Limiting DNA polymerase delta alters replication dynamics and leads to a dependence on checkpoint activation and recombination-mediated DNA repair

  1. Natasha C. Koussa
  2. Duncan J. Smith

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Abstract

DNA polymerase delta (Pol δ ) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol δ is responsible for the synthesis and processing of the lagging-strand. At replication origins, Pol δ has been proposed to initiate leading-strand synthesis by extending the first Okazaki fragment. Destabilizing mutations in human Pol δ subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol δ in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze how the depletion of Pol δ impacts replication origin firing and lagging-strand synthesis during replication elongation in vivo in S . cerevisiae . By analyzing nascent lagging-strand products, we observe a genome-wide change in both the establishment and progression of replication. S-phase progression is slowed in Pol δ depletion, with both globally reduced origin firing and slower replication progression. We find that no polymerase other than Pol δ is capable of synthesizing a substantial amount of lagging-strand DNA, even when Pol δ is severely limiting. We also characterize the impact of impaired lagging-strand synthesis on genome integrity and find increased ssDNA and DNA damage when Pol δ is limiting; these defects lead to a strict dependence on checkpoint signaling and resection-mediated repair pathways for cellular viability.

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  1. Version published to 10.1371/journal.pgen.1009322
  2. Version published to 10.1101/2019.12.17.879544v2 on bioRxiv
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    Reply to the reviewers

    We are grateful to all three reviewers for their careful analysis of the manuscript, and for their constructive comments. Two common critiques were:

    (1) that assaying origin firing via an independent method would strengthen the conclusions, and (2) that additional analysis of ribonucleotide incorporation to exclude the retention of lagging-strand primers would allow us to definitively determine whether Pol ɛ plays a role in lagging-strand synthesis.

    We will include experiments to address both critiques in a revised manuscript. To independently verify changes in origin efficiency, we will sequence nascent BrdU-containing DNA across a time course from cells released into S-phase: we will also use the last timepoint of our Okazaki sequencing analysis to control for potential cell-cycle differences. To further test the contribution of Pol ɛ and ascertain whether lagging-strand primers are retained, we will analyze ribonucleotide incorporation in both wild-type and *pol2-M644L *(Pol ɛ ribonucleotide hypo-incorporating) strains. We address individual specific comments and our planned revisions in more detail below.

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

    *This study examined the consequences of limiting levels of DNA polymerase d (Pol d) in yeast. The authors first reported multiple genome instability consequences following lowered Pol d level, including defect in S phase progression, growth defect, elevated spontaneous DNA damage, accumulation of ssDNA and activation of replication and DNA damage checkpoint. These observations are solid but not unexpected. By genome wide analysis using the Okazaki fragment (OF) mapping and ribonucleotide mapping (for polymerase usage), the authors claim a few potentially novel and striking observations that lowered Pol d differentially impact efficiencies of early vs. late origins, and that lowered Pol d results in Pol e participating in lagging strand synthesis around late origins. However, I remained unconvinced based on the data presented. These observations need to be further substantiated and alternative interpretations should be considered. *

    ***Main concerns:** *

    *One of major conclusions the authors tried to make is that the early vs. late origins are differentially affected by low level of Pol d. First, they used OF mapping data to examine origin efficiency. Asynchronous "Cultures were treated with IAA for 2h before the addition of rapamycin for 1h to deplete DNA ligase I (Cdc9) from the nucleus via anchor-away". IAA concentrations used were of 0, 0.2 mM, 0.6 mM, and 1 mM. The problem is that Figure S1 clearly showed that treating asynchronous cultures with >0.1 mM of IAA for as short as 30 min significantly alters the cell cycle profiles, mainly resulting in accumulation of S phase cells, to different extent. Presumably Okazaki fragments accumulated from these cultures suffering from the synchronizing effect may not be representative of the real change in global replication profile. For instance, it is not difficult to predict that the Okazaki fragments enrichment may be skewed towards late origins if more cells are accumulated in mid S phase following Pol d depletion. For this reason, I don't believe the result is conclusive. The experiment may be re-designed for samples at different time points following release from G1. *

    We agree that altered cell-cycle profiles might affect the number of Okazaki fragments sequenced in late vs early replicating regions of the genome. As noted by reviewer 3 in cross-commenting, these differences should not affect origin efficiency calculations as these are based on the ratio of reads on each strand (and therefore normalized). To more directly address this question, we will calculate origin firing efficiencies from the final timepoint of the arrest-release experiments shown in Figure 4 as suggested by the reviewer. We will also analyze origin efficiency using BrdU over a time course.

    *This concern also should preclude the authors from drawing conclusion about Pol e usage on lagging strands based on comparison between HydEn-seq data and OF mapping data shown in Figure 6. In fact, the rNMP incorporation change is very similar between early and late origins. The only evidence that the author rely on is the discrepancy in OF data between the two groups origins, which makes the reliability if origin efficiency measurement the central piece of data in this study. Thus, alternative approaches should also be considered to map origin efficiencies. *

    As noted above, we agree that an independent method of tracking origin firing efficiencies would be helpful to strengthen our conclusions. To this end, we will analyze time courses of BrdU incorporation from cultures released into S-phase.

    *Even if Pol e strand bias is lowered at late origins, as the authors tend to believe, there are still alternative models other than Pol e being used for lagging strand synthesis. For instance, if TLS polymerases are used on lagging strands, it could result in more ribonucleotide incorporation on the lagging strand, as they are lower-fidelity polymerases. Alternatively, if Pol d scarcity leads to more Pol e synthesis or lower RNA primer processing, it might also contribute to more apparent ribonucleotide incorporation on the lagging strands. *

    We feel that the widespread use of TLS polymerases is unlikely, especially given the data in figure 6A that show no growth or viability change upon deletion of all three TLS polymerases in the Pol ∂ depletion strain, even at very low levels of Pol ∂. We agree with the reviewer that our data do not conclusively rule out increased retention of lagging-strand primers – as we state in the text. We aim to test this possibility by analyzing ribonucleotide strand bias in a *pol2-M644L *strain that incorporates fewer ribonucleotides than the wild-type Pol ɛ. In this case, increased lagging-strand primer retention would lead to a lagging-strand bias of ribonucleotides upon Pol ∂ depletion, while increased Pol ɛ usage would not. An analogous experiment with wild-type POL2 is potentially harder to interpret because the wild-type polymerase is the predominant source of ribonucleotides in a wild-type strain (Nick McElhinny et al, 2010 - PMID:20729855), but we now have the data for this strain in hand and ready to analyze.

    *In Figure S5, the two HydEn-seq replicates are very different, where replicate1 shows very low strand bias. I suspect perhaps the strain used for replicate 1 does not contain pol2-M644G or rnh202 deletion. *

    The change in ribonucleotide incorporation is indeed substantially stronger in one replicate than the other. We have additional time-course data from a Pol ∂ depletion showing that ribonucleotide strand bias decreases over time as Pol ∂ is depleted, and will include this in a revised manuscript.

    *Reviewer #1 (Significance (Required)): *

    *Given that different aspects of Pol d deficiency have been implicated in various human diseases and cancer, this type of analysis is of interest to the field. *

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

    ***Summary** *

    *In this manuscript the authors explore the basis of the deleterious effects of reduced levels of the replicative Polymerase delta. This polymerase plays several important roles in the replication process: it synthesizes most of the lagging strand, but also extends the first primer during the synthesis of the lagging strand, and it contributes to the removal of the RNA and most of the DNA synthesized by primase/Polymerase alpha during Okazaki fragment maturation. In this study, the authors systematically analyze the impact of Pol delta depletion in S. cerevisiae. They use a degron-tagged allele to modulate the levels of the polymeraes and apply mainly NGS methods and classical genetics to explore the consequences for survival, checkpoint signalling and replication features such as fork speed, origin firing efficiency and Okazaki fragment length and distribution. * *They report that Pol delta depletion leads to a checkpoint activation via the Rad9-dependent damage signalling pathway (but not the Mrc1-dependent replisome-associated signalling) and an accumulation of single-stranded DNA. Phosphorylation of histone H2A is taken as a marker of DNA double-strand breaks, and from the observation that deletion of recombination factors, but not end-joining factors aggravate the fitness of Pol delta-depleted cells they conclude that homologous recombination is responsible for the repair of these breaks. Analysis of replication by Okazaki fragment sequencing indicates a slight decrease in the firing efficiency of early/efficient origins, but an increase in the firing efficiency of late origins. They also observe a reduction in fork speed, although they are not able to attribute this to either a globally slower fork movement or an increase in the stalling of individual forks. They find that Pol delta depletion does not change the size of Okazaki fragments, but causes defects in the nick translation during Okazaki fragment maturation. Finally, they use NGS technology to show that the leading strand Polymerase epsilon engages in lagging strand replication particularly at late origins when Pol delta is depleted. * *From their observations, the authors develop a model where depletion of Pol delta primarily affects late replicating regions. They explain this by invoking a stable association of Pol delta with early replisomes, which sequesters the enzyme, thus causing an under-supply at replisomes that assemble later during S phase. This then leads to the involvement of Pol epsilon on the lagging strand. Based on the observation that fork speed and Okazaki fragment maturation are both affected, they propose that these two reactions normally compete for Pol delta, suggesting that optimal replication would require two molecules of the polymerase per fork. *

    ***Major comments** *

    *The experiments shown here are largely clean and well controlled, and the manuscript is written nicely and well-structured. *

    *Compared to the Okazaki fragment analysis, the treatment of double-strand breaks appears somewhat cursory and remains inconclusive. Phosphorylation of H2A seems insufficient evidence for double-strand breaks, as other structures could also give rise to that signal. These lesions should be detected in a more direct manner, e.g. pulsed-field gel electrophoresis. The authors also don't provide a mechanism by which such breaks would emerge. Related to the minor effect of the ku mutant, I am wondering about the altered appearance of the colonies in Figure 2F (concerning both ku70 and rad51) - what is different about these, and could their „denser" appearance explain the slight suppression effect observed? *

    We agree that our treatment of double-strand breaks is limited: consistent with comments from all three reviewers about which aspects of this work are most novel, we intend to focus as much as possible on replication enzymology here. We will tone down the language around double-strand breaks in the manuscript.

    *Concerning the damage signalling: it is surprising to see a damage signal at concentrations of IAA that do not lead to a significant depletion of Pol delta yet (0.05 mM). At this point, it is hard to imagine DSBs to form. Could the authors explain this discrepancy? *

    We note that, as observed in Figure 1A and to a slightly lesser extent in Fig. 2E, Pol ∂ levels are already substantially reduced in 0.05 mM IAA. This reduction appears sufficient to induce damage

    *The notion that late origin firing is enhanced despite checkpoint activation is counterintuitive. Do the authors think that this effect overcomes the suppression of late origins that is normally associated with checkpoint activation? It would be helpful to test whether abolishing that phenomenon (e.g. by a mec1-100 mutant) would enhance the effect and render late origins even more active. *

    We thank the reviewer for this excellent suggestion: we will test the effects of a *mec1-100 *mutant and include the results in a revised manuscript.

    *It would be important to characterise the fork speed defect better, using alternative methods rather than just relying on Okazaki fragments. A differentiation between slower fork progression and more frequent fork stalling would be relevant and might help to evaluate the contribution of Pol epsilon. This might be accomplished by DNA fibre analysis. Alternatively, BrdU incorporation could serve to observe replication over the entire genome rather than only in the vicinity of replication origins. It would also be important to differentiate fork speeds in early versus late replicating regions - according to the authors' model, the defects should be most obvious in the late regions (Fig. 4 concerns only early origins). *

    As noted above in our response to reviewer 1, we will use BrdU incorporation to independently verify changes in fork speed and origin firing. Analysis of fork speed in late-replicating regions is challenging regardless of the methodology used, due to contributions from converging forks, but we will try to do this

    *Figure S3: Considering the differences in cell cycle progression, it would make more sense to compare equivalent stages of the cell cycle / S phase rather than identical time points. *

    We can include this analysis, although the changes in cell cycle progression and origin firing efficiency make such comparisons somewhat subjective

    *Considering that the Okazaki fragment analysis was done with non-synchronised cultures, is it possible that the skew in the cell cycle profile could influence the Okazaki fragment pattern? *

    (copy-pasted from our response to a similar query by reviewer 1)… We agree that altered cell-cycle profiles might affect the number of Okazaki fragments sequenced in late vs early replicating regions of the genome. As noted by reviewer 3 in cross-commenting, these differences should not affect origin efficiency calculations as these are based on the ratio of reads on each strand (and therefore normalized). To more directly address this question, we will calculate origin firing efficiencies from the final timepoint of the arrest-release experiments shown in Figure 4 as suggested by the reviewer.

    *Would it be possible to monitor not only total Pol delta levels, but also the level of Pol delta bound to the chromatin? It is shown that the level of Pol delta is depleted in the whole cell extracts, but it would be interesting to see how severe the depletion is on the chromatin. *

    We agree that the relative fraction of chromatin-bound vs free Pol ∂ is an interesting question, and will attempt this experiment. However, we note that extensive depletion of Pol3 makes it hard to detect by Western blot, so the results are likely to be most informative at modest depletion levels. Regardless, these data should give us an idea of the size of the ‘free’ Pol ∂ pool in cells with normal or mildly reduced Pol ∂.

    *Figure 6 is confusing and should be clarified: * *- Figure 6B: assigning the Watson and Crick strands in the schematic would make that figure easier to understand; * *- Figure 6B-C: the axes are labeled as 'Fraction of rNMP on Watson strand', but would it not make more sense if they were labeled 'Fraction of rNMP in Crick strand'? * *- Figure 6D-E: the right side scale is labelled as 'increase in rNMP on Crick strand' while in the figure legend is says it is 'change in the fraction of ribonucleotides mapping to the Watson strand. That description should be clarified; * *- Figure 6D: using 'Change in Okazaki fragments strand bias' to label the black line (description in the box above the figure) instead 'Change in Okazaki strand bias' would be more appropriate; * *- Figure 6F: the authors seem to have subtracted strand bias measured for Okazaki fragments from the strand bias measured for rNMP. It is valid to subtract these biases from each other, considering that the two structures arise independently and with different frequencies? *

    We can make changes to figure 6 as suggested. Regarding the validity of subtracting strand biases, we think this is sufficient to give at least a semi-quantitative view of Pol ɛ usage since both of our sequencing approaches produce quantitative readouts that directly report on replication direction or polymerase usage, respectively.

    ***Minor comments:** *

    *Can the authors conclude that Pol delta deficiency/ incompleteness of lagging strand synthesis affects the nucleosome deposition onto DNA? (Figure 5-A) *

    We cannot rule out that this is occurring, and we agree that this is an interesting question for future studies. But the changes that we observe Okazaki fragment terminus location are very similar to our previously published observations from cells lacking Rad27 function, consistent with decreased nick translation.

    *Why did the authors use rnh202Δ and not a mutant in the catalytic subunit of RNase H2? *

    Deletion of any subunit of the heterotrimeric RNase H2 complex completely abolishes its function in yeast, so *RNH202 *was a somewhat arbitrary choice

    *An extra control might be useful: comparing POL3-AID rnh202Δ with the POL3-AID pol2M644G rnh202Δ triple mutant could further confirm that the observed effect is Pol epsilon-dependent. *

    We agree (see also our response to reviewer 1). In addition to the wild-type, we will analyze pol2-M644L – a mutant in which Pol ɛ incorporates fewer than normal ribonucleotides. An increase in ribonucleotide density on the lagging strand in *pol2-M644L *would support increased primer retention on the lagging strand.

    *Figure 2H: It would be good to see the cell cycle distribution corresponding to the western blot images. *

    We can include this

    *Various spelling, grammar or precision of expression issues: * *- Pg. 4, line 4: endonucleolytically instead of nucleolytically. * *- Pg. 6, line 10: Remove 'was'. * *- Pg. 6, line 12: Remove 'in vivo' from the subtitle. * *- Pg. 6, line 14: 'an C-terminal' should be 'a C-terminal' * *- Pg 16, line 13: Phrasing implies that the synthesis of both leading and lagging strands by Pol delta in regions in the vicinity of replication origins is essential - please quote any paper testing its essentiality. * *- Please follow standard yeast genotype nomenclature, remove ';' when listing the yeast genotypes (e.g. POL3-AID mec1Δ sml1Δ instead of POL3-AID;mec1Δ;sml1Δ- example from Figure 2-B). * *- Concentrations of IAA are missing in few places (e.g. legend of Figure 1-C, page 24). * *- Figure 1A: add the label 'IAA (mM)' * *- Figure 2G: pleae provide a shorter exposure of the H4 blot in addition to the one shown. * *- Figure 6: adding a schematic presenting the events at actively and passively replicating late origins (and the predictions about leading and lagging strand bias) would help to understand the figure. * *- The format of the references is inconsistent. * *- 'On Watson/Crick strand' should be replaced with 'in Watson/Crick strand' * We will fix typos, etc

    *Reviewer #2 (Significance (Required)): *

    *This is a nice piece of work that provides in vivo confirmation of several observations that have been made in purified recombinant systems. In that sense, the overall novelty is limited, but this type of study is still important to do, as biochemical assays do not always reflect what is happening in cells, and this study gives insight into basic activities of the replisome. The participation of Pol epsilon in lagging strand synthesis is an interesting observation. Overall, the study will be of interest for the DNA replication field. * *My own expertise is in replication, predominantly in yeast. I have experience in NGS analysis of replication as well as in genetic analysis of the DNA damage response. I therefore feel competent to evaluate all aspects of the manuscript. *

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

    *This manuscript describes the consequences of reducing the cellular concentration of a pol-delta subunit in S. cerevisiae. Pol-delta plays multiple cellular roles at both the replication fork (it is one of two DNA polymerases responsible for lagging strand synthesis) and during repair synthesis after DNA damage. The authors combine genetic and genomic methodologies to characterise how reduction in pol-delta concentration impacts on cellular fitness and specifically lagging strand synthesis. Overall it is technically a well executed study that is clearly presented and the data are predominantly appropriately interpreted. *

    ***I have a number of major comments:** *

    *1) The authors apply OK-seq (a methodology first developed by the senior author and therefore they are clearly experts at this) to study the consequence of pol-delta depletion on genome replication. OK-seq requires isolation of Okazaki fragments and this in turn requires removal of DNA ligase (Cdc9) - the authors achieve this with the anchor away system. My concern is that in these experiments the authors are depleting two factors required for lagging strand synthesis: pol-delta and ligase; it is unclear to me how the authors can determine the relative contribution of each depletion to the observed phenotypes. Could some of the observed phenotypes (e.g. fork slowing, 5' and 3' ends of Okazaki fragments, etc) be a consequence of the double depletion, rather than just pol-delta depletion as concluded by the authors? The authors present this as a method to determine genome replication timing, but really it is an assay to look at fork direction. Given the need for an addition mutation in OK-seq (cdc9), I would encourage the authors to consider a more direct assay for replication dynamics upon pol-delta depletion, such as a copy number measure (or BrdU-ip) of DNA replication or DNA combing - these methods don't require Cdc9 depletion and could therefore ensure that observed phenotypes are a consequence of pol-delta depletion (rather than the double depletion). *

    As outlined in our response to the first two reviewers, we will do BrdU-IP experiments. We agree that the double depletion may have an effect on fork speed, and that BrdU-IP will allow us to test this possibility. However, we note that our analysis of Okazaki fragment initiation/processing requires the depletion of Cdc9, so for this we are limited to looking at differences between Cdc9 depletion alone vs Cdc9 depletion + Pol3 depletion.

    *2) One major conclusion reached by the authors is that pol-epsilon can contribute to lagging strand synthesis upon pol-delta depletion (at least during late replication). This conclusion comes from the authors use of HydEn-seq to measure rNTP incorporation from which the contribution of a polymerase (pol-epsilon in this case) to strand synthesis can be determined. In a manner analogous to OK-seq, this requires the introduction of additional mutations (both in the polymerase and by the removal of RNaseH activity). The authors interpretation that pol-epsilon can play a role in lagging strand synthesis is dependent upon there being no temporal change in pol-delta strand-displacement activity, despite continued pol-delta depletion through S phase. It is not clear to me that the data presented in Fig 5 & 6 has the sensitivity to conclude this (and the OK-seq data is also subject to the potential bias of the double depletion of pol-delta and Cdc9). I feel that a necessary control to support this conclusion, would be to undertake the HydEn-seq experiment in the absence of the pol-epsilon mutation (just pol-delta depletion in the absence of RNaseH activity). This would allow the authors to measure any increase is residual rNTPs (likely from pol-alpha primase) on the lagging strand as a consequence of pol-delta depletion and determine whether they are equally likely in early and late S phase. *

    As discussed in our response to the first two reviewers, we will analyze analogous data from both a POL2 wild-type and a pol2-M644L strain that incorporates fewer ribonucleotides than the wild-type.

    ***The following comments are more minor:** *

    *-for the experiment in Fig S1B, the growth in 1.0 mM IAA is somewhat surprising given how sick the cells appear on equivalent plates. I couldn't find in the methods a description of the experimental conditions. *

    The cells grow very slowly in 1 mM IAA (doubling time doubles). We think this is quite consistent with the poor growth on plates

    *-there is considerable variability in the S phase kinetics from bulk DNA analysis (flow cytometry) when comparing Fig 1C, 2D, S3. Fig 1C appears to be the exception, with all the other figures showing poor S phase progress by comparison. It would be useful for the authors to recognise these differences and comment upon them. E.g. they appear to all be identical experiments, but are there experimental differences that could explain the different kinetics? *

    We see some variability in our release, but generally cells enter S-phase at around 30 minutes. The release in figure S3 was carried out at 25 ˚C rather than 30 ˚C, which accounts for the additional delay in these data

    *-Fig 2F, why is the rad51-deletion less severe that rad52-deletion - should they not be identical? *

    We agree that these should logically be very similar: we do not know why the two mutants behave slightly differently at some (but not all) IAA concentrations

    *-Fig 2H - could the authors show the flow cytometry (in a supplemental figure) for this experiment? *

    We can show this

    *-Fig 3B-E: OEM is described as a measure of origin efficiency - how is possible for this to have negative values? *

    OEM describes Okazaki fragment strand bias around previously identified origins. If such an origin does not fire in our strain background, a negative OEM can result.

    *-pp9: "Analysis of Okazaki fragment strand bias across the genome suggested that the average direction of replication was relatively similar at most loci across all Pol3 depletion conditions". The authors data is quantitative and they should be able to quantify how similar their data are across the various conditions, rather than making a qualitative statement: "relatively similar". *

    We apologize and can re-phrase this. The intention of this statement is simply to draw the reader’s attention to the fact that global distributions of Okazaki fragments are not completely altered (e.g. only 1-2 origins per chromosome) as a prelude to the more quantitative analysis that follows in figure 3.

    *-pp9: "origin firing efficiency in S. cerevisiae correlates strongly with replication timing"; it would be useful for the authors to support this statement with a citation. *

    We will add 1-2 citations to support this statement

    *-Fig 4A: it would help the reader if the authors could show 'zoomed in' examples of the points that the authors make (in addition to the whole chromosome view): slowed fork progression, reduced early origin activity, increased late origin activity (e.g. an origin that is normally passively replicated, that upon pol-delta depletion is no longer passively replicated and therefore becomes more efficient), etc. *

    We agree that this would be helpful, and can add examples in the supplement

    *-pp11: "An analogous global decrease in replication-origin firing efficiency has been observed in Pol ∂-deficient human fibroblasts" - but the authors are reporting a global increase in origin firing efficiency (Fig 3B). *

    We can re-phrase this.

    *-the nucleosomal ladder in Fig 5A is only weakly apparent from the gel and not particularly apparent from the density trace, this makes it's disappearance upon IAA treatment hard to interpret. Is the weak nucleosomal ladder what the authors had anticipated (in the absence of IAA)? *

    We do not expect a weaker nucleosomal ladder than normal in the absence of IAA. In our experience these gels just sometimes give better ladders than others, and we hope that the traces help with interpretation

    *-I found the effects being described by the authors in Fig 5B & C difficult to see, particularly for the transcription factors. Furthermore, why are these data differently normalised to those in Fig 4B & C (median vs. maximum) *

    In figure 4 we normalize to maximum since all DNA should eventually be replicated, and we therefore think that showing coverage relative to a maximum value of 1 is most informative. In figure 5 we compare distributions of termini around obstacles, and therefore feel that normalizing to the median is a more appropriate way to compare enrichment around a given meta-element. The shapes of the graphs would be unchanged by choosing a different normalization point. In order to make changes easier to see, we can make the lines thinner in figure 5 and/or change the y-axis scale.

    *-the final sentence of the results section returns to an analysis of the OK-seq data and is essentially a temporally segregated analysis (Fig S6) otherwise equivalent to that presented in Fig 5B. Given the importance placed on these data by the authors in the interpretation of the HydEn-seq data, I feel that it would help the reader to have been presented with these data earlier in the results section. *

    We can move these data up

    *-p22: OK-seq methods. The authors should indicate the culture conditions for these experiments. *

    We can include this

    *-p22: Computational analyses: the authors should indicate which reference genome assembly they used. *

    We can include this

    *-Fig 6B & C: the y-axis labels are confusing - do the authors mean Crick strand here? *

    Oops. Yes, we do. We thank the reviewer for catching this

    ***REFEREE CROSS COMMENTING:** *

    *All three reviewers seems to be in broad agreement about this manuscript. There is one significant concern raised by the other reviewers that I'd missed: that some of the Okazaki fragment analysis was done with non-synchronised cultures. I agree with this concern, however I don't think that there is necessarily a problem with the alternative explanation suggested by reviewer #1 ('Okazaki fragments enrichment may be skewed towards late origins'). While the accumulation of S phase cells might well be expected to lead to a bias towards isolating more Okazaki fragments from around late origins, the authors calculate the fraction of reads (i.e. Okazaki fragments) mapping to each strand. The potential presence of more late S phase cells would give greater sequence coverage over late replicating regions, but alone would not alter the fraction of reads mapping to each strand. However, I agree that interpretation of this experiment is not as simple as suggested by the authors and there may well be alternative explanations along the lines suggested by reviewer #1. *

    *There was a subsequent Okazaki fragment experiment performed with synchronised cells (Fig 4) and it might be possible to use these data to assess any differential impact on early vs late origins. *

    We agree, and will do this analysis

    *Reviewer #3 (Significance (Required)): *

    My expertise is in DNA replication and genome stability, particularly replication timing and replication origin function.

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    Referee #3

    Evidence, reproducibility and clarity

    This manuscript describes the consequences of reducing the cellular concentration of a pol-delta subunit in S. cerevisiae. Pol-delta plays multiple cellular roles at both the replication fork (it is one of two DNA polymerases responsible for lagging strand synthesis) and during repair synthesis after DNA damage. The authors combine genetic and genomic methodologies to characterise how reduction in pol-delta concentration impacts on cellular fitness and specifically lagging strand synthesis. Overall it is technically a well executed study that is clearly presented and the data are predominantly appropriately interpreted.

    I have a number of major comments:

    1. The authors apply OK-seq (a methodology first developed by the senior author and therefore they are clearly experts at this) to study the consequence of pol-delta depletion on genome replication. OK-seq requires isolation of Okazaki fragments and this in turn requires removal of DNA ligase (Cdc9) - the authors achieve this with the anchor away system. My concern is that in these experiments the authors are depleting two factors required for lagging strand synthesis: pol-delta and ligase; it is unclear to me how the authors can determine the relative contribution of each depletion to the observed phenotypes. Could some of the observed phenotypes (e.g. fork slowing, 5' and 3' ends of Okazaki fragments, etc) be a consequence of the double depletion, rather than just pol-delta depletion as concluded by the authors? The authors present this as a method to determine genome replication timing, but really it is an assay to look at fork direction. Given the need for an addition mutation in OK-seq (cdc9), I would encourage the authors to consider a more direct assay for replication dynamics upon pol-delta depletion, such as a copy number measure (or BrdU-ip) of DNA replication or DNA combing - these methods don't require Cdc9 depletion and could therefore ensure that observed phenotypes are a consequence of pol-delta depletion (rather than the double depletion).

    2. One major conclusion reached by the authors is that pol-epsilon can contribute to lagging strand synthesis upon pol-delta depletion (at least during late replication). This conclusion comes from the authors use of HydEn-seq to measure rNTP incorporation from which the contribution of a polymerase (pol-epsilon in this case) to strand synthesis can be determined. In a manner analogous to OK-seq, this requires the introduction of additional mutations (both in the polymerase and by the removal of RNaseH activity). The authors interpretation that pol-epsilon can play a role in lagging strand synthesis is dependent upon there being no temporal change in pol-delta strand-displacement activity, despite continued pol-delta depletion through S phase. It is not clear to me that the data presented in Fig 5 & 6 has the sensitivity to conclude this (and the OK-seq data is also subject to the potential bias of the double depletion of pol-delta and Cdc9). I feel that a necessary control to support this conclusion, would be to undertake the HydEn-seq experiment in the absence of the pol-epsilon mutation (just pol-delta depletion in the absence of RNaseH activity). This would allow the authors to measure any increase is residual rNTPs (likely from pol-alpha primase) on the lagging strand as a consequence of pol-delta depletion and determine whether they are equally likely in early and late S phase.

    The following comments are more minor:

    -for the experiment in Fig S1B, the growth in 1.0 mM IAA is somewhat surprising given how sick the cells appear on equivalent plates. I couldn't find in the methods a description of the experimental conditions.

    -there is considerable variability in the S phase kinetics from bulk DNA analysis (flow cytometry) when comparing Fig 1C, 2D, S3. Fig 1C appears to be the exception, with all the other figures showing poor S phase progress by comparison. It would be useful for the authors to recognise these differences and comment upon them. E.g. they appear to all be identical experiments, but are there experimental differences that could explain the different kinetics?

    -Fig 2F, why is the rad51-deletion less severe that rad52-deletion - should they not be identical?

    -Fig 2H - could the authors show the flow cytometry (in a supplemental figure) for this experiment?

    -Fig 3B-E: OEM is described as a measure of origin efficiency - how is possible for this to have negative values?

    -pp9: "Analysis of Okazaki fragment strand bias across the genome suggested that the average direction of replication was relatively similar at most loci across all Pol3 depletion conditions". The authors data is quantitative and they should be able to quantify how similar their data are across the various conditions, rather than making a qualitative statement: "relatively similar".

    -pp9: "origin firing efficiency in S. cerevisiae correlates strongly with replication timing"; it would be useful for the authors to support this statement with a citation.

    -Fig 4A: it would help the reader if the authors could show 'zoomed in' examples of the points that the authors make (in addition to the whole chromosome view): slowed fork progression, reduced early origin activity, increased late origin activity (e.g. an origin that is normally passively replicated, that upon pol-delta depletion is no longer passively replicated and therefore becomes more efficient), etc.

    -pp11: "An analogous global decrease in replication-origin firing efficiency has been observed in Pol ∂-deficient human fibroblasts" - but the authors are reporting a global increase in origin firing efficiency (Fig 3B).

    -the nucleosomal ladder in Fig 5A is only weakly apparent from the gel and not particularly apparent from the density trace, this makes it's disappearance upon IAA treatment hard to interpret. Is the weak nucleosomal ladder what the authors had anticipated (in the absence of IAA)?

    -I found the effects being described by the authors in Fig 5B & C difficult to see, particularly for the transcription factors. Furthermore, why are these data differently normalised to those in Fig 4B & C (median vs. maximum)

    -the final sentence of the results section returns to an analysis of the OK-seq data and is essentially a temporally segregated analysis (Fig S6) otherwise equivalent to that presented in Fig 5B. Given the importance placed on these data by the authors in the interpretation of the HydEn-seq data, I feel that it would help the reader to have been presented with these data earlier in the results section.

    -p22: OK-seq methods. The authors should indicate the culture conditions for these experiments.

    -p22: Computational analyses: the authors should indicate which reference genome assembly they used.

    -Fig 6B & C: the y-axis labels are confusing - do the authors mean Crick strand here?

    REFEREE CROSS COMMENTING:

    All three reviewers seems to be in broad agreement about this manuscript. There is one significant concern raised by the other reviewers that I'd missed: that some of the Okazaki fragment analysis was done with non-synchronised cultures. I agree with this concern, however I don't think that there is necessarily a problem with the alternative explanation suggested by reviewer #1 ('Okazaki fragments enrichment may be skewed towards late origins'). While the accumulation of S phase cells might well be expected to lead to a bias towards isolating more Okazaki fragments from around late origins, the authors calculate the fraction of reads (i.e. Okazaki fragments) mapping to each strand. The potential presence of more late S phase cells would give greater sequence coverage over late replicating regions, but alone would not alter the fraction of reads mapping to each strand. However, I agree that interpretation of this experiment is not as simple as suggested by the authors and there may well be alternative explanations along the lines suggested by reviewer #1.

    There was a subsequent Okazaki fragment experiment performed with synchronised cells (Fig 4) and it might be possible to use these data to assess any differential impact on early vs late origins.

    Significance

    My expertise is in DNA replication and genome stability, particularly replication timing and replication origin function.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    In this manuscript the authors explore the basis of the deleterious effects of reduced levels of the replicative Polymerase delta. This polymerase plays several important roles in the replication process: it synthesizes most of the lagging strand, but also extends the first primer during the synthesis of the lagging strand, and it contributes to the removal of the RNA and most of the DNA synthesized by primase/Polymerase alpha during Okazaki fragment maturation. In this study, the authors systematically analyze the impact of Pol delta depletion in S. cerevisiae. They use a degron-tagged allele to modulate the levels of the polymeraes and apply mainly NGS methods and classical genetics to explore the consequences for survival, checkpoint signalling and replication features such as fork speed, origin firing efficiency and Okazaki fragment length and distribution. They report that Pol delta depletion leads to a checkpoint activation via the Rad9-dependent damage signalling pathway (but not the Mrc1-dependent replisome-associated signalling) and an accumulation of single-stranded DNA. Phosphorylation of histone H2A is taken as a marker of DNA double-strand breaks, and from the observation that deletion of recombination factors, but not end-joining factors aggravate the fitness of Pol delta-depleted cells they conclude that homologous recombination is responsible for the repair of these breaks. Analysis of replication by Okazaki fragment sequencing indicates a slight decrease in the firing efficiency of early/efficient origins, but an increase in the firing efficiency of late origins. They also observe a reduction in fork speed, although they are not able to attribute this to either a globally slower fork movement or an increase in the stalling of individual forks. They find that Pol delta depletion does not change the size of Okazaki fragments, but causes defects in the nick translation during Okazaki fragment maturation. Finally, they use NGS technology to show that the leading strand Polymerase epsilon engages in lagging strand replication particularly at late origins when Pol delta is depleted. From their observations, the authors develop a model where depletion of Pol delta primarily affects late replicating regions. They explain this by invoking a stable association of Pol delta with early replisomes, which sequesters the enzyme, thus causing an under-supply at replisomes that assemble later during S phase. This then leads to the involvement of Pol epsilon on the lagging strand. Based on the observation that fork speed and Okazaki fragment maturation are both affected, they propose that these two reactions normally compete for Pol delta, suggesting that optimal replication would require two molecules of the polymerase per fork.

    Major comments

    The experiments shown here are largely clean and well controlled, and the manuscript is written nicely and well-structured.

    Compared to the Okazaki fragment analysis, the treatment of double-strand breaks appears somewhat cursory and remains inconclusive. Phosphorylation of H2A seems insufficient evidence for double-strand breaks, as other structures could also give rise to that signal. These lesions should be detected in a more direct manner, e.g. pulsed-field gel electrophoresis. The authors also don't provide a mechanism by which such breaks would emerge. Related to the minor effect of the ku mutant, I am wondering about the altered appearance of the colonies in Figure 2F (concerning both ku70 and rad51) - what is different about these, and could their „denser" appearance explain the slight suppression effect observed?

    Concerning the damage signalling: it is surprising to see a damage signal at concentrations of IAA that do not lead to a significant depletion of Pol delta yet (0.05 mM). At this point, it is hard to imagine DSBs to form. Could the authors explain this discrepancy?

    The notion that late origin firing is enhanced despite checkpoint activation is counterintuitive. Do the authors think that this effect overcomes the suppression of late origins that is normally associated with checkpoint activation? It would be helpful to test whether abolishing that phenomenon (e.g. by a mec1-100 mutant) would enhance the effect and render late origins even more active.

    It would be important to characterise the fork speed defect better, using alternative methods rather than just relying on Okazaki fragments. A differentiation between slower fork progression and more frequent fork stalling would be relevant and might help to evaluate the contribution of Pol epsilon. This might be accomplished by DNA fibre analysis. Alternatively, BrdU incorporation could serve to observe replication over the entire genome rather than only in the vicinity of replication origins. It would also be important to differentiate fork speeds in early versus late replicating regions - according to the authors' model, the defects should be most obvious in the late regions (Fig. 4 concerns only early origins).

    Figure S3: Considering the differences in cell cycle progression, it would make more sense to compare equivalent stages of the cell cycle / S phase rather than identical time points.

    Considering that the Okazaki fragment analysis was done with non-synchronised cultures, is it possible that the skew in the cell cycle profile could influence the Okazaki fragment pattern?

    Would it be possible to monitor not only total Pol delta levels, but also the level of Pol delta bound to the chromatin? It is shown that the level of Pol delta is depleted in the whole cell extracts, but it would be interesting to see how severe the depletion is on the chromatin.

    Figure 6 is confusing and should be clarified:

    • Figure 6B: assigning the Watson and Crick strands in the schematic would make that figure easier to understand;
    • Figure 6B-C: the axes are labeled as 'Fraction of rNMP on Watson strand', but would it not make more sense if they were labeled 'Fraction of rNMP in Crick strand'?
    • Figure 6D-E: the right side scale is labelled as 'increase in rNMP on Crick strand' while in the figure legend is says it is 'change in the fraction of ribonucleotides mapping to the Watson strand. That description should be clarified;
    • Figure 6D: using 'Change in Okazaki fragments strand bias' to label the black line (description in the box above the figure) instead 'Change in Okazaki strand bias' would be more appropriate;
    • Figure 6F: the authors seem to have subtracted strand bias measured for Okazaki fragments from the strand bias measured for rNMP. It is valid to subtract these biases from each other, considering that the two structures arise independently and with different frequencies?

    Minor comments:

    Can the authors conclude that Pol delta deficiency/ incompleteness of lagging strand synthesis affects the nucleosome deposition onto DNA? (Figure 5-A)

    Why did the authors use rnh202Δ and not a mutant in the catalytic subunit of RNase H2?

    An extra control might be useful: comparing POL3-AID rnh202Δ with the POL3-AID pol2M644G rnh202Δ triple mutant could further confirm that the observed effect is Pol epsilon-dependent.

    Figure 2H: It would be good to see the cell cycle distribution corresponding to the western blot images.

    Various spelling, grammar or precision of expression issues:

    • Pg. 4, line 4: endonucleolytically instead of nucleolytically.
    • Pg. 6, line 10: Remove 'was'.
    • Pg. 6, line 12: Remove 'in vivo' from the subtitle.
    • Pg. 6, line 14: 'an C-terminal' should be 'a C-terminal'
    • Pg 16, line 13: Phrasing implies that the synthesis of both leading and lagging strands by Pol delta in regions in the vicinity of replication origins is essential - please quote any paper testing its essentiality.
    • Please follow standard yeast genotype nomenclature, remove ';' when listing the yeast genotypes (e.g. POL3-AID mec1Δ sml1Δ instead of POL3-AID;mec1Δ;sml1Δ- example from Figure 2-B).
    • Concentrations of IAA are missing in few places (e.g. legend of Figure 1-C, page 24).
    • Figure 1A: add the label 'IAA (mM)'
    • Figure 2G: pleae provide a shorter exposure of the H4 blot in addition to the one shown.
    • Figure 6: adding a schematic presenting the events at actively and passively replicating late origins (and the predictions about leading and lagging strand bias) would help to understand the figure.
    • The format of the references is inconsistent.
    • 'On Watson/Crick strand' should be replaced with 'in Watson/Crick strand'

    Significance

    This is a nice piece of work that provides in vivo confirmation of several observations that have been made in purified recombinant systems. In that sense, the overall novelty is limited, but this type of study is still important to do, as biochemical assays do not always reflect what is happening in cells, and this study gives insight into basic activities of the replisome. The participation of Pol epsilon in lagging strand synthesis is an interesting observation. Overall, the study will be of interest for the DNA replication field. My own expertise is in replication, predominantly in yeast. I have experience in NGS analysis of replication as well as in genetic analysis of the DNA damage response. I therefore feel competent to evaluate all aspects of the manuscript.

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    Referee #1

    Evidence, reproducibility and clarity

    This study examined the consequences of limiting levels of DNA polymerase d (Pol d) in yeast. The authors first reported multiple genome instability consequences following lowered Pol d level, including defect in S phase progression, growth defect, elevated spontaneous DNA damage, accumulation of ssDNA and activation of replication and DNA damage checkpoint. These observations are solid but not unexpected. By genome wide analysis using the Okazaki fragment (OF) mapping and ribonucleotide mapping (for polymerase usage), the authors claim a few potentially novel and striking observations that lowered Pol d differentially impact efficiencies of early vs. late origins, and that lowered Pol d results in Pol e participating in lagging strand synthesis around late origins. However, I remained unconvinced based on the data presented. These observations need to be further substantiated and alternative interpretations should be considered.

    Main concerns:

    One of major conclusions the authors tried to make is that the early vs. late origins are differentially affected by low level of Pol d. First, they used OF mapping data to examine origin efficiency. Asynchronous "Cultures were treated with IAA for 2h before the addition of rapamycin for 1h to deplete DNA ligase I (Cdc9) from the nucleus via anchor-away". IAA concentrations used were of 0, 0.2 mM, 0.6 mM, and 1 mM. The problem is that Figure S1 clearly showed that treating asynchronous cultures with >0.1 mM of IAA for as short as 30 min significantly alters the cell cycle profiles, mainly resulting in accumulation of S phase cells, to different extent. Presumably Okazaki fragments accumulated from these cultures suffering from the synchronizing effect may not be representative of the real change in global replication profile. For instance, it is not difficult to predict that the Okazaki fragments enrichment may be skewed towards late origins if more cells are accumulated in mid S phase following Pol d depletion. For this reason, I don't believe the result is conclusive. The experiment may be re-designed for samples at different time points following release from G1.

    This concern also should preclude the authors from drawing conclusion about Pol e usage on lagging strands based on comparison between HydEn-seq data and OF mapping data shown in Figure 6. In fact, the rNMP incorporation change is very similar between early and late origins. The only evidence that the author rely on is the discrepancy in OF data between the two groups origins, which makes the reliability if origin efficiency measurement the central piece of data in this study. Thus, alternative approaches should also be considered to map origin efficiencies.

    Even if Pol e strand bias is lowered at late origins, as the authors tend to believe, there are still alternative models other than Pol e being used for lagging strand synthesis. For instance, if TLS polymerases are used on lagging strands, it could result in more ribonucleotide incorporation on the lagging strand, as they are lower-fidelity polymerases. Alternatively, if Pol d scarcity leads to more Pol e synthesis or lower RNA primer processing, it might also contribute to more apparent ribonucleotide incorporation on the lagging strands.

    In Figure S5, the two HydEn-seq replicates are very different, where replicate1 shows very low strand bias. I suspect perhaps the strain used for replicate 1 does not contain pol2-M644G or rnh202 deletion.

    Significance

    Given that different aspects of Pol d deficiency have been implicated in various human diseases and cancer, this type of analysis is of interest to the field.

  7. Version published to 10.1101/2019.12.17.879544v1 on bioRxiv