Transcription-independent hold of the G1/S transition is exploited to cope with DNA replication stress

  1. Yue Jin
  2. Guoqing Lan
  3. Jiaxin Zhang
  4. Haoyuan Sun
  5. Li Xin
  6. Qinhong Cao
  7. Chao Tang
  8. Xiaojing Yang
  9. Huiqiang Lou
  10. Wenya Hou

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Abstract

RB1 (retinoblastoma) members control the G1/S commitment as transcriptional repressors in eukaryotic cells. Here we uncover that an extra copy of RB1 equivalent ( WHI7 or WHI5 ) is sufficient to bypass the indispensability of the central genomic checkpoint kinases Mec1 ATR -Rad53 CHK1 in Saccharomyces cerevisiae . Mec1-Rad53 directly phosphorylate Whi7/5, antagonizing their nuclear export or protein turnover upon replication stress. Through in vitro reconstitution, we show that Whi7 C-terminus directly binds and hinders S-CDK-Cks1 from processively phosphorylating Sic1. By microfluidic single-cell real-time quantitative imaging, we demonstrate that both Whi7 and Whi5 are required to flatten the degradation curve of the major S-CDK inhibitor Sic1 in vivo. These findings reveal an eclipsed transcription-independent role of Whi7 homologs, which is highlighted by genome integrity checkpoints to hold the G1/S transition instantly as a rapid response to unforeseeable replication threats.

Key points

  • Whi7 overexpression bypasses the essential function of Mec1 and Rad53 in a transcription-independent way.

  • Whi7 is stabilized by checkpoint-mediated phosphorylation.

  • Whi7 binds and hinders S-CDK-Cks1 from multi-phosphorylation of Sci1, thereby prolonging Sic1 degradation and G1/S transition.

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      Reply to the reviewers

      Manuscript number: RC-2022-01756

      Corresponding author(s): Wenya, Hou

      1. General Responses

      Dear Editors and Reviewers,

      We deeply appreciate all critical comments and constructive suggestion from all Reviewers, which have inspired us to conceive at least 8 new important experiments and mathematic analysis/modeling (shown in dark red). In addition, we will include more repeats with quantification for spot assays (with more HU doses) and biochemical experiments as well as language revision (shown in orange).

      Below we only list the general response to the Major Concerns raised by at least two Reviewers:

      • To perform mathematic analysis of the single-cell quantitative data (Fig 4, Fig 5 and Fig S4) (Analysis #1).

      __50% Sic1 degradation time from Sic1peak __

      WT SC

      7.62 min

      whi7 whi5 SC

      7.91 min

      WT HU

      36 min

      whi7 whi5 del HU

      7.49 min

      50% nuclear exit time of Whi5

      WT SC

      4.69 min

      rad53Δsml1Δ SC

      7.60 min

      WT HU

      22.33 min

      rad53Δsml1ΔHU

      13.41 min

      Table R1. 50% Sic1 degradation time calculated from Sic1peak and 50% nuclear exit time of Whi5 based on the experimental data shown in Fig 5 and Fig 4, respectively.

      (2) To reinterpret the HU-induced extension of G1/S transition with an updated model (Analysis #2).

      (3) predict that like WHI7/5 overexpression, CKS1 deletion (PMID: 7958905) or* sic1* mutants with longer destruction timing (T2,5S-VLLPP or T2,5S-RXL reported in Fig. 6C, PMID: 32296067), can suppress the HU sensitivity of rad53 mutants according to our model. Moreover, their suppression effects should be epistatic to WHI7/5 overexpression. Alternatively, the dosage suppression of WHI7/5 might be reversed by *CKS1 *overexpression or sic1 mutants with shorter destruction timing (unfortunately no such mutant has been reported yet). We will perform this set of genetic experiment to test these predictions and thereby functionally reinforce the Whi7/5-Cks1-Sic1 axis (Experiment #1).

      (4) do DNA replication profiling to examine the number of origin firing or replication capacity (Experiment #2).

      (5) To address the suppression effect of phosphorylation in Fig 2E. We agree that the phenotypes of the A-mutants of Whi7 have a weak difference compared with WT, but become much stronger (5-fold difference between two dilutions) compared with the D-mutants. As shown lately in Fig 3, phosphorylation solely facilitates protein stabilization/total levels, which can be masked by ectopic overexpression from an extra plasmid. Moreover, phosphorylation does NOT enhance Whi7’s interaction with Cks1. We should tune down the contribution of phosphorylation and focus more on the stability/protein level. Furthermore, we will do competition assays using A-/D- mutants with GFP and RFP labels (Experiment #3), and add back whi7 13A or 13D in its endogenous locus in the whi7Δwhi5Δ double mutant to test the effect on Sic1 turnover (Experiment #4).

      (6) To add more repeats with quantification for spot assays (with more HU doses) and biochemical experiments (shown in orange).

      Besides reinforcing the current model, these experiments, analysis and re-interpretation may help to clarify two concepts which remain elusive in current version:

      • S-CDK activation can switch from an abrupt/all-or-none pattern under normal condition to a gradually flattened one under replication stress.
      • Consequently, the Whi7/5-Cks1-S-CDKs axis may determine replication capacity and/or number of origin firing. Thus, we did not include a preliminary revision this time due to significant changes. We plan to request at least 6 months for an extensive full revision (e.g., from a short letter to a regular article) to improve this study to a higher level with more general significance. Therefore, we request a revision opportunity from The EMBO Journal.

      2. Point-to-point responses

      Reviewer #1 (Evidence, reproducibility and clarity (Required)):

      SUMMARY

      This work begins with a heterologous screen, introducing human genes in double mec1,sml1 yeast deletants, which are alive, but sensitive to hydroxyurea. The readout was mec1,sml1 proliferation in the presence of hydroxyurea. They found that mec1,sml1 yeast mutants carrying the human RB1 gene (a G1/S transcriptional repressor) proliferated on hydroxyurea. Then, they test if known yeast G1/S transcriptional repressors (Whi5 and Whi7) could have similar effects if provided at higher than normal levels (they did). With this initial result, followed up by a variety of experiments, the authors then go on to propose that replication stress, which activates Mec1 and Rad53, triggers the phosphorylation of Whi7 (by Mec1) and Whi5 (by both Rad53 and Mec1) blocking their eviction from the nucleus, allowing them instead to bind and inhibit Cks1, a Cdk processivity factor, needed for the complete phosphorylation and degradation of a Cdk inhibitor, Sic1. This is different from published work a decade earlier in mammalian cells (ref. 37; Bertoli et al.), which showed that upon replication stress, Chk1 phosphorylates G1/S transcriptional repressors to maintain G1/S transcription, which could help genome stability. Here, the authors propose that replication stress could block the G1/S transition. While the model and some of the experiments are interesting, the rationale for some experiments was shaky, and the data do not fully support the conclusions.

      MAJOR POINTS

        • Any cell that undergoes DNA replication must have already destroyed Sic1. It has been known for 25+ years that targeting Sic1 is the only necessary function of G1/Cdk to enable DNA replication (PMID: 8755551). Sic1 does not reappear until the M/G1 transition. Hence, in the authors' model, where cells are already in the S phase, how can multisite phosphorylation and degradation of Sic1 be the critical and final output of the pathway they propose when there shouldn't be any Sic1 around, to begin with? Why would a cell that has already completed Start and the G1/S transition, is in the S phase and experiencing replication stress, care about going through the G1/S?* A: Yes, S-CDK activity is regarded as an abrupt or so-called “all-or-none transition” due to a relative short half-life of Sic1 controlled by a robust double-negative feedback loop (PMID: 24130459; 23230424). Sic1 degradation requires multi-phosphorylation events including prime phosphorylation by G1-CDKs, two opposing multi-phosphorylation by S-CDK complex (Clb5–Cdk1–Cks1), one to trigger phosphodegrons and the other to terminate the degron route (PMID: 32296067). The timing and speed (or “sharpness”) of Sic1 degradation is determined by G1-CDKs and S-CDKs, respectively (PMID: 24130459 and PMID: 32296067). Sic1 degradation is not an instantaneous “all-or-none” event even under the optimal growth conditions. The Sic1 destruction timing calculated from Start (defined as 50% nuclear exit of Whi5) is about 14.2 min, whereas the time between Start and Sic1peak is about 5 min from independent studies (Fig 4G, PMID: 24130459; Fig. 6C, PMID: 32296067; Fig. 7B, 32976810). Similarly, the 50% Sic1 degradation time calculated from Sic1peak (50% of Sic1peak) is about 8 min for WT and whi7, in agreement with the results in Figure 2E, PMID: 24130459. However, in the presence of HU, the 50% of Sic1peak time remains constant (7.49 min) in whi7Δwhi5Δ cells but becomes greater than 36 min in WT. Meanwhile, the 50% nuclear exit time of Whi5 (Start) is about 22 min in WT compared to 13 min in rad53Δsml1Δ upon HU treatment.

      __50% Sic1 degradation time from Sic1peak __

      WT SC

      7.62 min

      whi7 whi5 SC

      7.91 min

      WT HU

      36 min

      whi7 whi5 del HU

      7.49 min

      50% nuclear exit time of Whi5

      WT SC

      4.69 min

      rad53Δsml1Δ SC

      7.60 min

      WT HU

      22.33 min

      rad53Δsml1ΔHU

      13.41 min

      Table R1. 50% Sic1 degradation time calculated from Sic1peak and 50% nuclear exit time of Whi5 based on the experimental data shown in Fig 5 and Fig 4, respectively.

      Therefore, G1/S transition is a “transition zone” (from Start to 50% of Sic1peak) rather than a single borderline. The key finding of this study is that in the presence of HU, Sic1 degradation speed/sharpness is significantly reduced (Figure 5), mechanistically due to the inhibition of S-CDK-Cks1 by Whi7/5. This eventually reflects a flattened S-CDK activity curve, no longer an “all-or-none activation” any more upon replication stress. S-CDKs phosphorylate the two essential targets (Sld2 and Sld3) to enable DNA replication. Therefore, the Sic1 levels determine the S-CDK activities, which in turn determine the DNA replication capacity (the maximal amount of DNA a cell can synthesize per unit time). In sum, under optimal conditions, the S-CDK activity appears an abrupt/sharp transition and cells replicate DNA in its maximum capacity (i.e., minimal S phase length). When cells encounter replication stress (HU), S-CDK is activated very slowly (very low Sic1 destruction speed) and replicate DNA with a low capacity (slow fork speed and/or few origin firing) to meet the limited resource. Recently, the de Bruin group demonstrates that replication capacity can be tuned by E2F-dependent transcription (includes S-Cyclin genes) in mammalian cells (PMID: 32665547).

      Inspired by these questions, we plan to

      (1) perform mathematic analysis of the single-cell quantitative data (Fig. 5 and S4) (Analysis #1).

      (2) reinterpret the HU-induced extension of G1/S transition with an updated model (Analysis #2).

      (3) predict that like WHI7/5 overexpression, CKS1 deletion (PMID: 7958905) or* sic1* mutants with longer destruction timing (T2,5S-VLLPP or T2,5S-RXL reported in Fig. 6C, PMID: 32296067), can suppress the HU sensitivity of rad53 mutants according to our model. Moreover, their suppression effects should be epistatic to WHI7/5 overexpression. Alternatively, the dosage suppression of WHI7/5 might be reversed by *CKS1 *overexpression or sic1 mutants with shorter destruction timing (unfortunately no such mutant has been reported yet). We will perform this set of genetic experiment to test these predictions and thereby functionally reinforce the Whi7/5-Cks1-Sic1 axis (Experiment #1).

      (4) do DNA replication profiling to examine the number of origin firing or replication capacity (Experiment #2).

      • The results in Figure 2C are confusing and difficult to interpret. For example, comparing lane 8 (WT without hydroxyurea) to lane 7 (WT with hydroxyurea), it appears that there is more phosphorylated Whi7 in lane 7 (hydroxyurea treatment) than in lane 8 (no treatment). But, the ratio of phosphorylated/unphosphorylated Whi7 is not that different (there is very little unphosphorylated Whi7 in lane 8). Same problem when comparing lanes 3 and 4. I understand that they later show that Whi7 is stabilized by hydroxyurea, but from the data in this figure, what exactly can they conclude here?*

      A: Yes, phosphorylation is a bit confusing according to the current statement. Without HU, Whi7 is phosphorylated by G1-CDKs with a much less total protein level as well. With HU, whi7 is phosphorylated by Mec1 and Rad53, because Whi7-P largely disappeared in rad53 mutant (lane 1) and 13A (with all putative Mec1-Rad53 sites mutated, lane 5). Lanes 3 and 4 are biological repeats of Lanes 7-8 with less loading. We will clarify our statement.

      • Their data in Figure 2E show that phosphorylation of Whi7 is not required for suppressing the lethality of rad53,sml1 cells treated with hydroxyurea. Cells carrying Whi7-41A (lacking all possible phosphorylations) suppressed nearly as well as wild-type Whi7 did. The purported differences in the suppression are minuscule at best and not evident at the dilutions tested. It is not clear at all how they can conclude that phosphorylation of Whi7 has anything to do with the ability of Whi7 overexpression to suppress the lethality of rad53,sml1 cells.*

      A: Yes, we agree that the phenotypes of the A-mutants of Whi7 have a weak difference compared with WT, but become much stronger (5-fold difference between two dilutions) compared with the D-mutants. As shown lately in Fig 3, phosphorylation solely facilitates protein stabilization/total levels, which can be masked by ectopic overexpression from an extra plasmid. Moreover, phosphorylation does NOT enhance Whi7’s interaction with Cks1.

      Anyway, we should tune down the contribution of phosphorylation and focus more on the stability/protein level. Furthermore, we will do competition assays using A-/D- mutants with GFP and RFP labels __(Experiment #3) __and add back whi7 13A or 13D in its endogenous locus in the whi7

      • For all the arguments they make about this new role of Whi5 and Whi7 at Start, they do not examine size homeostasis or the kinetics of cell cycle progression in any of their experiments and their mutants, with or without hydroxyurea treatment.*

      A: Good suggestion. We will examine size homeostasis, budding index or the cell cycle progression in the related experiments (Experiment #5). In Fig. S5, we only showed the cell cycle progression profiles in wild-type cells carrying an extra copy of Whi7 WIQ or Whi7 WIQ ΔC. WIQ mutant (without Swi6 binding activity) significantly slowed the cell cycle progression under normal conditions.

      • The Sic1 stability experiments they show in Figure 5 are nice. They would need to be extended to their various mutants, including their Whi7 phosphomutants, to make a case for phosphorylation by Rad53 and Mec1 in this process.*

      A: Thanks, very good suggestion, we will add back whi7 13A or 13D in its endogenous locus in the whi7Δwhi5Δ double mutant (Experiment #4), to avoid the effects of overexpression.

      MINOR POINTS

        • The language is awkward. Editing for style will be necessary.* A: We will request language editing.
      • They use different hydroxyurea doses in the experiments they show, making it difficult to conclude much when comparing different figures. Why aren't they consistent from experiment to experiment?*

      A: Sorry for the confusing. We used at least three HU concentration gradients in each experiment, but only showed one of them to save the space for a short article. Notably, S. cerevisiae has a much broader range of HU doses (up to 300 mM) than other species (less than 10 mM). We’ll add other Figures during revision.

      **Referees cross-commenting**

      Overall, all reviews are well-aligned. The points raised by the other reviewers are valid, and the reviews are thorough and detailed. I don't know whether the authors will be able to respond since the list is quite long. Even if they do, the manuscript will look very different. I do not have anything else to add.

      Reviewer #1 (Significance (Required)):

      The manuscript presents some interesting data, most notably the role of Whi7 and Whi5 in the stability of Sic1 in vivo and the various in vitro experiments the authors present. The advance is conceptual and mechanistic, offering a different and unanticipated model for the role of these proteins at Start, under replication stress. Unfortunately, the significance of the manuscript is limited. A convincing case for their model and its importance has not been made. For example, their data in Figure 2E, measuring the ability of phosphomutants to suppress the lethality of rad53,sml1 cells upon replication stress, is underwhelming and undermines the importance of the study, particularly to a wider audience.

      A: Thanks for the suggestion, we will improve the model as discussed above.

      Reviewer #2 (Evidence, reproducibility and clarity (Required)):

      Summary:

      Jin et al demonstrate a novel type of regulation of the G1/S transition in response to hydroxyurea stress. They approach this by first screening a library of human proteins (cDNA on yeast plasmids) for repressors of the mec1 or rad53 HU sensitivity. HU inhibits ribonucleotide reductase and thus lowers dNTP pools needed for S-phase. This slows replication and leads to stalled replication forks, triggering a "replication stress" response, which is executed by the kinases Mec1 and Rad53. Deletions of mec1 or rad53 are viable in unstressed conditions (with additional sml1 deletion), but are lethal on even low doses of HU. One main hit that rescued this lethality was the human G1/S inhibitor RB. They then went on to confirm that also the yeast analogs Whi5 and Whi7 can rescue mec1 or rad53 lethality when overexpressed. To track down the mechanism, the authors do a variety of genetic and biochemical assays. The resulting model is that Mec1 and Rad53 phosphorylate and stabilize Whi7, which binds to and inhibits the S-phase-CDK complex via the processivity factor Cks1. So on top of acting as a transcriptional repressor, Whi7 (and probably also Whi5) is also a direct interactor and inhibitor of CDK. The binding of Whi7 to Cks1-Clb5/6-CDK prevents the hyperphosphorylation and degradation of the inhibitor Sic1, and thus slows the G1/S transition in response to HU.

      Major comments:

      - Are the key conclusions convincing?

      ->Overall I think the sum of the evidence supports the suggested model, individual claims though are on somewhat shaky grounds based often on single replicates, see below.

      My main conceptual issue may be somewhat just a "semantic" problem. In my understanding "replication stress" refers to stalled replications forks and/or large stretches of single-strand DNA which then triggers a checkpoint response. So how would slowing the G1/S transition help to deal with "replication stress", if replication is not yet happening in these cells? I am assuming Mec1 senses dNTP depletion also in the absence of replication and that is how Mec1 and Rad53 become active in G1. But then maybe the model and the arguments can be phrased differently? What exactly is slowing down Sic1 degradation doing for the cell? Replenishing dNTP pools before the first origins fire? Or is maybe Sic1 not the most important target of this regulation? Maybe also during S-phase, partially inhibiting CDK is beneficial, maybe to stretch out origin firing... or?

      A: Thank you, very good suggestion. This also helps to address the Major Point 1 raised by Reviewer #1. This also reminds us about the work from Pasero’s group demonstrating that Mec1 is activated at the onset of normal S phase by low dNTPs (PMID: 32169162). We will revise the text, and do DNA replication profiling __(Experiment #2) __to examine the number of origin firing or replication speed. Also see response to Point 1 of Reviewer #1.

      - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

      ->Most of the work is done on Whi7 and then some Whi5 in the end, I would tone down on the Whi5 claims a bit.

      A: Very good suggestion. We have to include Whi5 in the story because it plays a redundant role with Whi7.

      - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

      -> Since the authors are clearly able to do quantitative live cell imaging, I do not understand why they do not quantify Whi7 concentrations and localization in response to HU instead of using Western blots of synchronized cells. This would make the whole thing much more credible, especially given the current lack of replicates (see below). This would also allow correlating the timing and amount of the Whi7 response with the stabilizing of Sic1 in single cells.

      A: Yes, we tried but did not see Whi7-GFP clearly because of its very low protein abundance, which is also not shown in literature as far as we know. Only overexpressed Whi7 fluorescence detection(PMID: 33443080).

      ->The causality of phosphorylation being required for stabilization seems plausible from the genetics, but is far from clear in the western blots. Here, concentration increase seems to precede phosphorylation. Could this due to induced Whi7 transcription?

      A: Good suggestion. We will detect Whi7 mRNA levels through qPCR (Experiment #6).

      ->Many if not most claims are based on single replicates. See below.

      - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

      -I am not suggesting any different types of experiments or new methods, so it should be doable within a few weeks.

      - Are the data and the methods presented in such a way that they can be reproduced?

      -I would suggest the authors spell out all of their experimental procedures instead of referring to "as described previously". I think everyone knows the pains of going on a wild goose chase of following references to the original method description.

      A: Good suggestion. I will described all experimental procedures to replace "as described previously".

      - Are the experiments adequately replicated and statistical analysis adequate?

      -The key weakness of this entire paper is imho that many claims are based on single experiments, that are neither replicated nor quantified. For example, all the co-IPs (such as 1E or 3F) should be replicated and the ratio of bait to target quantified and averaged.

      A: Good suggestion. We will show the biological repeats and quantification.

      -If a claim is made regarding increased phosphorylation in vivo, then again this should be replicated and the ratio of phosphorylated to unphosphorylated bands quantified. In many Whi7 gels it looks like it is mainly the total amount of the protein that is changing rather than the phosphorylation state. But again, by eye and from a single replicate, this is hard to tell.

      A: Good suggestion. We will add more repeats.

      -A similar thing holds true for the spot assays. Spot assays are great to show lethality and rescue as in the first figure. But making semi-quantitative claims of different degrees of "partial rescue" from a single spot assay is a bit speculative. This seems especially true since the authors are using different and seemingly random HU concentrations for every spot assay, which suggests that the effect is not very robust and can only be seen in very specific concentration ranges. If e.g. the degree of rescue between WT, A and D mutants or truncations matters for the model/the storyline, then more quantitative growth or competition assays should be added.

      A: Good suggestion. sorry for the confusing. We used at least three HU concentration gradients in each experiment, but only showed one of them to save the space for a short article. Notably, S. cerevisiae has a much broader range of HU doses (up to 300 mM) than other species (less than 10 mM). We’ll add other Figures during revision, and do competition assays using A-/D- mutants with GFP and RFP labels

      Minor comments:

      - Specific experimental issues that are easily addressable.

      ->At least some of the alpha-factor release experiments should contain infos on budding index and/or DNA content to understand see the delay in timing by HU addition.

      A: Good suggestion. We will examine size homeostasis, budding index or the cell cycle progression in the related experiments (Experiment #5).

      - Are prior studies referenced appropriately?

      ->Seems fine from the G1/S side, but I don't know the Mec1/Rad53 literature well enough to judge.

      - Are the text and figures clear and accurate?

      ->The authors could do another round of proofing figures and legends. For example, Fig 5C contains scale bars that are not defined, blot 3E has an asterix labeling that is not defined, the model in 5E has misspelled "degradation"...

      A: We will proofread and revise the full text again.

      - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

      -> The authors use a lot of different mutants (especially for Whi7). Even for someone who knows the proteins fairly well, it is hard to remember throughout the text which abbreviation is relating to which mutations and which function that is addressing. Maybe occasionally remind the reader of what the mutant is or use terms like Whi7non-binding rather than WIQ.

      A: Thank you for your suggestion. We will add (TF non-binding) after WIQ.

      ->The text could also use another round of proof-reading. The overall flow of the storyline is easily comprehensible, but sometimes there is a sudden switch of topics or new proteins come out of nowhere. Some expressions are used in a way that is not common English.

      A: We will request language editing.

      Reviewer #2 (Significance (Required)):

      - Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

      ->This study is a major conceptual contribution to understanding G1/S regulation in perturbed conditions (assuming the results can be replicated and quantified as detailed above). That Whi7 (and maybe Whi5) directly inhibit Clb5/Clb6-CDK through Cks1 binding is an important addition/modification to the current model and may well be important beyond genotoxic stress.

      A: Thanks and we’ll reinforce it with more repeats and quantification.

      - Place the work in the context of the existing literature (provide references, where appropriate).

      ->The authors do this reasonably well.

      - State what audience might be interested in and influenced by the reported findings.

      -> Anyone in the yeast cell cycle/replication field should find this interesting. It should also have important implications for the mammalian cell cycle/replication/DNA damage field.

      - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

      ->I am well familiar with G1/S control and all the methods used in the study. I am not an expert on replication stress/DNA damage/ checkpoint signaling.

      Reviewer #3 (Evidence, reproducibility and clarity (Required)):

      Summary

      In their manuscript "Transcription-independent hold of the G1/S transition is exploited to cope with DNA replication stress", Jin et al. intend to show that Retinoblastoma-like G1/S transcriptional repressors can also work as S-CDK-Cks1 inhibitors in response to DNA replication stress, hence prolongating the G1/S transition to enable cells to deal with replication stress. In particular, they aim to identify the mechanism by which Whi7/Srl3 (Suppressor of Rad53 Lethality) rescues the lethality of rad53 yeast mutants. Even though their very first experiment is performed using human RB1, the remainder or the work is performed in the yeast model organism. Experimental methods used include mostly immunoprecipitation experiments (Western blots), spot assays, and some single cell microscopy (not specified if widefield or confocal).

      Major comments

      1) the authors refer to a cross-species screen where they aim to detect human proteins that rescue, upon overexpression, the yeast mec1Dsml1D and rad53Dsml1D lethality (of note, not mec1D/rad53D: why?). They identify hRB1 this way. But the entire screen data is missing, either is the analysis pipeline and "hit selection thresholds" (if applicable). Then no more experiments are performed on human cells or using human proteins. In my opinion this cross-specie approach is not necessary, or not developed enough.

      A: Yes, we have only performed a pilot screen based on the growing on 4 mM HU. We consider removing it. The reason to use *mec1Dsml1D *for genetic screen is that mec1D/rad53D cells are dead even without HU, whereas dissection assays do not fit for large-scale screening.

      2) Moreover, the interpretation of the data provided as a whole is strongly complicated by the variability in the HU doses used to trigger the Mec1/Rad53 response. While most immunoprecipitation experiments are performed with 200mM, spot assays are performed at various HU concentrations ranging from 3 to 21mM (and exploring the entire range). Sometimes HU concentrations differ on the same Figure panels. Downstream effects of such diverse HU concentrations might also be very diverse and due to this it is difficult to get an understanding of how the different experiments fit together.

      A: Sorry for the confusing. We used at least three HU concentration gradients in each experiment, but only showed one of them to save the space for a short article. Notably, S. cerevisiae has a much broader range of HU doses (up to 300 mM) than other species (less than 10 mM). Spot assays (HU are persistent) are mostly done in the mec1Dsml1D and rad53Dsml1D background (sensitive to 4 mM HU), whereas the IP experiments (only 2-3 h treatment and then removal) are mainly performed in WT or at least in comparison with WT background (resistant up to 250 mM HU). We’ll add other Figures during revision.

      3) Likewise, some experiments are performed only on rad53D backgrounds, or only on mec1D backgrounds (e.g. Fig1B and Fig1F, respectively), while results are claimed valid for the two gene deletion backgrounds.

      A: Thank you. We will add some “not shown data” and remove the invalid claims without data.

      4) Finally, the experiments performed in this study and/or their quantitative analysis are insufficient to support several of the claims, and results are often "over-interpreted". Below I have listed some of such insufficient experiments/analyses, in regard of the interpretation that the authors make of each piece of data.

      - Fig1B could indeed show that Whi7 could rescue rad53D lethality but it is hard to judge from just one tetrad. Many tetrads should be shown to exclude "random sampling" effects.

      A: Thank you. We will add more repeats and remove over-statements. Fig 1B was carried out for at least 12 tetrads but the original picture has been unintentionally lost. We can repeat it if necessary, but the result was validated by the plasmid shuffling experiment (Fig 1C).

      - Fig1F indeed shows that the rescue effect of Whi7 overexpression on mec1Dsml1D lethality in HU does not require its G1/S transcription factor-binding motif (GTB); however, it does not prove that it is independent on any putative effects that Whi7 could have on transcription (it could affect other transcription factors, or even the same ones via other domains).

      A: Good suggestion. As far as we know, there are no reports proving that Whi7 binds to other transcription factors. To rule out this possibility, we will detect whether overexpression of WHI7 affects the transcription of representative G1/S genes (Experiment #7).

      - FigS2A does not really support the authors' claim that Whi7 is hyperphosphorylated upon HU-treatment: the first lane before HU treatment already show the same hyperphosphorylated bands than the second lane (see "darker exposure"); however, the signal intensity is clearly lower so the overall levels of Whi7 are clearly increased by HU, rather than the relative fractions of phosphorylated species.

      A: Yes, we will modify the statement as suggested.

      - Fig2B shows that HU-dependent increase in Whi7 levels is partially abrogated in rad53Dsml1D and mec1Dsml1D mutant backgrounds, which demonstrates that Whi7 upregulation requires either Rad53 or Sml1, and Mec1 or Sml1, but not Rad53/Mec1 as claimed by the authors.

      A: Thank you, we will revise the statement. The only known function of Sml1 is a small unstructured protein inhibitor of Rnr1.

      - Likewise, Fig2B does not show any significant Whi7 phosphorylation following HU-treatment in the whi7-13AP mutant with all CDK consensus sites mutated to alanine. There is indeed a slightly slower migrating band appearing as acknowledge by the authors, which also appears in the mec1Dsml1D and rad53Dsml1D backgrounds. Again here, higher Whi7 levels in the WT background make the comparison with mec1Dsml1D and rad53Dsml1D backgrounds almost impossible. Quantification of the blots, including normalization of the signals of each phosphorylated band to the total signal, could help. But overall this figure does not demonstrate any Mec1/Rad53-dependent Whi7 phosphorylation following HU treatment. The phostag gel Fig2C might show the same result, as the differences in phosTag signals between different conditions might just simply reflect the differences in total amount of Whi7 between those same conditions. However, I acknowledge that Figs 2D and S2C shows Rad53- and Mec1-triggered Whi7 phosphorylation in vitro, but the conditions of this experiments likely differ a lot from in vivo context (kinase levels, competing substrates, presence of co-factors...).

      A: Thank you, we will quantify the blotting as suggested.

      - Along the same lines, Fig3E seems to show that truncation of Whi7 C terminus slightly reduces its efficiency in pulling down Cks1 (indicating reduced interaction). However, the total amount of WT Whi7 in the pull down seems to exceed the total amount of Whi7-DeltaC protein, which could in part explain the difference in Cks1 signal. Here again, quantification of the WB signals and adequate normalization would maybe make this figure more convincing.

      A: Good suggestion. We will show the biological repeats and quantification.

      - Fig4A-B (Whi5 GFP data): the cell representing the absence of HU shows Whi5 nuclear export and therefore likely passes through G1/S; the HU-treated cell shown as example does not export Whi5 from the nucleus, certainly because it does not pass G1/S. IMHO this demonstrates that the G1/S transition is delayed in HU-treated cells (as shown previously), irrespective of any role of Whi5 or Whi7 in this delay.

      - Likewise, Fig4C shows the absence of HU-induced delay in Whi5 nuclear export in rad53Dsml1D cells; however, while the authors claim this indicates "Rad53-dependent nuclear detention of Whi5", it is equally plausible that it indicates that rad53Dsml1D cells do not delay the G1/S transition under HU treatment.

      A: good comments. We should claim both possibilities at this stage. Previous studies mainly show delays in the Start stage (e.g., down-regulate SBF transcription). CLN1/2 deletion is known to delay DNA replication in a Sic1-dependent manner albeit with unknown mechanism in the S-CDK activation stage.

      - The same ambiguity holds for Fig5A,B (Sic1-GFP quantification in whi5Dwhi7D double deletion strain following release from alpha factor block): indeed Sic1 is degraded fast after release from alpha factor block both in presence of HU, while in WT cells Sic1 is not immediately degraded in presence of HU. While authors claim that "Whi7 and Whi5 significantly slow down the Sic1 degradation", this result could also likely reflect that whi5Dwhi7D cells pass G1/S even in this context, and therefore that whi5 or whi7 or both have a role in maintaining cells in G1, not showing any direct implication of Whi5/Whi7 in Sic1 degradation.

      A: good comments. It only provides some indirect hints. For instance, whi5Dwhi7D cells pass G1/S in a same timing as WT in the absence of HU (Fig. S4), indicating that the role of Whi5/7 in the G1/S delay is related to additional checkpoint function, not normal G1 maintaining function. Moreover, it should be combined with other results, for example, dosage suppression effects in the presence of HU and inhibitory effects in the absence of HU. Direct evidence of Whi5/Whi7 in Sic1 degradation and Cks1 inhibition comes only from the biochemical experiments shown in Fig 3E-3H.

      - FigS5: the authors show here that overexpression of Whi7-WIQ (that does not bind SBF) slows down the G1/S transition following release from alpha factor blockade, but this data does not demonstrate anything related to the role of Whi7 in the DNA replication stress response. Indeed, since Whi7 sequesters Cln3 in the ER (independent of any putative role on transcription regulation), its overexpression could simply reflect an increased sequestering of Cln3 pool. What does this result become in a cln3D background?

      A: Very good suggestion. We will check whether cln3Δ affects the suppression effect of Whi7 (Experiment #8).

      Due to the fundamental concerns raised above in the interpretation of the data, it is difficult to predict the outcome of more controlled experiments that would aim to prove the same statements. This makes the estimation of the time and resources required to complete the study almost impossible.

      Minor comments

      Owing to the major comments above, an important re-structuration of the study is required, and minor comments I may have on this version are likely to be irrelevant to the revised manuscript.

      Reviewer #3 (Significance (Required)):

      The study aims to establish a molecular link between the progression through the G1/S transition and the DNA damage and DNA replication stress responses. Establishing molecular links between different phases of the cell cycle is an important question in basic research, and might be of interest for a broad range of cell biologists, even though the study is conducted in a model organism (budding yeast). The link proposed involves G1/S inhibitors Whi5 and Whi7, that would bind and inhibit the Cks1 subunit of S-CDK complexes, downstream of Rad53 and Mec1 signaling. The authors confirm some known results (e.g., Whi7 overexpression bypasses rad53 lethality in presence of HU) and gather new pieces of data using well-established methods (immunoprecipitation, spot assays, fluorescence microscopy). However, many experiments reported in this study are not sufficient to support the authors' claims, and therefore the novel mechanistic insight that this study ambitions to provide is not established.

      My scientific background being more in bio-imaging than in biochemistry, it is possible that I missed some hands-on experience to correctly interpret artefacts on western blots, however I do not feel like I missed sufficient expertise to evaluate any section of the manuscript.

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      Referee #3

      Evidence, reproducibility and clarity

      Summary

      In their manuscript "Transcription-independent hold of the G1/S transition is exploited to cope with DNA replication stress", Jin et al. intend to show that Retinoblastoma-like G1/S transcriptional repressors can also work as S-CDK-Cks1 inhibitors in response to DNA replication stress, hence prolongating the G1/S transition to enable cells to deal with replication stress. In particular, they aim to identify the mechanism by which Whi7/Srl3 (Suppressor of Rad53 Lethality) rescues the lethality of rad53 yeast mutants. Even though their very first experiment is performed using human RB1, the remainder or the work is performed in the yeast model organism. Experimental methods used include mostly immunoprecipitation experiments (Western blots), spot assays, and some single cell microscopy (not specified if widefield or confocal).

      Major comments

      1. the authors refer to a cross-species screen where they aim to detect human proteins that rescue, upon overexpression, the yeast mec1Dsml1D and rad53Dsml1D lethality (of note, not mec1D/rad53D: why?). They identify hRB1 this way. But the entire screen data is missing, either is the analysis pipeline and "hit selection thresholds" (if applicable). Then no more experiments are performed on human cells or using human proteins. In my opinion this cross-specie approach is not necessary, or not developed enough.
      2. Moreover, the interpretation of the data provided as a whole is strongly complicated by the variability in the HU doses used to trigger the Mec1/Rad53 response. While most immunoprecipitation experiments are performed with 200mM, spot assays are performed at various HU concentrations ranging from 3 to 21mM (and exploring the entire range). Sometimes HU concentrations differ on the same Figure panels. Downstream effects of such diverse HU concentrations might also be very diverse and due to this it is difficult to get an understanding of how the different experiments fit together.
      3. Likewise, some experiments are performed only on rad53D backgrounds, or only on mec1D backgrounds (e.g. Fig1B and Fig1F, respectively), while results are claimed valid for the two gene deletion backgrounds.
      4. Finally, the experiments performed in this study and/or their quantitative analysis are insufficient to support several of the claims, and results are often "over-interpreted". Below I have listed some of such insufficient experiments/analyses, in regard of the interpretation that the authors make of each piece of data.
      • Fig1B could indeed show that Whi7 could rescue rad53D lethality but it is hard to judge from just one tetrad. Many tetrads should be shown to exclude "random sampling" effects.
      • Fig1F indeed shows that the rescue effect of Whi7 overexpression on mec1Dsml1D lethality in HU does not require its G1/S transcription factor-binding motif (GTB); however, it does not prove that it is independent on any putative effects that Whi7 could have on transcription (it could affect other transcription factors, or even the same ones via other domains).
      • FigS2A does not really support the authors' claim that Whi7 is hyperphosphorylated upon HU-treatment: the first lane before HU treatment already show the same hyperphosphorylated bands than the second lane (see "darker exposure"); however, the signal intensity is clearly lower so the overall levels of Whi7 are clearly increased by HU, rather than the relative fractions of phosphorylated species.
      • Fig2B shows that HU-dependent increase in Whi7 levels is partially abrogated in rad53Dsml1D and mec1Dsml1D mutant backgrounds, which demonstrates that Whi7 upregulation requires either Rad53 or Sml1, and Mec1 or Sml1, but not Rad53/Mec1 as claimed by the authors.
      • Likewise, Fig2B does not show any significant Whi7 phosphorylation following HU-treatment in the whi7-13AP mutant with all CDK consensus sites mutated to alanine. There is indeed a slightly slower migrating band appearing as acknowledge by the authors, which also appears in the mec1Dsml1D and rad53Dsml1D backgrounds. Again here, higher Whi7 levels in the WT background make the comparison with mec1Dsml1D and rad53Dsml1D backgrounds almost impossible. Quantification of the blots, including normalization of the signals of each phosphorylated band to the total signal, could help. But overall this figure does not demonstrate any Mec1/Rad53-dependent Whi7 phosphorylation following HU treatment. The phostag gel Fig2C might show the same result, as the differences in phosTag signals between different conditions might just simply reflect the differences in total amount of Whi7 between those same conditions. However, I acknowledge that Figs 2D and S2C shows Rad53- and Mec1-triggered Whi7 phosphorylation in vitro, but the conditions of this experiments likely differ a lot from in vivo context (kinase levels, competing substrates, presence of co-factors...).
      • Along the same lines, Fig3E seems to show that truncation of Whi7 C terminus slightly reduces its efficiency in pulling down Cks1 (indicating reduced interaction). However, the total amount of WT Whi7 in the pull down seems to exceed the total amount of Whi7-DeltaC protein, which could in part explain the difference in Cks1 signal. Here again, quantification of the WB signals and adequate normalization would maybe make this figure more convincing.
      • Fig4A-B (Whi5 GFP data): the cell representing the absence of HU shows Whi5 nuclear export and therefore likely passes through G1/S; the HU-treated cell shown as example does not export Whi5 from the nucleus, certainly because it does not pass G1/S. IMHO this demonstrates that the G1/S transition is delayed in HU-treated cells (as shown previously), irrespective of any role of Whi5 or Whi7 in this delay.
      • Likewise, Fig4C shows the absence of HU-induced delay in Whi5 nuclear export in rad53Dsml1D cells; however, while the authors claim this indicates "Rad53-dependent nuclear detention of Whi5", it is equally plausible that it indicates that rad53Dsml1D cells do not delay the G1/S transition under HU treatment.
      • The same ambiguity holds for Fig5A,B (Sic1-GFP quantification in whi5Dwhi7D double deletion strain following release from alpha factor block): indeed Sic1 is degraded fast after release from alpha factor block both in presence of HU, while in WT cells Sic1 is not immediately degraded in presence of HU. While authors claim that "Whi7 and Whi5 significantly slow down the Sic1 degradation", this result could also likely reflect that whi5Dwhi7D cells pass G1/S even in this context, and therefore that whi5 or whi7 or both have a role in maintaining cells in G1, not showing any direct implication of Whi5/Whi7 in Sic1 degradation.
      • FigS5: the authors show here that overexpression of Whi7-WIQ (that does not bind SBF) slows down the G1/S transition following release from alpha factor blockade, but this data does not demonstrate anything related to the role of Whi7 in the DNA replication stress response. Indeed, since Whi7 sequesters Cln3 in the ER (independent of any putative role on transcription regulation), its overexpression could simply reflect an increased sequestering of Cln3 pool. What does this result become in a cln3D background? Due to the fundamental concerns raised above in the interpretation of the data, it is difficult to predict the outcome of more controlled experiments that would aim to prove the same statements. This makes the estimation of the time and resources required to complete the study almost impossible.

      Minor comments

      Owing to the major comments above, an important re-structuration of the study is required, and minor comments I may have on this version are likely to be irrelevant to the revised manuscript.

      Significance

      The study aims to establish a molecular link between the progression through the G1/S transition and the DNA damage and DNA replication stress responses. Establishing molecular links between different phases of the cell cycle is an important question in basic research, and might be of interest for a broad range of cell biologists, even though the study is conducted in a model organism (budding yeast). The link proposed involves G1/S inhibitors Whi5 and Whi7, that would bind and inhibit the Cks1 subunit of S-CDK complexes, downstream of Rad53 and Mec1 signaling. The authors confirm some known results (e.g., Whi7 overexpression bypasses rad53 lethality in presence of HU) and gather new pieces of data using well-established methods (immunoprecipitation, spot assays, fluorescence microscopy). However, many experiments reported in this study are not sufficient to support the authors' claims, and therefore the novel mechanistic insight that this study ambitions to provide is not established.

      My scientific background being more in bio-imaging than in biochemistry, it is possible that I missed some hands-on experience to correctly interpret artefacts on western blots, however I do not feel like I missed sufficient expertise to evaluate any section of the manuscript.

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      Referee #2

      Evidence, reproducibility and clarity

      Summary:

      Jin et al demonstrate a novel type of regulation of the G1/S transition in response to hydroxyurea stress. They approach this by first screening a library of human proteins (cDNA on yeast plasmids) for repressors of the mec1 or rad53 HU sensitivity. HU inhibits ribonucleotide reductase and thus lowers dNTP pools needed for S-phase. This slows replication and leads to stalled replication forks, triggering a "replication stress" response, which is executed by the kinases Mec1 and Rad53. Deletions of mec1 or rad53 are viable in unstressed conditions (with additional sml1 deletion), but are lethal on even low doses of HU. One main hit that rescued this lethality was the human G1/S inhibitor RB. They then went on to confirm that also the yeast analogs Whi5 and Whi7 can rescue mec1 or rad53 lethality when overexpressed. To track down the mechanism, the authors do a variety of genetic and biochemical assays. The resulting model is that Mec1 and Rad53 phosphorylate and stabilize Whi7, which binds to and inhibits the S-phase-CDK complex via the processivity factor Cks1. So on top of acting as a transcriptional repressor, Whi7 (and probably also Whi5) is also a direct interactor and inhibitor of CDK. The binding of Whi7 to Cks1-Clb5/6-CDK prevents the hyperphosphorylation and degradation of the inhibitor Sic1, and thus slows the G1/S transition in response to HU.

      Major comments:

      • Are the key conclusions convincing?

      Overall I think the sum of the evidence supports the suggested model, individual claims though are on somewhat shaky grounds based often on single replicates, see below.

      My main conceptual issue may be somewhat just a "semantic" problem. In my understanding "replication stress" refers to stalled replications forks and/or large stretches of single-strand DNA which then triggers a checkpoint response. So how would slowing the G1/S transition help to deal with "replication stress", if replication is not yet happening in these cells? I am assuming Mec1 senses dNTP depletion also in the absence of replication and that is how Mec1 and Rad53 become active in G1. But then maybe the model and the arguments can be phrased differently? What exactly is slowing down Sic1 degradation doing for the cell? Replenishing dNTP pools before the first origins fire? Or is maybe Sic1 not the most important target of this regulation? Maybe also during S-phase, partially inhibiting CDK is beneficial, maybe to stretch out origin firing... or?

      • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

      Most of the work is done on Whi7 and then some Whi5 in the end, I would tone down on the Whi5 claims a bit.

      • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

      Since the authors are clearly able to do quantitative live cell imaging, I do not understand why they do not quantify Whi7 concentrations and localization in response to HU instead of using Western blots of synchronized cells. This would make the whole thing much more credible, especially given the current lack of replicates (see below). This would also allow correlating the timing and amount of the Whi7 response with the stabilizing of Sic1 in single cells.

      The causality of phosphorylation being required for stabilization seems plausible from the genetics, but is far from clear in the western blots. Here, concentration increase seems to precede phosphorylation. Could this due to induced Whi7 transcription?

      Many if not most claims are based on single replicates. See below.

      • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

      I am not suggesting any different types of experiments or new methods, so it should be doable within a few weeks.

      • Are the data and the methods presented in such a way that they can be reproduced?

      I would suggest the authors spell out all of their experimental procedures instead of referring to "as described previously". I think everyone knows the pains of going on a wild goose chase of following references to the original method description.

      • Are the experiments adequately replicated and statistical analysis adequate?

      The key weakness of this entire paper is imho that many claims are based on single experiments, that are neither replicated nor quantified. For example, all the co-IPs (such as 1E or 3F) should be replicated and the ratio of bait to target quantified and averaged.

      If a claim is made regarding increased phosphorylation in vivo, then again this should be replicated and the ratio of phosphorylated to unphosphorylated bands quantified. In many Whi7 gels it looks like it is mainly the total amount of the protein that is changing rather than the phosphorylation state. But again, by eye and from a single replicate, this is hard to tell.

      A similar thing holds true for the spot assays. Spot assays are great to show lethality and rescue as in the first figure. But making semi-quantitative claims of different degrees of "partial rescue" from a single spot assay is a bit speculative. This seems especially true since the authors are using different and seemingly random HU concentrations for every spot assay, which suggests that the effect is not very robust and can only be seen in very specific concentration ranges. If e.g. the degree of rescue between WT, A and D mutants or truncations matters for the model/the storyline, then more quantitative growth or competition assays should be added.

      Minor comments:

      • Specific experimental issues that are easily addressable.

      At least some of the alpha-factor release experiments should contain infos on budding index and/or DNA content to understand see the delay in timing by HU addition.

      • Are prior studies referenced appropriately?

      Seems fine from the G1/S side, but I don't know the Mec1/Rad53 literature well enough to judge.

      • Are the text and figures clear and accurate?

      The authors could do another round of proofing figures and legends. For example, Fig 5C contains scale bars that are not defined, blot 3E has an asterix labeling that is not defined, the model in 5E has misspelled "degradation"...

      • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

      The authors use a lot of different mutants (especially for Whi7). Even for someone who knows the proteins fairly well, it is hard to remember throughout the text which abbreviation is relating to which mutations and which function that is addressing. Maybe occasionally remind the reader of what the mutant is or use terms like Whi7non-binding rather than WIQ.

      The text could also use another round of proof-reading. The overall flow of the storyline is easily comprehensible, but sometimes there is a sudden switch of topics or new proteins come out of nowhere. Some expressions are used in a way that is not common English.

      Significance

      • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

      This study is a major conceptual contribution to understanding G1/S regulation in perturbed conditions (assuming the results can be replicated and quantified as detailed above). That Whi7 (and maybe Whi5) directly inhibit Clb5/Clb6-CDK through Cks1 binding is an important addition/modification to the current model and may well be important beyond genotoxic stress.

      • Place the work in the context of the existing literature (provide references, where appropriate).

      The authors do this reasonably well.

      • State what audience might be interested in and influenced by the reported findings.

      Anyone in the yeast cell cycle/replication field should find this interesting. It should also have important implications for the mammalian cell cycle/replication/DNA damage field.

      • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

      I am well familiar with G1/S control and all the methods used in the study. I am not an expert on replication stress/DNA damage/ checkpoint signaling.

    8. Review Commons

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      Referee #1

      Evidence, reproducibility and clarity

      Summary

      This work begins with a heterologous screen, introducing human genes in double mec1,sml1 yeast deletants, which are alive, but sensitive to hydroxyurea. The readout was mec1,sml1 proliferation in the presence of hydroxyurea. They found that mec1,sml1 yeast mutants carrying the human RB1 gene (a G1/S transcriptional repressor) proliferated on hydroxyurea. Then, they test if known yeast G1/S transcriptional repressors (Whi5 and Whi7) could have similar effects if provided at higher than normal levels (they did). With this initial result, followed up by a variety of experiments, the authors then go on to propose that replication stress, which activates Mec1 and Rad53, triggers the phosphorylation of Whi7 (by Mec1) and Whi5 (by both Rad53 and Mec1) blocking their eviction from the nucleus, allowing them instead to bind and inhibit Cks1, a Cdk processivity factor, needed for the complete phosphorylation and degradation of a Cdk inhibitor, Sic1. This is different from published work a decade earlier in mammalian cells (ref. 37; Bertoli et al.), which showed that upon replication stress, Chk1 phosphorylates G1/S transcriptional repressors to maintain G1/S transcription, which could help genome stability. Here, the authors propose that replication stress could block the G1/S transition. While the model and some of the experiments are interesting, the rationale for some experiments was shaky, and the data do not fully support the conclusions.

      Major points

      1. Any cell that undergoes DNA replication must have already destroyed Sic1. It has been known for 25+ years that targeting Sic1 is the only necessary function of G1/Cdk to enable DNA replication (PMID: 8755551). Sic1 does not reappear until the M/G1 transition. Hence, in the authors' model, where cells are already in the S phase, how can multisite phosphorylation and degradation of Sic1 be the critical and final output of the pathway they propose when there shouldn't be any Sic1 around, to begin with? Why would a cell that has already completed Start and the G1/S transition, is in the S phase and experiencing replication stress, care about going through the G1/S?
      2. The results in Figure 2C are confusing and difficult to interpret. For example, comparing lane 8 (WT without hydroxyurea) to lane 7 (WT with hydroxyurea), it appears that there is more phosphorylated Whi7 in lane 7 (hydroxyurea treatment) than in lane 8 (no treatment). But, the ratio of phosphorylated/unphosphorylated Whi7 is not that different (there is very little unphosphorylated Whi7 in lane 8). Same problem when comparing lanes 3 and 3. I understand that they later show that Whi7 is stabilized by hydroxyurea, but from the data in this figure, what exactly can they conclude here?
      3. Their data in Figure 2E show that phosphorylation of Whi7 is not required for suppressing the lethality of rad53,sml1 cells treated with hydroxyurea. Cells carrying Whi7-41A (lacking all possible phosphorylations) suppressed nearly as well as wild-type Whi7 did. The purported differences in the suppression are minuscule at best and not evident at the dilutions tested. It is not clear at all how they can conclude that phosphorylation of Whi7 has anything to do with the ability of Whi7 overexpression to suppress the lethality of rad53,sml1 cells.
      4. For all the arguments they make about this new role of Whi5 and Whi7 at Start, they do not examine size homeostasis or the kinetics of cell cycle progression in any of their experiments and their mutants, with or without hydroxyurea treatment.
      5. The Sic1 stability experiments they show in Figure 5 are nice. They would need to be extended to their various mutants, including their Whi7 phosphomutants, to make a case for phosphorylation by Rad53 and Mec1 in this process.

      Minor points

      1. The language is awkward. Editing for style will be necessary.
      2. They use different hydroxyurea doses in the experiments they show, making it difficult to conclude much when comparing different figures. Why aren't they consistent from experiment to experiment?

      Referees cross-commenting

      Overall, all reviews are well-aligned. The points raised by the other reviewers are valid, and the reviews are thorough and detailed. I don't know whether the authors will be able to respond since the list is quite long. Even if they do, the manuscript will look very different. I do not have anything else to add.

      Significance

      The manuscript presents some interesting data, most notably the role of Whi7 and Whi5 in the stability of Sic1 in vivo and the various in vitro experiments the authors present. The advance is conceptual and mechanistic, offering a different and unanticipated model for the role of these proteins at Start, under replication stress. Unfortunately, the significance of the manuscript is limited. A convincing case for their model and its importance has not been made. For example, their data in Figure 2E, measuring the ability of phosphomutants to suppress the lethality of rad53,sml1 cells upon replication stress, is underwhelming and undermines the importance of the study, particularly to a wider audience.

    9. Version published to 10.1101/2022.11.10.515958v1 on bioRxiv