it a quantitatively significant sink for ORC2 and what regulates ORC2 release?

    Minor points:

    All blots should include size markers

    Some use of language is not sufficiently precise. For instance:

    • the meaning of 'DNA lesions' at the end of the first paragraph of the introduction needs to be more explicit.
    • the approach to measurement of these 'lesions' (monitoring gamma-H2Ax) needs to be spelled out explicitly, e.g. line 4 of the last paragraph of the introduction.
    • 'we observed that the interaction between GNL3-dB and ORC2 was stronger' ... I do not see how number of foci indicates necessarily the strength of an interaction.
    • in many places throughout 'replication origins firing' should be 'replication origin firing' (or 'firing of replication origins').

    Significance

    The model put forward here has the potential to shed light on an important facet of the cellular response to DNA damage, namely the control of origin firing in response to replication stress that will certainly be of interest to the DNA repair / replication community and possibly more widely. The roles of GNL3 are poorly understood and this study could improve this state of affairs. However, the gaps in the mechanism outlined above and somewhat confusing conclusions do limit the ability of the paper to achieve this at present.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript Lebdy et al. describe a new role of GNL3 in DNA replication. They show that GNL3 controls replication fork stability in response to replication stress and they propose this is due to the regulation of ORC2 and the licensing of origins of replication. Their data suggest that GNL3 regulates the sub nuclear localization of ORC2 to limit the number of licensed origins of replication and to prevent resection of DNA at stalled forks in the presence of replication stress.

    While many of the points of the manuscript are proven and well supported by the results, there are some experiments that could improve the quality and impact of the manuscript. The main issue is that the connection between the role of GNL3 in controlling ORC2, the firing of new origins and the protection of replication forks is not clearly established. At the moment the model relies on mainly correlative data. In order to further substantiate the model, we propose to address some of the following issues:

    1. The authors indicate that RPA and RAD51 accumulation at stalled forks is not affected by GNL3 depletion. These data should be included and other proteins should be analysed. In addition, the role of helicases could be explored through the depletion of the main helicases involved in the remodelling of the forks.
    2. The proposed model implies that GNL3 depletion leads to increased origin licensing. FThe authors should address if the primary effect of GNL3 depletion is on origin firing by using CDC7 inhibition in the absence of stress (Rodríguez-Acebes et al., JBC 2018).
    3. A way to prove that origin firing mediates the effect of GNL3 on fork protection would be to reduce the number of available origins. The depletion of MCM complexes has been shown to limit the number of back-up origins that are licensed and leads to sensitivity to replication stress (Ibarra et al., PNAS 2008). If GNL3 depletion results in increased number of origins, this effect should be prevented by the partial depletion of MCM complexes.
    4. Alternatively, the authors could try to modulate the depletion of GNL3. Origin licensing takes place in the G1 phase and thus the depletion of GNL3 by siRNA could affect the following S phase. Using an inducible degron for GNL3 depletion would allow to deplete GNL3 in G1 or S phase specifically. If the model is correct, the removal of GNL3 in S phase should not affect fork protection but removing GNL3 in the previous G2/M phase should reduce the number of licensed origins and lead to impaired fork protection. In addition to the connection GNL3-origin firing-fork protection, it is unclear how the lack of GNL3 in the nucleolus and the change in the sub nuclear localization of ORC2 controls origin firing and resection. The strong interaction observed between GNL3-dB and ORC2, and the subsequent change in ORC2 localization does not explain how origin licensing can be affected. In this sense, the authors could address:
      1. Does the depletion of GNL3 and the expression of GNL3-dB affect the formation of the ORC complex, its subnuclear localization or its binding to chromatin? The authors have not explored if the interaction of GNL3 with ORC2 is established in the context of the ORC complex. An IF showing NOP1 with PLA data from GNL3-dB and ORC2 is needed to analyse how the expression of increasing amounts of GNL3-dB affects ORC2.
      2. In order to confirm if the mislocalization of ORC2 by the expression of GNL3-dB increases origin firing and mediates the effects on fork protection the authors could check DNA resection levels inhibiting CDC7 in high GNL3-dB conditions. Also, the levels of MCM2, phosphor-MCM2, CDC45, have not been analysed upon expression of GNL3-dB.
      3. The data in the paper suggest that GNL3 may affect the role of ORC2 in centromeres. Since depletion of GNL3 leads to increased levels of H2AX, it would be interesting to address if this damage is due to incomplete replication in centromeres by analysing the co-localization of H2AX and centromeric markers both in unstressed conditions and upon the induction of replication stress.

    Minor points:

    1. In the initial esiRNA screen the basal levels of H2AX should also be shown.
    2. Figure EV1B: I think the rank needs another RS mark to see better the effect of each esiRNA on DNA lesions (high variability in all the conditions showed).
    3. Figure 1C and Figure EV1D/E: the quantification of the pCHK1/CHK1 levels could be included to show that there are no changes in phosphorylation upon GNL3 depletion.
    4. In the first section of the results, at the end Figure 4B is incorrectly called for.
    5. The levels of GLN3 expression in 293 cells should be already included in section GNL3 interacts with ORC2.
    6. The full MS data needs to be included for both GNL3 and ORC2.
    7. Figure 4B should be improved, since there is a faint band in the IgG mouse control.

    Significance

    The work is nicely written, the figures are well presented and the experiments have the necessary controls. It provides relevant information to understand how replication stress is controlled and linked to replication fork protection through origin firing. These results are relevant to the field, linking GNL3 to origin firing and with potential to help understand the role of GNL3 in cancer. They provide new information and can give rise to new studies in the future. Many of the conclusions of the manuscript are well supported. Additional support for some of the main claims would strengthen the results and also increase the impact providing a bigger conceptual advance by performing some of the suggested experiments.

  6. Version published to 10.1101/2022.10.27.514025v2 on bioRxiv
  7. Version published to 10.1101/2022.10.27.514025v1 on bioRxiv