CDKs-mediated phosphorylation of PNKP is required for end-processing of single-strand DNA gaps on Okazaki Fragments and genome stability

  1. Kaima Tsukada
  2. Tomoko Miyake
  3. Rikiya Imamura
  4. Kotaro Saikawa
  5. Mizuki Saito
  6. Naoya Kase
  7. Masamichi Ishiai
  8. Yoshihisa Matsumoto
  9. Mikio Shimada

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Abstract

Polynucleotide kinase phosphatase (PNKP) has enzymatic activities as 3′ -phosphatase and 5′ - kinase of DNA ends to promote DNA ligation and repair. Here, we show that cyclin-dependent kinases (CDKs) regulate the phosphorylation of threonine 118 (T118) in PNKP. This phosphorylation allows recruitment to the gapped DNA structure found in single-strand DNA nicks and/or gaps between Okazaki fragments (OFs) during DNA replication. T118A (alanine)-substituted PNKP-expressing cells exhibited accumulation of single-strand DNA gaps in S phase and accelerated replication fork progression. Furthermore, PNKP is involved in poly (ADP-ribose) polymerase 1 (PARP1)-dependent replication gap filling as a backup pathway in the absence of OFs ligation. Altogether, our data suggest that CDK-mediated PNKP phosphorylation at T118 is important for its recruitment to single-strand DNA gaps to proceed with OFs ligation and its backup errors via the gap-filling pathway to maintain genome stability.

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    __Below is our point-by-point reply to the reviewer's comments __

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

    PNKP is one of critical end-processing enzymes for DNA damage repair, mainly base excision & single strand break repair, and double strand break repair to a certain extent. This protein has dual enzyme function: 3' phosphatase and 5' kinase to make DNA ends proper for ligation. It has been demonstrated that PTM of PNKP (e.g., S114, S126), particularly phosphorylation by either ATM or DNAPK, is important for PNKP function in DNA damage repair. The authors found a new phosphorylation site, T118, of PNKP which might be modified by CDK1 or 2 during S phase. This modification of phosphorylation is involved in maintenance and stability of the lagging strand, particularly Okazaki fragments. Loss of this phosphorylation could result in increased single strand gaps, accelerated speed of fork progression, and eventually genomic instability. And for this process, PNKP enzyme activity is not that important. And the authors concluded that PNKP T118 phosphorylation is important for lagging strand stability and DNA damage repair.

    Major comments

    In general, enzymes have protein interactions with its/their substrates. If PNKP is phosphorylated by either/both CDK1/2, the protein interaction between these would be expected. However, the authors did not provide any protein interactions in PNKP and CDKs. *Thank you for your suggestion. We will perform GFP-pulldown assays using cell extracts from HEK293 cells expressing GFP-WT-PNKP, GFP-T118A-PNKP. And then to confirm the interaction of PNKP and CDK1/2, we will blot with CDK1 and CDK2 antibodies. *

    It is not clear how T118 phosphorylation is involved in DNA damage repair itself as the authors suggested. The data presenting the involvement of T118 phosphorylation in this mechanism are limited. This claim opens more questions than answers. CDK1/2 still phosphorylates T118 in this DNA damage repair process? What would happen to DNA damage repair in which PNKP involves outside of S phase in terms of T118 phosphorylation?

    Thank you for your comment. We have investigated how T118 phosphorylation is important in DNA damage repair by several experiments. In figure S8, we tested SSB and DSB repair abilities of PNKP KO cells expressing PNKP T118A mutant, in which PNKP T118 phosphorylation has critical roles in both SSB and DSB repair pathways. Interestingly, the result of SSB repair assay (figure S8A & B) may indirectly indicate that T118 phosphorylation is important for SSB repair throughout cell cycle as these SSBs are instantly induced by IR exposure and recovered only for 30 mins that is presumably not enough time for cells to go through cell cycle. Along with the repair abilities, we also analyzed a recruitment kinetics/ability to DNA damage in PNKP T118A and T118D mutants using laser micro-irradiation assay in figure S9. This result indicates that the phosphorylation of PNKP at T118 is controlling its recruitment to at least laser-induced DNA damage sites. Moreover, we have analyzed recruitment of PNKP to a single-strand DNA gap structure, which mimics intermediates of some DNA repair pathways and incomplete Okazaki fragment maturation, using cell extracts from PNKP KO cells expressing PNKP T118A and T118D mutants and biochemical assay in figure 4H. This assay is much cleaner and shows that loss of T118 phosphorylation impairs PNKP recruitment to the ssDNA gap structure. We believe that these data sufficiently support our model that the phosphorylation of T118 on PNKP is involved in DNA repair in general. However, we agree with that we have not yet directly tested DNA repair ability of PNKP T118A in outside of S-phase. Therefore, in addition to these data, we will perform H2O2-induced SSB and IR-induced DSB repair assay using EdU (S phase) pulse labelling in PNKP KO cells expressing PNKP T118A mutant, then we will measure the ADP-ribose intensity and pH2AX foci in EdU negative cells (outside of S phase as the reviewer suggested).

    Along the same line with #1/2 comments, the recruitment of PNKP to the damage sites is XRCC1 dependent. Is not clear whether PNKP recruitment to gaps on the lagging strand is XRCC1 independent or dependent. It might be interesting to examine (OPTIONAL)

    *Thank you for an important suggestion. XRCC1 acts as a scaffold of PNKP and is required for recruitment of PNKP for canonical SSB repair, although we propose that PNKP is involved in two pathways in DNA replication: PARP1-XRCC1-dependent ssDNA gap filling pathway and Okazaki fragment maturation pathway working with FEN1. It is still important to address how XRCC1 is required for PNKP recruitment to the single-strand gaps on nascent DNA. Therefore, we will perform iPOND analysis in XRCC1 knock down + GFP-WT-PNKP expressed HEK293 cells. *

    Minor comments

    In results: 'Generation of PNKP knock out U2OS cell line'

    • In figure S2A; There are no data regarding diminishing the phosphorylation of g-H2AX.

    Thank you for your suggestion. We will add pH2AX blot data in fig S2A (all reviewers requested).

    • By showing data in figure S2B/C/D/E, the authors describe 'PNKP KO cells impaired the SSBs repair activity'. However, as the authors mentioned in this manuscript, PNKP could bind to either XRCC1 or XRCC4. Also for this experiment, IR had been applied, which induces DNA double strand breaks. Therefore, it is not certain that the authors' description is fully supported by these data presented. Perhaps, SSB inducing reagents should be used instead of IR.

    In figure S2B/C/D/E, we used gamma-ray as IR source, which classified as low energy transfer irradiation. which mainly act as indirect effect to the DNA. It is estimated gamma-ray induce DNA damage as 60-80% SSBs and 20-40 % DSBs. We believe our results are reasonable. In addition to these results, we will perform poly-ADP-ribose assay with H2O2 treatment to more specifically assess SSBs repair activity.

    • Is there any FACS analysis data to support the description of the last sentence 'especially the phosphorylation of PNKP T118, is required for S phase progression and proper cell proliferation'?

    Thank you for your suggestion. We will add the FACS analysis data of cell cycle profiles in PNKP KO cells expressing GFP, GFP-PNKP WT, T118A.

    In results: 'CDKs phosphorylate T118 of PNKP ~~~ replication forks'

    • In figure 3A, Is there any change in total PNKP (both GFP-tagged & endogenous) level?

    *Thank you for your suggestion. We agree with your comment. We will add the PNKP expression analysis in different cell cycle population in asynchronized and synchronized cells (G1, S, G2/M samples). *

    In results: 'Phosphorylation of PNKP at T118 ~~~ between Okazaki fragments'

    • In figure 4D, What happens in the ADP-ribose level, when T118D PNKP is expressed?

    *Thank you for your suggestion. This is interesting question. We will perform ADP-ribosylation assay in PNKP KO cells and PNKP KO cells expressing PNKP WT and T118D, and add data of ADP-ribose levels in those cells. *

    In results: 'PNKP is involved in postreplicative single-strand DNA gap-filling pathway'

    • The description regarding data presented in figure 6 is not clear enough. These data might suggest that wildtype U2OS does not have SSB which is a substrate for S1 nuclease (except under FEN1i and PARPi treatment), whereas PNKP KO has SSB during both IdU and CIdU incorporation, so that S1 nuclease treatment dramatically reduces the speed of fork formation in PNKP KO cells. Also In figure 6B/C/D, adding an experimental group of PNKP KO with S1 nuclease + PARPi might help to understand the role of PNKP during replication better. Also these additional data could support the description in discussion 'Furthermore, PNKP is required for the PARP1-dependent single-strand gap-filling pathway ~~~ DNA gap structure'.

    *We agree with reviewer's comment and suggestion. Since this point is also raised by reviewer 3, we will add the rationale of the experiment and more detailed description about the results, which would substantially improve this manuscript. We will also revise our representation in text followed by the comment. In addition to revising the text, we will add experiment groups of PNKP KO with S1 nuclease with/without PARPi as the reviewer suggested. *

    In results: 'Phosphorylation of PNKP at T118 is essential for genome stability'

    • In figure S8C, Did you measure g-H2AX foci disappearance for later time point, such as 24 hrs after DNA damage? Is not clear whether non-phosphorylated PNKP at T118 inhibit DNA damage repair or make it slower? How does T114A-PNKP behave in this experimental condition? T114 is well known target of ATM/DNAPK for DDR & DSB repair.

    Thank you for your suggestion. We agree with your point. It is very important to analyze whether T118A mutant shows delayed or total loss of DSB repair ability. We will add the measurement of pH2AX foci at 24 hrs after IR in PNKP KO cells expressing GFP, WT-PNKP, T118A-PNKP. Although the analysis of pS114 PNKP is previously reported (Segal-Raz et al., EMBO reports, 2011 and Zolner et al., Nucleic Acids Research, 2011), we will also perform pH2AX assay in PNKP KO cells expressing S114A-PNKP as a control.

    The result shown in figure S9 should be described in the result section, not in the discussion section.

    Thank you for your suggestion. This is a point also raised by Reviewer 3. Since we are going to re-consider the layout of the manuscript upon the planned revision (as reviewer 3 suggested), we will move these points to the appropriate result section from the discussion.

    **Referees cross-commenting**

    I could see a similar degree of positive tendency toward the manuscript. I agree with the comments and suggestions in additional experiments made by reviewers 2 and 3. Those suggestions will improve an impact of the manuscript in the DNA damage repair field.

    Reviewer #1 (Significance (Required)):

    Significance

    The authors discovered new phosphorylation site (T118) of PNKP which is an important DNA repair protein. This modification seems to play a role in maintenance of the lagging strand stability in S phase. This discovery is something positive in DNA repair field to expand the canonical and non-canonical functions of DNA repair factors.

    The data presented to support PNKP functions and T118 phosphorylation in S phase seem solid in general, yet it is not sure how much PNKP is critical in the Okazaki fragment maturation process which is known that several end processing enzymes (like FEN1, EXO1, DNA2 etc which leave clean DNA ends.) are involved.

    These finding might draw good attentions from researchers interested broadly in cell cycle, DNA damage repair, replication, and possibly new tumor treatment.

    My field and research interest: DNA damage response (including cell cycle arrest and programmed cell death), DNA damage repair (including BER, SSBR, DSBR)

    Thank you very much for your positive comment. As you mentioned, there are several other end processing enzymes that seem to be involved in Okazaki fragment maturation, however, none of those enzymes is reported as a protein involved in the gap-filling pathway as well. Therefore, the role(s) of PNKP in DNA replication are very outstanding as PNKP could be involved in two separate pathways, Okazaki fragment maturation and a back-up gap-filling repair process. As you suggested, we will add several experiments such as iPOND experiments using XRCC1-depleted cells, analysis of DNA repair ability of PNKP T118A mutant throughout cell cycle and S1 nuclease DNA fiber assays in PNKP KO cells with/without PARP inhibitor treatment, to reveal how much PNKP is critical in the Okazaki fragment maturation. We believe that performing those experiments makes the conclusion and this manuscript more solid and convincing.

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

    Polynucleotide kinase phosphatase (PNPK) participates in multiple DNA repair processes, where it acts on DNA breaks to generate 5'-phosphate and 3'-OH ends, facilitating the downstream activities of DNA ligases or polymerases.

    This manuscript identifies a CDK-dependent phosphorylation site on threonine 118 in PNKP's linker region. The authors provide some convincing evidence that this modification is important to direct the activity of PNPK towards ssDNA gaps between Okazaki fragments during DNA replication. The authors monitored protein expression levels, enzymatic activity, the growth rate and replication fork speed, as well as the presence of ssDNA damage to make a comprehensive overview of the features of PNKP necessary for its function.

    Overall, the conclusions are sufficiently supported by the results and this manuscript is relevant and of general interest to the DNA repair and genome stability fields. Some level of revision to the experimental data and text would help strengthen its message and conclusions.

    Major points:

    In an iPOND experiment the authors detect the wt PNKP and the T118 phosphorylated form at the forks and conclude that this phosphorylation promotes interaction with nascent DNA (Figure 3E). An informative sample to include here would have been the T118A mutant. Based on the model proposed, the prediction would be that it would not be associated with the forks, or at least, associated at reduced levels compared to the wt. *Thank you for your suggestion. We agree with your comment. We will add the iPOND analysis in PNKP KO cells expressing T118A mutant to confirm that pT118 is important for recruitment of PNKP at nascent DNA. *

    The quality of the gels showing the phosphatase and kinase assays in Figure 5 could be improved to facilitate quantification of the results. The gel showing the phosphatase activity has a deformed band corresponding to K378A mutant. The gel showing the kinase activity seems to be hitting the detection limits, and the overall high background might influence the quantification of D171A mutant in the area of interest. The authors should provide a better quality of these gels, focusing on better separation (running them longer, eventually with a slightly increased electric current) and higher signal of the analyzed bands (longer incubation phosphatase/kinase prior to quenching or loading higher amount of DNA).

    We agree with your suggestion. This phosphatase and kinase assay could be improved. We will perform this assay again followed by reviewer's suggestions.

    The authors sometimes make statements like: "a slight increase, slightly increased, relatively high" without an evaluation of the statistical significance for the presented data. An example of such a statement is: "T118A mutant-expressing cells exhibited a marked delay in cell growth, which was not observed for S114A, although T122A, S126A, and S143A were slightly delayed," based on the figure 2E. A similar comment applies also to figures 4A, 5A, 5E. Whenever possible, the authors should include also an evaluation of the statistical significance in the statement.

    Thank you for your suggestion. We will check manuscript and revise representation as reviewer's suggestion.

    Minor revisions:

    I could not find a gH2AX blot for figure S2A.

    Thank you for your suggestion. We will add pH2AX blot data in fig S2A.

    The authors established two PNKP-/- clones and supported it with sequencing and several functional observations However, the C-terminal antibody appears to detect lower-intensity bands (Figure 1A). Can authors comment on those bands?

    Thank you for your comment. One possibility of this band is artificially recognized bands. To improve this problem, we will try electrophoresis for longer time to separate this band.

    Why the S1 nuclease data on DNA fibers do not show the same level of epistasis with the Fen1i, as do those on ADP-ribosylation?

    Because FEN1 dependent Okazaki fragment maturation and PARP1-XRCC1 dependent gap-filling pathway are different pathways, FEN1i and PARPi treatment resulted in an additive effect in S1 nuclease data in PNKP WT cells. To facilitate better understanding, we will add graphical scheme in figure 6 (a similar problem was raised by Reviewer 3 below) and revise the description of the result.

    **Referees cross-commenting**

    I agree with all the comments from the reviewers 1 and 3.

    Reviewer #2 (Significance (Required)):

    Significance:

    The manuscript identifies a CDK phosphorylation site in a relevant DNA repair protein. The experiments on this part are elegant and convincing. It seems that this phosphorylation is important during DNA replication and there is some supporting evidence in this point, although not as robust, meaning that it is not clear whether this phosphorylation is controlling specifically the recruitment to Okazaki fragments, or a general role in DNA repair. Maybe if they see a reduced recruitment of the T118A mutant to the forks (iPOND experiment) this would further increase the impact.

    This work will be relevant to the basic research, especially in the fields of DNA repair and DNA replication.

    My expertise: DNA replication, genome stability, telomere biology.

    Thank you very much for your positive comment. As you suggested, we will perform an iPOND assay using PNKP T118A mutant. In addition of the T118A iPOND assay, we will also analyze the DNA repair function of PNKP T118A mutant throughout cell cycle as reviewer 1 suggested. We believe that results of these experiments will pin down whether the phosphorylation of PNKP on T118 is controlling its recruitment to Okazaki fragments specifically or single-strand DNA gaps in general, and solidify the conclusion of the manuscript.

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

    Tsukada and colleagues studied the role of PNKP phosphorylation in processing single-strand DNA gaps and its link to fork progression and processing of Okazaki fragments.

    They generated two PNKP KO human clonal cell lines and described defects in cell growth, accumulation in S-phase, and faster fork progression. With some elegant experiments, they complement the KO cell lines with deletion and point mutants for PNKP, identifying a critical phosphorylation site (T118) in the linker regions, which is important for cell growth and DNA replication.

    They show that phosphorylation of PNKP peaks in the mid-S phase. CDK1 and CDK2/ with Cyclin A2 are the two main CDK complexes responsible for this modification. With the IPOND experiment, the author shows that PNKP is recruited at nascent DNA during replication.

    They described increased parylation activity in PNKP KO cells, and by using HU and emetin, they concluded that this increased activity depends on replication and synthesis of Okazaki fragments.

    Interfering with Okazaki fragment maturation by FEN1 inhibition is epistatic with PNKP KO (and T118A) in influencing parylation activity in the S phase and fork progression. The authors try to understand by mutant complementation which of the two functions (Phosphatase vs Kinase) is important in processing OF, and they propose a primary role for the phosphatase activity of PNKP. They also show that T118 is important in controlling genome stability following different genotoxic stress. Finally, by coupling the measurement of fork progression with PARP/FEN1 inhibitors and S1 treatment, they propose a role of PNKP in the post-replicative repair of single-strand gaps due to unligated OF.

    Here are my major points:

    The authors use a poly ADP ribose deposition measurement to estimate SSB nick/gap formation. Even if PARP activity is strictly linked to SSB repair, ADP ribosylation does not directly estimate SSB/nick gap formation. In addition, in Figs S2A, B, and C, the authors use IR and PARG inhibition to measure poly-ADP ribosylation in WT and PNKP KO cells. IR produces both SSB and DSB. A better and cleaner experiment would be to directly measure SSB formation (with alkaline comet assay, for example) in combination with treatments that are known to mainly cause SSB (H2O2, or low doses of bleomycin). Thank you for your suggestion. The main purpose of this manuscript is to clarify the potential role of PNKP in DNA replication. Therefore, we generated PNKP KO human cells and figure S2 showed confirmation of function of established role of PNKP in SSBs and DSBs repair. In addition, previous our report published in EMBO Journal (Shimada et al., 2015), we showed SSBs and DSBs repair defect in PNKP KO MEF with comet assay (both alkaline and neutral) after IR and H2O2 treatment. In addition to those observations, we will also perform BrdU incorporation assay in PNKP WT and KO cells treated with H2O2. BrdU staining under an undenatured condition has now been commonly used and is a more direct method to detect ssDNA nick/gap formation. We believe that the importance of PNKP in SSB repair is sufficiently supported by all data such as previous comet assays in PNKP KO MEF cells and two SSB repair assays in human cells using ADP-ribose staining or BrdU incorporation, which will be provided in the revised manuscript.

    The manuscript would benefit from substantially restructuring the figures' order and panels. Before starting the T118 part, the authors could create several figures to explain the main consequences of the loss of PNKP. A figure could be focused on DSB-driven genome instability (fig1 + fig S8 and S9). Then, a figure for the single-strand break and link to the S-phase. For example, by using data from Figure 6 and showing only WT vs PNKP KO +- Nuclease S1 (without FEN1 or PARP inhibitors), the authors could easily convince the readers that loss of PNKP leads to the accumulation of single-strand gaps. Only in the second part of the manuscript could they introduce all the T118 parts. Thank you for your suggestion. The layout of this manuscript makes reviewers feeling confusing. After performing all planned experiments, we will carefully re-consider the total layout of the revised manuscript.

    I understand the use of a FEN1 inhibitor to link the PNKP KO phenotype to OF processing, but this drug does not either rescue or exacerbate any of the phenotypes described by the authors. It seems to have just an epistatic effect everywhere. So, what other conclusion can we have if not that PNKO has a similar effect to FEN1? I think that the presence of this inhibitor in many plots complicates the digestion of several figures a little bit. Maybe clustering the data in a different way (DMSO on one side FEN1i on the other) would help. Thank you for your suggestion. We agree that this data set is complicate. To facilitate better understanding, we will change organization of the data according to your suggestion and add graphical scheme in figure 6.

    In terms of the other conclusion we can have from those experiments, the other conclusion is that PNKP might plays two important roles in DNA replication: Okazaki fragment maturation, which seems an epistatic effect with FEN1, and PARP1-XRCC1 dependent single-strand gap filling pathway, which is required for repairing single-strand gaps between Okazaki fragments when Okazaki fragment maturation pathway does not work properly (e.g., loss of FEN1 or PNKP). In figure 6D, we show that a double treatment of FEN1i and PARPi in PNKP WT cells with S1 nuclease treatment shows extensive amount of digested DNA fibers, although a single treatment of either FEN1i or PARPi in PNKP WT cells with S1 nuclease treatment leads to only limited amount of digested DNA fibers, which indicates that two pathways regulated by FEN1 or PARP are coordinately required for preventing eruption of ssDNA gaps in DNA replication. On the other hand, PNKP KO cells with S1 nuclease treatment cause extensive amount of digested DNA fibers even without FEN1i and PARP1i treatments, also it is not further increased by FEN1i and PARPi treatment. Those results indicate that PNKP itself is involved in two pathways mentioned above. Therefore, loss of PNKP has a similar phenotype with loss of FEN1 in terms of Okazaki fragment maturation, but also there is an additional effect in repairing ssDNA nicks/gaps, which is created in FEN1 loss condition, but FEN1 seems not dealing with it.

    Fig S9 should be removed from the discussion. Additionally, the authors should consider whether they want to keep that piece of data in a manuscript that is already pretty dense. Why should we focus on additional linker residues and microirradiation data at the end of this manuscript? *Thank you for your suggestion. This is a point also raised by Reviewer 1. Since we are going to re-consider the layout of the manuscript upon the planned revision, we will move these points to the appropriate result section from the discussion. *

    I suggest using a free AI writing assistant. I think this manuscript would substantially benefit from one. As a non-native English speaker, I personally use one of them and find it extremely useful. Thank you for your suggestion. Our manuscript was revised by a native speaker from an English correction company. However, for revised manuscript, we will discuss with native speakers as well as use a free AI writing assistant to improve the quality of the manuscript.

    Minor points:

    In Figure S1A, the author refers to P-H2AX, but I do not see this marker in the western blot. Thank you for your suggestion. We will add pH2AX blot data in fig S2A.

    **Referees cross-commenting**

    I agree with all comments from reviewer 1 and 2.

    Reviewer #3 (Significance (Required)):

    This is an interesting paper with generally solid data and proper statistical analysis. The figures are pretty straightforward. Unfortunately, the manuscript is dry, and the reader needs help to follow the logical order and the rationale of the experiments proposed. This is also complicated by the enormous amount of data the authors have generated. The authors should improve their narrative, explaining better why they are performing the experiment and not simply referring to a previous citation. Reordering panels and figures would help in this regard. Overall, with some new experiments, tone-downs over strong claims and a better explanation of the rationale behind experiments the authors could create a fascinating paper.

    Thank you very much for your positive comment about the data/analysis and the logic behind the experiments provided in the manuscript. We agree with that a manner and a structure of the manuscript could be improved by reordering figures, cutting down some redundant experiments, adding better explanation of the rationale behind experiments, and toning-down some claims. With rewriting the manuscript as stated above and performing several additional experiments suggested by the reviewers, we believe that the revised manuscript will be more convincing and fascinating.

    1. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    Reviewer #1:

    Minor comments

    • Is there any difference (except for PARGi exposure time?!) between figure S2B/C and S2D/E? Both data show increased ADP ribose after IR. It seems redundancy. Also it is hard to imagine that there is absolutely no sign of ADP ribose after IR w/o PARGi treatment (figure S2D).

    Figure S2B/C show spontaneous single strand DNA breaks (SSBs) in PNKP KO cells, on the other hand, figure 2S/E show ectopic SSBs induced by IR exposure in PNKP KO cells. We believe these data help for readers to understand the effect of endo or exo damage in PNKP KO cells. Poly-ADP ribosylations are immediately removed from SSB sites after repair as demonstrated previously (Tsukada, et al., PLoS One 2019, Kalasova et al., Nucleic Acids Research, 2020), although not zero (low level), it is very difficult to detect without PARGi treatment.

    Legend for figure S3 - typo!

    Thank you for your suggestion about typo. The legend for figure S3 is corrected as "Protein expression of PNKP mutants in U2OS cells".

    • In figure S3A/B, it is quite interesting that the PNKP antibody used for this analysis can detect all truncated and alanine substituted PNKP proteins. It might be helpful to indicate for other researchers which antibody used (Novus; epitope - 57aa to 189 aa or Abcam; epitope not revealed).

    In S3A/B, Novus PNKP antibody was used for all blots. We indicated this in the figure legend as "PNKP antibody (Novus: NBP1-87257) was used for comparing expression levels of endogenous and exogenous PNKP".

    In results: 'PNKP phosphorylation, especially of T118 ~~~ proliferation'

    • In the fork progression experiment (figure 2C), is there any statistical difference between D2 and D3/4 expressing cells?

    *Thank you for your suggestion. We performed statistical analysis as the reviewer suggested. Statistical analysis shows that there are no significant differences between D2 and D3/D4. Meanwhile, there are significant differences between WT and D3(P- What is the basis of the description 'Since the linker region of PNKP is considered to be involved in fork progression'? Any reference?

    This sentence was considered based on the above sentences "Furthermore, D2 mutant-expressing cells also showed an increased speed of the replication fork compared to WT and D1 mutant-expressing cells, although D3 and D4 showed mildly high-speed fork progression.". The D2 mutant lacks a whole linker region, which shows increased speed of DNA fiber in figure 2C. Therefore, we originally explained as the sentence above. We have revised the sentence to "Since these results may indicate the linker region of PNKP is involved in proper fork progression".

    • In figure 3B: pS114-PNKP (also pS15-p53) is DNA damage inducible. In this experiment, was DNA damage introduced? Roscovitine could hinder DNA repair process, but not inducing DNA damage itself.

    Thank you for your suggestion. DNA damage induction was not applied in this experiment. We agree that this panel makes confusing. We think that endogenously S114-PNKP (also S15-p53) might be phosphorylated slightly but not significant, although this is not the scope of this manuscript. This result showing that phosphorylated-T118 is reduced by Roscovitine treatment maybe redundant as we also have a result of in vitro phosphorylation assay using several combinations of CDKs and Cyclin proteins, which is a cleaner experiment to prove which CDK/Cyclin complex is directly controlling the T118 phosphorylation. Since the manuscript already contains enough amount of data to support the conclusion (as reviewer 3 also stated), we removed those blots result from the panel to avoid complicating the conclusion.

    In results: 'Phosphatase activity of PNKP is ~~~ of Okazaki fragments'

    • In figure 5C, any statistical analysis between WT-PNKP KO vs D171A-PNKP KO or K378A-PNKP KO has been done?

    Thank you for your comment. Statistical analysis shows P *

    In discussion, 'In contrast, the T118A mutants showed the absence of both SSBs and DSBs repair (Fig. S7) : figure S7 does not indicate what the authors describe.

    Thank you for pointing out this. This should refer to figure S8 instead of figure S7. We have corrected this error.

    In addition, the same sentence in discussion: No evidence demonstrate that 'the absence of both SSBs and DSBs repair', and the following sentence is not clear.

    *This is same point with above. We have corrected this mis-referencing and revised the sentence to "In contrast, the T118A mutants showed the impaired abilities of both SSBs and DSBs repair (Fig. S8).". We also revised the following sentence to "However, residual SSBs due to impaired SSB repair ability (e.g., in PARPi-treated cells and T118A cells) sometimes cause DNA replication-coupled DSBs formation in S phase, and the phenotype in DSB repair assay of the T118A mutant may be caused by an accumulated formation of DNA replication-coupled DSBs. Future works will be needed to distinguish whether the T118 phosphorylation directly regulate PNKP recruitment to DSBs as well as SSBs." for better explanation of the result. *

    In discussion, 'Because both CDK1/cyclin A2 and CDK2/cyclin A2 are involved in PNKP phosphorylation, cyclin A2 is likely important for these activities': It is not clear what this description intends? Is 'cyclin A2' important in what stance?

    This description is coming from Fig3C observation. Since both CDK1 and CDK2 activities are cyclin A2 dependent, we speculated cyclin A2 is important for CDK1/CDK2 dependent PNKP T118 phosphorylation. We revised the description to "Since both CDK1/Cyclin A2 and CDK2/Cyclin A2 phosphorylate T118 of PNKP, we speculated that PNKP T118 is phosphorylated in S phase to G2 phase in CDK1/Cyclin A2- and CDK2/Cyclin A2-dependent manner (Fig. 3B and C)".

    In discussion, 'This may be explained by the fact that mutations in the phosphorylated residue in the linker region are embryonic lethal': any reference to support this embryonic lethality?

    Thank you for your suggestion. We agree with that this sentence is overwriting. We revise the sentence to "This observation may indicate that mutations in the phosphorylated residue (T118) in the linker region are potentially embryonic lethal due to the importance of T118 in DNA replication, which is revealed in the present study.".

    Reviewer #2:

    Minor comments

    Sometimes there are incorrect references to the figures in the discussion (e.g. FigS9A, B, and C, are called out instead of E, F and G), a similar issue is found 4 lines below in the same page.

    Thank you for pointing out these errors. We checked the references in the discussion and corrected to the appropriate references.

    Based on the data in Figure 3A the authors suggest that pT118-PNKP follows Cyclin A2 levels, but this does not appear very clearly in the gel, especially for the last point. Even though the results are convincing, the authors should rephrase the conclusions of Figure 3A to reflect better the results.

    Thank you for your suggestion. We agree that this phrase is overwriting. We revised the conclusion to "pT118-PNKP was detected in asynchronized cells but increased particularly in the S phase, similar to Cyclin A2 expression levels, although the reduction of pT118, possibly dephosphorylation of T118, seems not as robust as the reduction of the Cyclin A2 expression level at the 12 hours time point. However, this effect was very weak during mitosis, suggesting that T118 phosphorylation plays a specific role in the S phase.".

    I did not find a reference to what seems to be a relevant work in this topic: PMID: 22171004

    Thank you for your suggestion. We have added the ref (Coquelle et al., PNAS, 2011) in Introduction section.

    Reviewer #3:

    Major comments

    The authors should consider and discuss the potential role of PNKP KO outside of the S-phase. In Figure 4C, while it is clear that poly ADP ribosylation is higher in S-phase, the effects of PNKP KO and complementation by WT or T118A are equally present. This would be more immediate if comparison, fold change, and statistical significance calculation were done within the same cell cycle phase instead of between cell stages. This is also clear by IF in Figure 4B. How do the authors explain this? Thank you for your suggestion. We agree with reviewer's suggestion. We compared intensities of ADP-ribose between cell lines in same cell cycle rather than between different cell cycles in a same cell line and added the respective statistics in figure 4C. Also, we agree with that poly ADP-ribose intensity is changed outside of S phase between WT and T118A PNKP expressing PNKP KO cells. As shown in figure S8, PNKP pT118 is also involved in DNA repair. These results might reflect of PNKP function outside of S phase. We have added the sentence "Of note, PNKP−/−*cells and PNKP T118A cells showed markedly higher ADP-ribose intensity in outside the S phase as well, which indicate that PNKP and T118 may have an endogenous role to prevent SSBs formation in outside the S phase. Since FEN1 has been reported to function in R-loop processing, PNKP could also be involved in this process. Future studies of a role of PNKP in different cell cycle will be able to address this question." to discuss about the function of PNKP outside the S phase. We have added the ref (Cristini et al., Cell Reports, 2019, and Laverde et al., Genes, 2022). *

    In connection with the previous point, can the author provide the same quantification in Figure 4E also for G2/M and not only the S phase? This should give an estimate of the activity of FEN1 outside the S-phase. This is important because FEN1 has other functions apart from OF maturation, such as R loop processing (Cristini 2019; Laverde 2023) Thank you for your suggestion. Here attached is the data of ADP-ribose intensity in cells outside the S phase as you suggested. FEN1i treatment still induces increased ADP-ribose intensity in outside the S phase as well, although the difference between with/without FEN1i treatment is much smaller than that in S phase, indicating that FEN1 has other functions outside the S phase. This finding is very interesting. However, the function of FEN1 in outside the S phase is outside the scope of this manuscript. Therefore, we would like to not put this data in the manuscript to avoid complicating the conclusion (as reviewer 3 also suggested).

    Why does FEN1 inhibition induce a faster fork progression in Fig4 but not in Fig5 and Fig6? Yes, it does in figure 4 and figure 5. In PNKP WT cells, FEN1i-treated fibers (CldU) show an increased speed of forks compared to non-treated fibers (IdU). However, loss of PNKP and T118 phosphorylation themselves cause a faster fork progression even if without FEN1i treatment, therefore the difference of speeds of forks before/after FEN1i treatment in PNKP KO and T118A cells is disappeared as both fibers grow faster than intact fibers in normal cells. In regard to figure 6, as you mentioned in a latter comment about figure 6, the title of vertical axis of the graph showing CldU length should not be speeds of replication forks as those DNA fibers are potentially digested by S1 nuclease, which is modified in the revised manuscript. Even so, DNA fibers from FEN1i-treated cells (CldU) with S1 nuclease shows similar length with fibers from untreated cells with S1 nuclease, whereas FEN1 inhibitor treatment accelerates a speed of forks in general (figure 4 and figure 5, assays without S1 nuclease), indicating that FEN1i treatment induces remaining of some ssDNA nicks/gaps which are substrates of S1 nuclease.

    How do the authors explain the impaired DNA gap binding activity of the phospho-mimetic T118D? Thank you for your suggestion. We think that the appropriate timing of phosphorylation of PNKP T118 is important, while the phosphor-mimetic mutant T118D mimics consecutively phosphorylated situation that may result in incomplete complementation of PNKP function.

    I would like to see a representative fiber image from Fig 6. Additionally, in Figure 6, the author should not label the y-axis as CldU-fork speed. Nuclease S1 treatment destroys single-strand gaps (in vitro) and does not affect the fork speed (in vivo) Thank you for your suggestion. We have added a representative fiber image. We also agree with that CldU fork speed is not a right label of y-axis as CldU fibers are potentially digested by S1 nuclease. We changed the y-axis label to "CldU tract length [kb/min]" in figure 6.

    Figure 5E: both mutants (kinase vs phosphatase) increase polyADP ribose intensity, while the title of this figure only emphasizes the phosphatase activity. We agree with your comment. We have changed this subtitle to "Enzymatic activities of PNKP is important for the end-processing of Okazaki fragments".

    Minor comments

    The authors refer to Hoch Nature 2017 when referring to polyADP ribose IF + PARG inhibition. Should they not refer to Hanzlikova Mol Cell 2018?

    Thank you for your suggestion. We have added the ref (Hanzlikova et al., Mol Cell 2018).

    Statistical analysis should be performed on the cell cycle profile in Figure 1B

    We performed statistical analysis to check whether there are significant differences of S phase population between WT and PNKP KO cells. There were significant differences between WT vs PNKP KO C1 (PThe authors should not refer to fork degradation or protection as a given fact without assessing it in these conditions. Thank you for your suggestion. We assume that this comment refers to the result section of figure 1 and figure 4. We have added a sentence "although future studies will be needed to investigate whether PNKP−/−* cells has the fork protection phenotype" in the result section of figure 1. We have changed representation in the section according to the reviewer's suggestion in the result section of figure 4.

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    Referee #3

    Evidence, reproducibility and clarity

    Tsukada and colleagues studied the role of PNKP phosphorylation in processing single-strand DNA gaps and its link to fork progression and processing of Okazaki fragments.

    They generated two PNKP KO human clonal cell lines and described defects in cell growth, accumulation in S-phase, and faster fork progression. With some elegant experiments, they complement the KO cell lines with deletion and point mutants for PNKP, identifying a critical phosphorylation site (T118) in the linker regions, which is important for cell growth and DNA replication.

    They show that phosphorylation of PNKP peaks in the mid-S phase. CDK1 and CDK2/ with Cyclin A2 are the two main CDK complexes responsible for this modification. With the IPOND experiment, the author shows that PNKP is recruited at nascent DNA during replication.

    They described increased parylation activity in PNKP KO cells, and by using HU and emetin, they concluded that this increased activity depends on replication and synthesis of Okazaki fragments.

    Interfering with Okazaki fragment maturation by FEN1 inhibition is epistatic with PNKP KO (and T118A) in influencing parylation activity in the S phase and fork progression. The authors try to understand by mutant complementation which of the two functions (Phosphatase vs Kinase) is important in processing OF, and they propose a primary role for the phosphatase activity of PNKP. They also show that T118 is important in controlling genome stability following different genotoxic stress. Finally, by coupling the measurement of fork progression with PARP/FEN1 inhibitors and S1 treatment, they propose a role of PNKP in the post-replicative repair of single-strand gaps due to unligated OF.

    Here are my major points:

    • The authors use a poly ADP ribose deposition measurement to estimate SSB nick/gap formation. Even if PARP activity is strictly linked to SSB repair, ADP ribosylation does not directly estimate SSB/nick gap formation. In addition, in Figs S2A, B, and C, the authors use IR and PARG inhibition to measure poly-ADP ribosylation in WT and PNKP KO cells. IR produces both SSB and DSB. A better and cleaner experiment would be to directly measure SSB formation (with alkaline comet assay, for example) in combination with treatments that are known to mainly cause SSB (H2O2, or low doses of bleomycin).
    • The manuscript would benefit from substantially restructuring the figures' order and panels. Before starting the T118 part, the authors could create several figures to explain the main consequences of the loss of PNKP. A figure could be focused on DSB-driven genome instability (fig1 + fig S8 and S9). Then, a figure for the single-strand break and link to the S-phase. For example, by using data from Figure 6 and showing only WT vs PNKP KO +- Nuclease S1 (without FEN1 or PARP inhibitors), the authors could easily convince the readers that loss of PNKP leads to the accumulation of single-strand gaps. Only in the second part of the manuscript could they introduce all the T118 parts.
    • The authors should consider and discuss the potential role of PNKP KO outside of the S-phase. In Figure 4C, while it is clear that poly ADP ribosylation is higher in S-phase, the effects of PNKP KO and complementation by WT or T118A are equally present. This would be more immediate if comparison, fold change, and statistical significance calculation were done within the same cell cycle phase instead of between cell stages. This is also clear by IF in Figure 4B. How do the authors explain this?
    • In connection with the previous point, can the author provide the same quantification in Figure 4E also for G2/M and not only the S phase? This should give an estimate of the activity of FEN1 outside the S-phase. This is important because FEN1 has other functions apart from OF maturation, such as R loop processing (Cristini 2019; Laverde 2023)
    • I understand the use of a FEN1 inhibitor to link the PNKP KO phenotype to OF processing, but this drug does not either rescue or exacerbate any of the phenotypes described by the authors. It seems to have just an epistatic effect everywhere. So, what other conclusion can we have if not that PNKO has a similar effect to FEN1? I think that the presence of this inhibitor in many plots complicates the digestion of several figures a little bit. Maybe clustering the data in a different way (DMSO on one side FEN1i on the other) would help.
    • Why does FEN1 inhibition induce a faster fork progression in Fig4 but not in Fig5 and Fig6?
    • How do the authors explain the impaired DNA gap binding activity of the phospho-mimetic T118D?
    • Fig S9 should be removed from the discussion. Additionally, the authors should consider whether they want to keep that piece of data in a manuscript that is already pretty dense. Why should we focus on additional linker residues and microirradiation data at the end of this manuscript?
    • I would like to see a representative fiber image from Fig 6. Additionally, in Figure 6, the author should not label the y-axis as CldU-fork speed. Nuclease S1 treatment destroys single-strand gaps (in vitro) and does not affect the fork speed (in vivo)
    • Figure 5E: both mutants (kinase vs phosphatase) increase polyADP ribose intensity, while the title of this figure only emphasizes the phosphatase activity.
    • I suggest using a free AI writing assistant. I think this manuscript would substantially benefit from one. As a non-native English speaker, I personally use one of them and find it extremely useful.

    Minor points:

    • In Figure S1A, the author refers to P-H2AX, but I do not see this marker in the western blot.
    • The authors refer to Hoch Nature 2017 when referring to polyADP ribose IF + PARG inhibition. Should they not refer to Hanzlikova Mol Cell 2018?
    • Statistical analysis should be performed on the cell cycle profile in Figure 1B
    • The authors should not refer to fork degradation or protection as a given fact without assessing it in these conditions.

    Referees cross-commenting

    I agree with all comments from reviewer 1 and 2.

    Significance

    This is an interesting paper with generally solid data and proper statistical analysis. The figures are pretty straightforward. Unfortunately, the manuscript is dry, and the reader needs help to follow the logical order and the rationale of the experiments proposed. This is also complicated by the enormous amount of data the authors have generated. The authors should improve their narrative, explaining better why they are performing the experiment and not simply referring to a previous citation. Reordering panels and figures would help in this regard. Overall, with some new experiments, tone-downs over strong claims and a better explanation of the rationale behind experiments the authors could create a fascinating paper.

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    Referee #2

    Evidence, reproducibility and clarity

    Polynucleotide kinase phosphatase (PNPK) participates in multiple DNA repair processes, where it acts on DNA breaks to generate 5'-phosphate and 3'-OH ends, facilitating the downstream activities of DNA ligases or polymerases.

    This manuscript identifies a CDK-dependent phosphorylation site on threonine 118 in PNKP's linker region. The authors provide some convincing evidence that this modification is important to direct the activity of PNPK towards ssDNA gaps between Okazaki fragments during DNA replication. The authors monitored protein expression levels, enzymatic activity, the growth rate and replication fork speed, as well as the presence of ssDNA damage to make a comprehensive overview of the features of PNKP necessary for its function.

    Overall, the conclusions are sufficiently supported by the results and this manuscript is relevant and of general interest to the DNA repair and genome stability fields. Some level of revision to the experimental data and text would help strengthen its message and conclusions.

    Major points:

    1. In an iPOND experiment the authors detect the wt PNKP and the T118 phosphorylated form at the forks and conclude that this phosphorylation promotes interaction with nascent DNA (Figure 3E). An informative sample to include here would have been the T118A mutant. Based on the model proposed, the prediction would be that it would not be associated with the forks, or at least, associated at reduced levels compared to the wt.
    2. The quality of the gels showing the phosphatase and kinase assays in Figure 5 could be improved to facilitate quantification of the results. The gel showing the phosphatase activity has a deformed band corresponding to K378A mutant. The gel showing the kinase activity seems to be hitting the detection limits, and the overall high background might influence the quantification of D171A mutant in the area of interest. The authors should provide a better quality of these gels, focusing on better separation (running them longer, eventually with a slightly increased electric current) and higher signal of the analyzed bands (longer incubation phosphatase/kinase prior to quenching or loading higher amount of DNA).
    3. The authors sometimes make statements like: "a slight increase, slightly increased, relatively high" without an evaluation of the statistical significance for the presented data. An example of such a statement is: "T118A mutant-expressing cells exhibited a marked delay in cell growth, which was not observed for S114A, although T122A, S126A, and S143A were slightly delayed," based on the figure 2E. A similar comment applies also to figures 4A, 5A, 5E. Whenever possible, the authors should include also an evaluation of the statistical significance in the statement.

    Minor revisions:

    1. I could not find a gH2AX blot for figure S2A.
    2. Sometimes there are incorrect references to the figures in the discussion (e.g. FigS9A, B, and C, are called out instead of E, F and G), a similar issue is found 4 lines below in the same page.
    3. The authors established two PNKP-/- clones and supported it with sequencing and several functional observations However, the C-terminal antibody appears to detect lower-intensity bands (Figure 1A). Can authors comment on those bands?
    4. Based on the data in Figure 3A the authors suggest that pT118-PNKP follows Cyclin A2 levels, but this does not appear very clearly in the gel, especially for the last point. Even though the results are convincing, the authors should rephrase the conclusions of Figure 3A to reflect better the results.
    5. Why the S1 nuclease data on DNA fibers do not show the same level of epistasis with the Fen1i, as do those on ADP-ribosylation?
    6. I did not find a reference to what seems to be a relevant work in this topic: PMID: 22171004

    Referees cross-commenting

    I agree with all the comments from the reviewers 1 and 3.

    Significance

    The manuscript identifies a CDK phosphorylation site in a relevant DNA repair protein. The experiments on this part are elegant and convincing. It seems that this phosphorylation is important during DNA replication and there is some supporting evidence in this point, although not as robust, meaning that it is not clear whether this phosphorylation is controlling specifically the recruitment to Okazaki fragments, or a general role in DNA repair. Maybe if they see a reduced recruitment of the T118A mutant to the forks (iPOND experiment) this would further increase the impact.

    This work will be relevant to the basic research, especially in the fields of DNA repair and DNA replication.

    My expertise: DNA replication, genome stability, telomere biology.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    PNKP is one of critical end-processing enzymes for DNA damage repair, mainly base excision & single strand break repair, and double strand break repair to a certain extent. This protein has dual enzyme function: 3' phosphatase and 5' kinase to make DNA ends proper for ligation. It has been demonstrated that PTM of PNKP (e.g., S114, S126), particularly phosphorylation by either ATM or DNAPK, is important for PNKP function in DNA damage repair. The authors found a new phosphorylation site, T118, of PNKP which might be modified by CDK1 or 2 during S phase. This modification of phosphorylation is involved in maintenance and stability of the lagging strand, particularly Okazaki fragments. Loss of this phosphorylation could result in increased single strand gaps, accelerated speed of fork progression, and eventually genomic instability. And for this process, PNKP enzyme activity is not that important. And the authors concluded that PNKP T118 phosphorylation is important for lagging strand stability and DNA damage repair.

    Major comments

    1. In general, enzymes have protein interactions with its/their substrates. If PNKP is phosphorylated by either/both CDK1/2, the protein interaction between these would be expected. However, the authors did not provide any protein interactions in PNKP and CDKs.
    2. It is not clear how T118 phosphorylation is involved in DNA damage repair itself as the authors suggested. The data presenting the involvement of T118 phosphorylation in this mechanism are limited. This claim opens more questions than answers. CDK1/2 still phosphorylates T118 in this DNA damage repair process? What would happen to DNA damage repair in which PNKP involves outside of S phase in terms of T118 phosphorylation?
    3. Along the same line with #1/2 comments, the recruitment of PNKP to the damage sites is XRCC1 dependent. Is not clear whether PNKP recruitment to gaps on the lagging strand is XRCC1 independent or dependent. It might be interesting to examine (OPTIONAL)

    Minor comments

    1. In results: 'Generation of PNKP knock out U2OS cell line'
      • In figure S2A; There are no data regarding diminishing the phosphorylation of g-H2AX.
      • Is there any difference (except for PARGi exposure time?!) between figure S2B/C and S2D/E? Both data show increased ADP ribose after IR. It seems redundancy. Also it is hard to imagine that there is absolutely no sign of ADP ribose after IR w/o PARGi treatment (figure S2D).
      • By showing data in figure S2B/C/D/E, the authors describe 'PNKP KO cells impaired the SSBs repair activity'. However, as the authors mentioned in this manuscript, PNKP could bind to either XRCC1 or XRCC4. Also for this experiment, IR had been applied, which induces DNA double strand breaks. Therefore, it is not certain that the authors' description is fully supported by these data presented. Perhaps, SSB inducing reagents should be used instead of IR.
    2. Legend for figure S3 - typo!
      • In figure S3A/B, it is quite interesting that the PNKP antibody used for this analysis can detect all truncated and alanine substituted PNKP proteins. It might be helpful to indicate for other researchers which antibody used (Novus; epitope - 57aa to 189 aa or Abcam; epitope not revealed).
    3. In results: 'PNKP phosphorylation, especially of T118 ~~~ proliferation'
      • In the fork progression experiment (figure 2C), is there any statistical difference between D2 and D3/4 expressing cells?
      • What is the basis of the description 'Since the linker region of PNKP is considered to be involved in fork progression'? Any reference?
      • Is there any FACS analysis data to support the description of the last sentence 'especially the phosphorylation of PNKP T118, is required for S phase progression and proper cell proliferation'?
    4. In results: 'CDKs phosphorylate T118 of PNKP ~~~ replication forks'
      • In figure 3A, Is there any change in total PNKP (both GFP-tagged & endogenous) level?
      • In figure 3B: pS114-PNKP (also pS15-p53) is DNA damage inducible. In this experiment, was DNA damage introduced? Roscovitine could hinder DNA repair process, but not inducing DNA damage itself.
    5. In results: 'Phosphorylation of PNKP at T118 ~~~ between Okazaki fragments'
      • In figure 4D, What happens in the ADP-ribose level, when T118D PNKP is expressed?
    6. In results: 'Phosphatase activity of PNKP is ~~~ of Okazaki fragments'
      • In figure 5C, any statistical analysis between WT-PNKP KO vs D171A-PNKP KO or K378A-PNKP KO has been done?
    7. In results: 'PNKP is involved in postreplicative single-strand DNA gap-filling pathway'
      • The description regarding data presented in figure 6 is not clear enough. These data might suggest that wildtype U2OS does not have SSB which is a substrate for S1 nuclease (except under FEN1i and PARPi treatment), whereas PNKP KO has SSB during both IdU and CIdU incorporation, so that S1 nuclease treatment dramatically reduces the speed of fork formation in PNKP KO cells. Also In figure 6B/C/D, adding an experimental group of PNKP KO with S1 nuclease + PARPi might help to understand the role of PNKP during replication better. Also these additional data could support the description in discussion 'Furthermore, PNKP is required for the PARP1-dependent single-strand gap-filling pathway ~~~ DNA gap structure'.
    8. In results: 'Phosphorylation of PNKP at T118 is essential for genome stability'
      • In figure S8C, Did you measure g-H2AX foci disappearance for later time point, such as 24 hrs after DNA damage? Is not clear whether non-phosphorylated PNKP at T118 inhibit DNA damage repair or make it slower? How does T114A-PNKP behave in this experimental condition? T114 is well known target of ATM/DNAPK for DDR & DSB repair.
    9. The result shown in figure S9 should be described in the result section, not in the discussion section.
    10. In discussion, 'In contrast, the T118A mutants showed the absence of both SSBs and DSBs repair (Fig. S7) : figure S7 does not indicate what the authors describe.
    11. In addition, the same sentence in discussion: No evidence demonstrate that 'the absence of both SSBs and DSBs repair', and the following sentence is not clear.
    12. In discussion, 'Because both CDK1/cyclin A2 and CDK2/cyclin A2are involved in PNKP phosphorylation, cyclin A2 is likely important for these activities': It is not clear what this description intends? Is 'cyclin A2' important in what stance?
    13. In discussion, 'This may be explained by the fact that mutations in the phosphorylated residue in the linker region are embryonic lethal': any reference to support this embryonic lethality?

    Referees cross-commenting

    I could see a similar degree of positive tendency toward the manuscript. I agree with the comments and suggestions in additional experiments made by reviewers 2 and 3. Those suggestions will improve an impact of the manuscript in the DNA damage repair field.

    Significance

    The authors discovered new phosphorylation site (T118) of PNKP which is an important DNA repair protein. This modification seems to play a role in maintenance of the lagging strand stability in S phase. This discovery is something positive in DNA repair field to expand the canonical and non-canonical functions of DNA repair factors.

    The data presented to support PNKP functions and T118 phosphorylation in S phase seem solid in general, yet it is not sure how much PNKP is critical in the Okazaki fragment maturation process which is known that several end processing enzymes (like FEN1, EXO1, DNA2 etc which leave clean DNA ends.) are involved. These finding might draw good attentions from researchers interested broadly in cell cycle, DNA damage repair, replication, and possibly new tumor treatment.

    My field and research interest: DNA damage response (including cell cycle arrest and programmed cell death), DNA damage repair (including BER, SSBR, DSBR)

  5. Version published to 10.1101/2021.07.29.452278v2 on bioRxiv
  6. Version published to 10.1101/2021.07.29.452278v1 on bioRxiv