Molecular dependencies and genomic consequences of a global DNA damage tolerance defect

  1. Daniel de Groot
  2. Aldo Spanjaard
  3. Ronak Shah
  4. Maaike Kreft
  5. Ben Morris
  6. Cor Lieftink
  7. Joyce J.I. Catsman
  8. Shirley Ormel
  9. Matilda Ayidah
  10. Bas Pilzecker
  11. Olimpia Alessandra Buoninfante
  12. Paul C.M. van den Berk
  13. Roderick L. Beijersbergen
  14. Heinz Jacobs

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Abstract

DNA damage tolerance (DDT) enables replication to continue in the presence of fork stalling lesions. To determine the molecular and genomic impact of a global DDT defect, we studied Pcna K164R/- ;Rev1 -/- compound mutants. Double mutant (DM) cells displayed increased replication stress, hypersensitivity to genotoxic agents, replication speed, and repriming. A whole genome CRISPR-Cas9 screen revealed a strict reliance of DM cells on the CST complex, where CST promotes fork stability. Whole genome sequencing indicated that this DM DDT defect favors the generation of large, replication-stress inducible deletions of 0.4-4.0kbp, defined as type 3 deletions. Junction break sites of these deletions revealed preferential microhomology preferences of 1-2 base pairs, differing from the smaller type 1 and type 2 deletions. These differential characteristics suggest the existence of molecularly distinct deletion pathways. Type 3 deletions are abundant in human tumors, can dominate the deletion landscape and are associated with DNA damage response status and treatment modality. Our data highlight the essential contribution of the DDT system to genome maintenance and type 3 deletions as mutational signature of replication stress. The unique characteristics of type 3 deletions implicate the existence of a novel deletion pathway in mice and humans that is counteracted by DDT.

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    Reply to the reviewers

    Manuscript number: RC-2023-02247

    Corresponding author(s): Heinz Jacobs

    Description of the planned revisions

    Reviewer #1

    This manuscript by de Groot et al. is focused on investigating the role of the DNA damage tolerance (DDT) pathway for maintaining genomic stability in mammalian cells. All experiments are well designed and executed, and the conclusions are strongly supported by the experimental data. The authors generate a pair of congenic T cell lymphoma cell lines with either WT or PcnaK164R/-Rev1-/- genotype (the latter are referred to as double-mutant [DM] cells throughout the proposal). The DDT-deficient DM cells are surprisingly normal under standard growth conditions but show a strongly increased sensitivity to DNA damaging agents. Thus, the authors conclude that in the absence of exogenous stress a backup pathway exists to allow for normal growth, and a CRISPR screen reveals the CDC13/CTC1, STN1, and TEN1 (CST) complex as central for cell survival and cell cycle progression. Subsequent DNA fiber assays reveal that this survival relies on increased repriming of replication, and that exogenous stress overwhelms this backup pathway. Finally, the consequences of DDT deficiency were tested by whole genome sequencing of the WT and DM cells were exposed after a single round of UV stress. and subsequent whole genome sequencing was used to identify DNA alterations. The absence of DDT led to a striking increase in the number of deletions ranging in size from 0.4 to 4.0 kbp (called to a type 3 deletions). Importantly, such mutations are also present in many human tumor genomes and their level appears to be linked to alterations in DNA repair pathways (but no clear causal relationship was shown). The main take home message is that repriming after the lesion is the last resort when a replication forks stalls at a DNA lesion as this leads to the loss of 400-4000 bp of genome information at every such event. The DDT pathway serves to channel the responses towards less deleterious (or even error-free) replication outcomes.

    The authors like to thank this reviewer for the very positive summary of our study.

    Major points:

    1. The authors rely on a genetically modifed cell line in which the Pcna and Rev1 genes are altered (the latter by CRISPR/Cas9 technology). To rule out that any additional inadvertent genetic changes occurred that may influence the phenotypes see here, it would be important to show for at least a subset of the experiments that ectopic re-expression of Rev1 and WT PCNA can rescue the survival defects seen here.

    To rule out any inadvertent genetic changes, we opted for an isogenic system and used two independent clones which provided consistent phenotypes. Furthermore, the double mutant model has been often and independently published, showing very similar phenotypes as observed in this study (PMID: 36669105, PMID: 18498753, PMID: 37498746, PMID: 17105346). Additionally, in a p53-WT setting, Rev1-deficient and PCNA-K164R mutant mice are viable, develop normally, and are born at the expected mendelian frequencies, indicating a rescue in the absence of stress would not show an effect. In our published NAR paper (PMID: 35819193), we do not observe sensitivity of REV1-ko lymphomas (which is an isogenic SM clone of the DM and WT lymphoma), indicating that a rescue experiment would likely not help us much; and in the context of a single PCNA-K164R mutant, these become sensitive to genotoxic agents, in line with widely available literature. These aspects will be clarified by textual amendments.

    1. It is unclear whether all experiments were conducted with a single clone of each genotype or if different clones were tested. This should be clarified.

    A valid point, to exclude inter-clonal variations, two different isogenic clones were used for both DM and WT. This important aspect will be clarified in the results section.

    1. The increase in the type 3 deletions in human cancers is very obvious, and as the authors clearly demonstrate DDT-deficiency results in the very same type 3 deletions. Although there is no data for this shown here, I'm assuming that a single deficiency in Rev1 would show a distinctly different mutation pattern. Given alterations of DNA repair/DNA damage tolerance gene mutations in the human tumors, it appears very unlikely to me that all tumors lack the DDT in its entirety. So why would the type 3 deletions then emerge? The authors should provide a clearer model of how this might work that could be tested in the future.

    Indeed, the human tumors analyzed in the manuscript are unlikely to have DDT defects but do have the indicated alteration in other DNA repair genes. This led us to hypothesize that these type 3 deletions are not specific for DDT defects but more a general phenotype that results from replication stress, a hallmark of tumors and especially those suffering from specific DDR defects. We consider that future studies should address this important aspect in more detail and will extend the discussion section accordingly.

    1. The authors only assess the genome alterations after a single dose of UV irradiation. Do the type 3 deletions also accumulate (albeit at a much lower rate) when these cells are grown for an extended period of time under normal conditions and do such cultures ultimately undergo senescence once too many deletions have been acquired?

    We did culture these cells for an extended period of 5 months and compared the mutation profiles of pre-cultured and post cultured WT and DM cells. On the genomic level no major differences appeared between the pre- and post-cultured samples, indicating that these deletions likely do not accumulate easily over time. Furthermore, DM grow indefinitely and do not display any signs of senescence. We will clarify this relevant point and further extend on hypothesis that replication stress is likely to underlie the generation of type 3 deletions.

    Minor points:

    1. In the methods section the description of how the cell lines that are central to this work were generated is not clear. The authors start with a p53-/-PcnaK164R/loxP Rev1wt/wt background. Then the Rev1 was inactivated using CRISPR technology, but how the Pcna wt/- genotype in the WT was restored is unclear. It would be helpful to provide a schematic drawing of the gene targeting strategies as a supplementary figure.

    An important point, we will add a schematic figure and legend to clarify the generation of the isogenic cell lines.

    1. The authors should describe whether (and how many) independent clones of the DM (and may be WT) cell lines were tested and used in the experiments.

    As stated above, two independent clones were studied to exclude inter-clonal variation, and this info will be added to the result and material and methods sections.

    1. The approach by which the genome-wide mutation load was assessed for each genotype is not described in sufficient detail. Did the authors compare WT before and after UV exposure and DM before and after UV exposure separately or were just the genomes of WT and DM after UV exposure compared.

    We extensively analyzed the data in both pre- and post-UVC exposures. Based on these analyses we chose to display our data as revealed in figure 4, where 4B indicates the deletions prior to UVC exposure, and figure 4C the deletions acquired upon UVC exposure. Additional analyses can be provided upon request.

    Reviewer #2

    The manuscript by de Groot and colleagues investigates the cellular and mutational phenotypes of mouse cells that are mutant for REV1 and also carry a PCNA-K164R mutation that prevents post-translational modifications at this residue. This double mutant (DM) likely removes all mechanisms for the recruitment of canonical translesion synthesis polymerases (Y family and pol zeta), thus the authors use it as a general DNA damage tolerance (DDT) deficient model. Using the cell line, they find signs of increased replication stress and a reliance on repriming. A whole genome CRISPR screen revealed a genetic dependence of the DM cells on the CST complex. Sequencing the genome of DM cells showed a specific increase in a distinct category of large deletions, which were also shown to be present in cancer genomes. While the study raises interesting points and contains much valuable data, I find major issues with both the study design and especially with the methodology, which appear to make it unsuitable for publication in its present form.

    We like to thank this reviewer for the careful analyses of our data. Remarkably, while reviewer 1 praised the study design and methodology, this reviewer raised some concerns which feel are addressed and clarified appropriately as outlined below.

    Major points:

    __ __Study design:

    The paper focuses on the double mutant PCNAK164R/- REV1-/- cells throughout, without testing the single mutants. This is a major drawback. It is unclear whether such single mutant cell lines were available to the authors. A PCNA-K164R appears to have been published previously (Ref.46) but do they also have a REV1 mutant lymphoma in a tp53 muntant background? By comparing a double mutant to the wild type the authors miss the opportunity to assign phenotypes to either mutation. For example, large deletions very similar to those found here have been recently reported in human cells (Ref79, noted at the end of this manuscript). That paper shows that these are due to the loss of REV1 or REV3, and the concurrent loss of PCNA ubiquitination does not contribute to this phenotype (partially?).

    We do have the WGS data of single mutants, but as this data did not show significant mutational differences, we felt like it would distract from the main story and decided to leave these data out. The major difference of our findings compared to Gyüre et al. (PMID: 37498746) is lack of a specific deletion phenotype in REV1 single mutant clones. With this independent study, we consider the overlap of our findings as most relevant.

    A second example is the interesting observation that the DM cells rely on repriming even during unperturbed DNA replication. However, this could also potentially be the consequence of the inactivation of REV1. Again, single REV1 mutants should be assayed, and REV1-related literature discussed.

    The role of REV1 in repriming and replication fidelity has been studied extensively in multiple systems (PMID: 31178121, PMID: 3797129, PMID: 31607544, PMID: 32330130, PMID: 32577513, PMID: 36669105, PMID: 34508659, PMID: 34624216, and others). Given the fact that this has been firmly elucidated, we decided to focus on the DM. However, we agree that this important aspect deserves to be discussed in detail and will add this to the discussion section.

    Mutation detection methodology:

    The analysis of small scale mutations shows some unexpected results. Not only is there no effect of the DDT mutations, there is also no effect of UV irradiation (Fig. S6E). Several papers have described in vitro experiments with UV treatment showing the clear mutation spectra that are also seen in cancers (SBS7). UV induces these spectra in mice even in vivo (PMID: 34210801 - though this paper used UVB). So it is difficult to believe that there would be no mutagenic effect in the cells used in this manuscript. Could there be an analysis problem instead?

    The lack of UV signature has also come to our attention, but we clearly see an effect of the UV in the large deletions and cell viability, indicating these cells were exposed to UV. Additionally, we also provided the data to several independent bioinformaticians confirming our results. This excluded an experimental and analytical bias. Given these assurances, we theorized that the lack of a UV-mutation signature relates to the very low UVC dose these cells were exposed to. This is an experimental limitation caused by the marked UVC sensitivity of the DM cells. Of note, other published data employing DDT deficient systems also accumulated very low numbers of de novo mutations (PMID: 29323295, PMID: 37498746, PMID: 32330130). We agree that the surprising observation regarding the lack of a UVC signature deserves detailed explanation, which will be argued in the discussion section.

    The mutagenesis experiment and the mutation calling are incompletely described. Precisely how many clones were sequenced? A table should be provided with such data, and sequencing data must be uploaded to an accessible database.

    As mentioned for reviewer one, for the mutagenesis analyses two independent clones have been used for both genotypes, these provided very similar results. This relevant aspect will be indicated in the revised manuscript. The sequencing data have been uploaded and the link will be provided upon acceptance. Furthermore, we will extend the method section to clarify mutation calling.

    Most importantly, how was the mutation calling done? Did the authors sequence an initial cell clone, to which the post-treatment clones could be compared? Without doing that, detected 'mutations' include many heterozygous SNPs which are differently called in different samples due to stochastic read count differences. Indeed, the mutation spectra in Fig. S6D and E look precisely like standard SNP spectra: flat in the C>T and T>C segment. If this is indeed the issue, mutation calling can be improved somewhat by filtering against mouse SNP databases, but the experiment cannot be fully rescued.

    The mutation calling has been performed with the use of a standard (MM10, from a C57BL/6 mouse, the same background as our cell lines) mouse reference genome for all samples. We opted for this method as it is widely used (similar method as used by Gyüre et al., PMID: 37498746, but for human). Additionally, we used alternative filtering strategies to call mutations with high confidence, such as joint genotype calling. Importantly, we also used the untreated WT lymphoma as a reference, all of these methods provided very similar results that did not change the interpretation of our results. We agree that the original mouse genome sample would have served as the most ideal reference genome, however given the above outline of steps taken, we are confident in our conclusions. We will address these points in an extended discussion and method section.

    The detection of large deletions is equally problematic. Fig. S5 suggests that the deletions are found in the same locations in the WT and the mutant cells. The probable reason for this is that the authors are finding the exact same deletions in both cell lines, which pre-existed even the making of the mutant cells, and are simply differences compared to whatever reference genome they are using! The DM appears to produce enough extra deletions to be detectable, but the real difference between the WT and the DM is likely much stronger than found here.

    We thank this reviewer for pointing out this relevant comment. In accordance with the data gathered, we hypothesize that replication stress favors the formation of type 3 deletions. Consequently, our p53 deficient WT cell lines experience replication stress and thus will generate type 3 deletions. Using this cell line to generate the DM cell lines, we agree that these pre-existing type 3 deletions will be present in subsequent sequencing analyses. However, due to the enormous increase of replication in the DM additional type 3 deletions will accumulate. This aspect was the intended message of figure S5 A&B. We have also figures that only depict the differences between the type 3 deletions in WT and DM in predefined genomic regions (bins of 1 million bp).

    Figure:

    (A) Genome wide distribution of the difference in the number of type 3 deletions comparing DM minus WT in untreated conditions.

    (B) Genome wide distribution of the difference in the number of type 3 deletions comparing DM minus WT after 0.4 J/m2 UV-C exposure.

    (A)

    Figure could not be uploaded in this portal.

    (B)

    Figure could not be uploaded in this portal.

    We feel that generally, the issue that is put forward is that the use of a widely used standard reference would increase the background and thereby prevent the detection of small mutational changes. This however does not subtract from the mutational changes we did detect, leaving the core of the story and results unchanged. We agree that the effect size is likely to be stronger and we will address this aspect in the results and discussion section.

    Some specific comments:

    The CRISPR screen in the DM cells no doubt provided very valuable data, and CST is an interesting hit. The authors found that the STN1 gene could not be knocked out in the DM, but it could be when apoptosis was inhibited by Bcl2 overexpression. Unexpectedly, Bcl2 overexpression reduced the increased replication speed in the DM, thus interfering with the very effect the authors were trying to measure. Without understanding the mechanism of this effect, it is difficult to draw conclusions from this cell line. And again, the effect of Bcl2 and Stn1 should have also been assayed in a WT background as controls, not just in triple/quadruple mutant combinations.

    Would single CST mutants also affect the cell cycle profile? The authors conclude that CST appears to have a role in tolerance of endogenous replication impediments, but without seeing the effect of the single mutant on the cell cycle they can only conclude about such impediments that are created in the absence of REV1 and PCNA-Ub. These may well be the breaks that result in the large deletions shown later.

    Our prime interest in CST is only in the context of DDT. This means that we conclude that in the absence of DDT, CST seems to have a role in damage tolerance of endogenous replication impediments. We will highlight this better in the revised text to prevent confusion. Indeed, we initially speculated that CST would have a role in forming these deletions but due to lack of evidence we decided not to make this connection.

    Additionally previous studies reported extensively on the role of CST in telomere maintenance (PMID: 28934486 and many others), DSB repair (PMID: 29768208), maintenance of genetically unstable regions (PMID: 34520548, PMID: 29481669) and its role in DDT (PMID: 35150303, PMID: 37590191).

    The analysis of deletion size distribution in tumors is interesting and does appear to show that the 'type 3' deletions are a general phenomenon. However, the last point seems tautological: those tumors with a higher proportion of type 3 deletions have 'a sizeable increase of type 3 deletions'? (Fig. 5C). The fact that these deletions were also abundant in the WT cell line (Fig. 4B), where they are likely pre-existing genomic variations, suggests that such deletions can arise as part of spontaneous mutagenic processes even in normal cells. Their presence in all tumor types to similar degrees agrees with this.

    Indeed, we agree that the process that leads to these types of deletions would be present in WT or normal cells. The increase in replication stress is likely underlying the formation of type 3 deletions. Their accumulation in the DDT deficient system is likely because this system generates replication stress similarly as in the presented human tumors, which in both cases appears to favor the formation of type 3 deletions.

    Figure 5C is meant to give an overview of 5B using the density profiles similarly as shown in the previous figures. Additionally, this figure provides a direct comparison between the mouse and human density profiles of large deletions. Tumors with the cutoff of 25% of deletions that fall in type 3 range, have density profile similar to those of DM cells. We will alter the text accordingly to explain this relevant issue more clearly.

    Minor comments:

    __ __- The model organism for the DM cells (mouse) should be mentioned in the abstract.

    Will be done.

    • In the introduction, 4 modes of DDT are described including template switching without the formation of a post-replicative gap, but there is little evidence for this. Ref18 is the authors' own review, which cites further reviews.

    We agree that this specific mode of DDT is less well documented, we will adjust the text to clarify this point and provide additional references.

    • In the first Methods item, the plasmid used for transfection is not specified. "To obtain WT and PcnaK164R/-;Rev1-/- lymphoma cell lines, 10 x 106 lymphoma cells from a p53-KO, PcnaK164R/loxP mouse(46) were nucleofected."

    The plasmid used was pX333. We will provide the info and reference.

    • The y axis label of Figure 4F appears to be wrong (frequency vs. percentage)

    We will change this legend to percentage.

    Reviewer #3

    The manuscript by De Groot et al investigates the impact of the PCNA(K164R)-REV1 double inactivation on genome integrity in lymphoma cells. The group had previously demonstrated that the double mutant is lethal in mice (papers in PNAS and NAR) but here, in lymphoma cells, additional mechanistic work could be performed. Chiefly, they were able to conduct a CRISPR screen to investigate backup mechanisms in these double mutant lymphoma cells and they identified a specific complex (CST). Mutating one of the CST complex proteins within double mutant cells led to lethality that was rescued by Bcl2 overexpression, allowing for further mechanistic studies. WGS on such cells identified specific types of structural variants that would normally kill cells (mostly large deletions). Finally, they identify similar type deletions in databases of human tumours, with specific preferences with regard to treatment modality (more deletions with chemo, immunotherapy and hormonal therapy, and fewer in tumours treated with tamoxifen, imatinib and some other small molecule inhibitors).

    The authors like to thank the reviewer for the time invested and the appreciation of our novel insights.

    Please find below our responses to the remaining specific comments.

    Specific comments:

    1. can the authors comment on the physiological relevance of the screen, considering that the double mutation is lethal in normal tissues?

    The screen was performed to understand how cancer cells can survive in a DDT deficient setting. This increases our understanding of the general function of DDT and identify alternative pathways that enable cancerous as well as normal cells to cope with DNA damage. Furthermore, tumors can have defects in the DDT system, knowledge on DDT function may help to target these tumors with specific inhibitors and chemotherapeutics. A relevant aspect, that we will elaborate on in the revised discussion.

    1. can the authors suggest a mechanism regarding how CST would work to maintain the viability of the double knockout lymphoma cells?

    An important point, our insights gathered so far favor a model where the CST prevents the formation of single stranded DNA gaps. As the double mutant DDT deficient cells already accumulate a high number of post-replicative gaps, the lack of CST complex further increases those, leading to genomic instability and eventually cell death. To clarify our model, we will extend our discussion section.

    1. it is implied that STN1 deletion would only kill double mutant lymphoma cells but is this actually the case? (a similar deletion in wildtype and single mutant cells is a necessary control).

    The role of CST, and its ablation, has been extensively studied by many others. Our main interest in CST is in the context of DDT and how CST maintains cells in a DDT deficient setting. Because the single DDT mutants have been studied in detail, we here focused on the role of CST in our double mutant DDT deficient setting. This setting enabled us to identify CST as a potential novel back up mechanisms to cope with replication impediments.

    1. I really liked the data from human tumour databases (figure 5) but are the deletions there correlated with the same DDT profiles as investigated here?

    The human tumors discussed in the manuscript do not contain similar specific DDT defects as in the lymphomas. However, we do see that human tumors with varied DDR defects have an increase in these deletions. This led us to speculate that the type 3 deletions arise due to general replication stress, present in both the human tumors and the DM lymphomas.

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by De Groot et al investigates the impact of the PCNA(K164R)-REV1 double inactivation on genome integrity in lymphoma cells. The group had previously demonstrated that the double mutant is lethal in mice (papers in PNAS and NAR) but here, in lymphoma cells, additional mechanistic work could be performed. Chiefly, they were able to conduct a CRISPR screen to investidate backup mechanisms in these double mutant lymphoma cells and they identified a specific complex (CST). Mutating one of the CST complex proteins within double mutant cells led to lethality that was rescued by Bcl2 overexpression, allowing for futher mechanistic studies. WGS on such cells identified specific types of structural variants that would normally kill cells (mostly large deletions). Finally, they identify similar type deletions in databases of human tumours, with specific preferences with regard to treatment modality (more deletions with chemo, immunotherapy and hormonal therapy, and fewer in tumours treated with tamoxifen, imatinib and some other small molecule inhibitors).

    Specific comments:

    1. can the authors comment on the physiological relevance of the screen, considering that the double mutation is lethal in normal tissues?
    2. can the authors suggest a mechanism regarding how CST would work to maintain the viability of the double knockout lymphoma cells?
    3. it is implied that STN1 deletion would only kill double mutant lymphoma cells but is this actually the case? (a similar deletion in wildtype and single mutant cells is a necessary control).
    4. I really liked the data from human tumour databases (figure 5) but are the deletions there correlated with the same DDT profiles as investigated here?

    Referees cross-commenting

    Nice to see that most of the comments are aligned.

    Significance

    Reasonable significance, potentially ablated by the (lack of) physiological relevance of the screen (see comments above).

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by de Groot and colleagues investigates the cellular and mutational phenotypes of mouse cells that are mutant for REV1 and also carry a PCNA-K164R mutation that prevents post-translational modifications at this residue. This double mutant (DM) likely removes all mechanisms for the recruitment of canonical translesion synthesis polymerases (Y family and pol zeta), thus the authors use it as a general DNA damage tolerance (DDT) deficient model. Using the cell line, they find signs of increased replication stress and a reliance on repriming. A whole genome CRISPR screen revealed a genetic dependence of the DM cells on the CST complex. Sequencing the genome of DM cells showed a specific increase in a distinct category of large deletions, which were also shown to be present in cancer genomes. While the study raises interesting points and contains much valuable data, I find major issues with both the study design and especially with the methodology, which appear to make it unsuitable for publication in its present form.

    Study design:

    The paper focuses on the double mutant PCNAK164R/- REV1-/- cells throughout, without testing the single mutants. This is a major drawback. It is unclear whether such single mutant cell lines were available to the authors. A PCNA-K164R appears to have been published previously (Ref.46) but do they also have a REV1 mutant lymphoma in a tp53 muntant background? By comparing a double mutant to the wild type the authors miss the opportunity to assign phenotypes to either mutation. For example, large deletions very similar to those found here have been recently reported in human cells (Ref79, noted at the end of this manuscript). That paper shows that these are due to the loss of REV1 or REV3, and the concurrent loss of PCNA ubiquitination does not contribute to this phenotype. A second example is the interesting observation that the DM cells rely on repriming even during unperturbed DNA replication. However, this could also potentially be the consequence of the inactivation of REV1. Again, single REV1 mutants should be assayed, and REV1-related literature discussed.

    Mutation detection methodology:

    The analysis of small scale mutations shows some unexpected results. Not only is there no effect of the DDT mutations, there is also no effect of UV irradiation (Fig. S6E). Several papers have described in vitro experiments with UV treatment showing the clear mutation spectra that are also seen in cancers (SBS7). UV induces these spectra in mice even in vivo (PMID: 34210801 - though this paper used UVB). So it is difficult to believe that there would be no mutagenic effect in the cells used in this manuscript. Could there be an analysis problem instead? The mutagenesis experiment and the mutation calling are incompletely described. Precisely how many clones were sequenced? A table should be provided with such data, and sequencing data must be uploaded to an accessible database. Most importantly, how was the mutation calling done? Did the authors sequence an initial cell clone, to which the post-treatment clones could be compared? Without doing that, detected 'mutations' include many heterozygous SNPs which are differently called in different samples due to stochastic read count differences. Indeed, the mutation spectra in Fig. S6D and E look precisely like standard SNP spectra: flat in the C>T and T>C segment. If this is indeed the issue, mutation calling can be improved somewhat by filtering against mouse SNP databases, but the experiment cannot be fully rescued. The detection of large deletions is equally problematic. Fig. S5 suggests that the deletions are found in the same locations in the WT and the mutant cells. The probable reason for this is that the authors are finding the exact same deletions in both cell lines, which pre-existed even the making of the mutant cells, and are simply differences compared to whatever reference genome they are using! The DM appears to produce enough extra deletions to be detectable, but the real difference between the WT and the DM is likely much stronger than found here.

    Some specific comments:

    The CRISPR screen in the DM cells no doubt provided very valuable data, and CST is an interesting hit. The authors found that the STN1 gene could not be knocked out in the DM, but it could be when apoptosis was inhibited by Bcl2 overexpression. Unexpectedly, Bcl2 overexpression reduced the increased replication speed in the DM, thus interfering with the very effect the authors were trying to measure. Without understanding the mechanism of this effect, it is difficult to draw conclusions from this cell line. And again, the effect of Bcl2 and Stn1 should have also been assayed in a WT background as controls, not just in triple/quadruple mutant combinations. Would single CST mutants also affect the cell cycle profile? The authors conclude that CST appears to have a role in tolerance of endogenous replication impediments, but without seeing the effect of the single mutant on the cell cycle they can only conclude about such impediments that are created in the absence of REV1 and PCNA-Ub. These may well be the breaks that result in the large deletions shown later.

    The analysis of deletion size distribution in tumours is interesting, and does appear to show that the 'type 3' deletions are a general phenomenon. However, the last point seem tautological: those tumours with a higher proportion of type 3 deletions have 'a sizeable increase of type 3 deletions'? (Fig. 5C). The fact that these deletions were also abundant in the WT cell line (Fig. 4B), where they are likely pre-existing genomic variations, suggests that such deletions can arise as part of spontaneous mutagenic processes even in normal cells. Their presence in all tumour types to similar degrees agrees with this.

    Minor comments:

    • The model organism for the DM cells (mouse) should be mentioned in the abstract.
    • In the introduction, 4 modes of DDT are described including template switching without the formation of a post-replicative gap, but there is little evidence for this. Ref18 is the authors' own review, which cites further reviews.
    • In the first Methods item, the plasmid used for transfection is not specified. "To obtain WT and PcnaK164R/-;Rev1-/- lymphoma cell lines, 10 x 106 lymphoma cells from a p53-KO, PcnaK164R/loxP mouse(46) were nucleofected."
    • The y axis label of Figure 4F appears to be wrong (frequency vs. percentage)

    Referees cross-commenting

    I agree with comments by the other reviewers.

    Significance

    General assessment:

    My assessment is provided above, the manuscript is not suitable for publication in its present form. If I am correct about the faults of the experimental design, it would be advisable to repeat the entire mutagenesis experiment starting from newly isolated and sequenced single cell clones. Ideally, single mutants should also be included. Even if this is done, the novelty of the expected results is unfortunately compromised by the very similar recent data published from human cell lines. Alternatively, the mutation data could be left out, and the CST-based data could be expanded with more controls and hopefully more mechanistic insight.

    Advance:

    The CST-dependence of PCNAK164R/- REV1-/- cells and the general presence of 400-4000 bp deletions in tumours are both significant findings, but limited mechanistic insight is provided.

    Audience:

    DNA repair field.

    I have expertise in the field of DNA repair and mutagenesis.

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    Referee #1

    Evidence, reproducibility and clarity

    This manuscript by de Groot et al. is focused on investigating the role of the DNA damage tolerance (DDT) pathway for maintaining genomic stability in mammalian cells. All experiments are well designed and executed, and the conclusions are strongly supported by the experimental data. The authors generate a pair of congenic T cell lymphoma cell lines with either WT or PcnaK164R/-Rev1-/- genotype (the latter are referred to as double-mutant [DM] cells throughout the proposal). The DDT-deficient DM cells are surprisingly normal under standard growth conditions but show a strongly increased sensitivity to DNA damaging agents. Thus the authors conclude that in the absence of exogenous stress a backup pathway exists to allow for normal growth, and a CRISPR screen reveals the CDC13/CTC1, STN1, and TEN1 (CST) complex as central for cell survival and cell cycle progression. Subsequent DNA fiber assays reveal that this survival relies on increased repriming of replication, and that exogenous stress overwhelms this backup pathway. Finally, the consequences of DDT deficiency were tested by whole genome sequencing of the WT and DM cells were exposed after a single round of UV stress. and subsequent whole genome sequencing was used to identify DNA alterations. The absence of DDT led to a striking increase in the number of deletions ranging in size from 0.4 to 4.0 kbp (called to a type 3 deletions). Importantly, such mutations are also present in many human tumor genomes and their level appears to be linked to alterations in DNA repair pathways (but no clear causal relationship was shown). The main take home message is that repriming after the lesion is the last resort when a replication forks stalls at a DNA lesion as this leads to the loss of 400-4000 bp of genome information at every such event. The DDT pathway serves to channel the responses towards less deleterious (or even error-free) replication outcomes.

    Major points:

    1. The authors rely on a genetically modifed cell line in which the Pcna and Rev1 genes are altered (the latter by CRISPR/Cas9 technology). To rule out that any additional inadvertent genetic changes occurred that may influence the phenotypes see here, it would be important to show for at least a subset of the experiments that ectopic re-expression of Rev1 and WT PCNA can rescue the survival defects seen here.
    2. It is unclear whether all experiments were conducted with a single clone of each genotype or if different clones were tested. This should be clarified.
    3. The increase in the type 3 deletions in human cancers is very obvious, and as the authors clearly demonstrate DDT-deficiency results in the very same type 3 deletions. Although there is no data for this shown here, I'm assuming that a single deficiency in Rev1 would show a distinctly different mutation pattern. Given alterations of DNA repair/DNA damage tolerance gene mutations in the human tumors, it appears very unlikely to me that all tumors lack the DDT in its entirety. So why would the type 3 deletions then emerge? The authors should provide a clearer model of how this might work that could be tested in the future.
    4. The authors only assess the genome alterations after a single dose of UV irradiation. Do the type 3 deletions also accumulate (albeit at a much lower rate) when these cells are grown for an extended period of time under normal conditions and do such cultures ultimately undergo senescence once too many deletions have been acquired?

    Minor points:

    1. In the methods section the description of how the cell lines that are central to this work were generated is not clear. The authors start with a p53-/-PcnaK164R/loxP Rev1wt/wt background. Then the Rev1 was inactivated using CRISPR technology, but how the Pcnawt/- genotype in the WT was restored is unclear. It would be helpful to provide a schematic drawing of the gene targeting strategies as a supplementary figure.
    2. The authors should describe whether (and how many) independent clones of the DM (and may be WT) cell lines were tested and used in the experiments.
    3. The approach by which the genome-wide mutation load was assessed for each genotype is not described in sufficient detail. Did the authors compare WT before and after UV exposure and DM before and after UV exposure separately or were just the genomes of WT and DM after UV exposure compared.

    Referees cross-commenting

    It appears that our comments are mostly overlapping.

    Significance

    This manuscript is a continuation of the very systematic work by the Jacobs lab to dissect the molecular mechanisms by which DDT factors act and the role of DDT in DNA damage responses. Here the authors demonstrate that in complete absence of DDT factors is not lethal, but reveals the priming of replication as the last resort response to avoid cell death. The analyses of the human tumor genome sequences suggest that dysfunctional DDT response are likely intimately involved in the generation of a distinct set of genetic lesions found in many tumors. What remains unclear is how the DDT pathway is inactivated in tumors as there is no consistent pattern of DDT factors being mutated. Overall this manuscript is of broad general interest far beyond the DNA repair community. The novelty of this manuscript is how reduced by the fact that another group published similar data in the human RPE cells 2023 (see reference 79 as acknowledged by the authors), but observing the same phenomena in two distinct system provides additional weight to these discoveries.
    Expertise: My expertise is in the gene diversification processes that assemble and alter TCR and immunoglobulin genes. They involve a broad range of DNA repair factors and DDT plays a unique role in somatic hypermutation that allows for the generation of high affinity antibodies.

  5. Version published to 10.1101/2023.10.11.561854v2 on bioRxiv
  6. Version published to 10.1101/2023.10.11.561854v1 on bioRxiv