DNA replication in primary hepatocytes without the six-subunit ORC

  1. Róża K. Przanowska
  2. Yuechuan Chen
  3. Takayuki-Okano Uchida
  4. Etsuko Shibata
  5. Xiaoxiao Hao
  6. Isaac Segura Rueda
  7. Kate Jensen
  8. Piotr Przanowski
  9. Anthony Trimboli
  10. Yoshiyuki Shibata
  11. Gustavo Leone
  12. Anindya Dutta

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Abstract

The six subunit ORC is essential for initiation of DNA replication in eukaryotes. Cancer cell-lines in culture can survive and replicate DNA replication after genetic inactivation of individual ORC subunits, ORC1, ORC2 or ORC5. In primary cells, ORC1 was dispensable in the mouse liver for endo-reduplication, but this could be explained by the ORC1 homolog, CDC6, substituting for ORC1 to restore functional ORC. Here, we have created mice with a conditional deletion of ORC2, which does not have a homolog. Although mouse embryo fibroblasts require ORC2 for proliferation, mouse hepatocytes synthesize DNA in cell culture and endo-reduplicate in vivo without ORC2. Mouse livers endo-reduplicate after simultaneous deletion of ORC1 and ORC2 both during normal development and after partial hepatectomy. Since endo-reduplication initiates DNA synthesis like normal S phase replication these results unequivocally indicate that primary cells, like cancer cell lines, can load MCM2-7 and initiate replication without ORC.

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  1. Version published to 10.1101/2024.04.04.588006v2 on bioRxiv
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    Reviewer #1

    __Evidence, reproducibility and clarity __

    The work by Przanowska et al., sought to understand the role of ORC2 in murine development and further wanted to discover its role in liver endo-reduplication. The overall methods used is sufficient enough to address its role but is not very conclusive based on their overall results and data provided as elaborated in below comments.

    Major Comments:

    1. The major issue of the paper is how well is ORC2 depleted in perinatal liver (Fig. 2C) and is not very clear from the data as all the western blots are at very low exposure levels and bands are very weak (still weak bands seen). There are good antibodies of ORC2 which can be used for IHC staining and can be used to address the extent of ORC2 depletion.

    We have now shown that ORC2 protein is significantly decreased in the hepatocytes of the Orc2 KO and DKO livers (New Fig. 2C and 6D). The decrease is consistent, with 4-5 mice examined, and all showing the depletion. We have been unable to do immunohistochemistry on tissue sections of the mouse livers with the anti-ORC antibodies we have tried, and this could be a reflection of the low level of the proteins. On hepatocytes in culture we have obtained faint signal with the anti-ORC2 antibody in WT cells, and this is clearly absent in 100% of the hepatocytes. See Fig. R1 below.

    __Reviewer Fig R1: __

    A) Immunofluorescence of hepatocytes in culture from livers of WT and two DKO mice.

    B) Quantitation of A) from counting 70-100 cells from each specimen.

    However, the calculations in the methods and the discussion are very compelling that at least the last 6-9 cell divisions in normal development start with 2n nuclei in the livers at baseline (Fig. 3B-G and 6I).

    Why in Fig 2C, the M2 mice is showing an equivalent level of ORC2 protein compared to mice M1 with NO CRE expression (compare lane1 and lane5). So, the results are based on one mouse which I do not think is significant enough to come to the conclusion. The authors need to add more data from different mice for statistical significance. Please use IHC to show the depletion of ORC2 protein in the liver sections.

    We had used total liver and had pointed out that residual ORC2 protein will be seen from stromal cells (endothelia, blood vessels and blood cells). We have therefore removed the figure which measured ORC2 levels in total liver and have now shown that when hepatocytes are isolated from five animals there was a massive depletion of ORC2 in all five animals (new Fig. 3C).

    As nicely demonstrated in the previous paper by Okano-Uchida et al., 2018 that ORC1 depletion in the liver shows an DNA ploidy effect from 6-week onwards. The authors need to demonstrate in this paper also when the 16N phenotype is observed starting from week1 to 12 months.

    Based on the results from our previous paper (Okano-Uchida et al., 2018) we decided to measure 16N phenotype at 6 weeks of age. The endoreduplication occurs at a stage when ORC2 protein is undetectable during normal development or during regeneration.

    In the double knockout experiments (ORC1 and ORC2) the authors are not even bothered to demonstrate that how much are both the proteins are actually depleted from the cells, so on the results obtained from these mice experiments are not conclusive or explanatory.

    We have performed immunoblotting of isolated hepatocytes and immunohistochemistry of livers for ORC1 and ORC2. Our data shows that both proteins are depleted in all four mice tested (New Fig. 6D).

    Minor points:

    1. Why are scale bars missing in right panel of Fig. 2G, Fig. 6D Supp Fig. 2B KO studies. The authors need to confirm that that all the large nuclei have NO or less significant ORC2 protein through IHC H&E staining.

    The scale bars are missing from the right panels to avoid redundancy. We have added “Both panels are at the same scale.” in the figure legend, according to https://doi.org/10.1371/journal.pbio.3001161.

    1. Please explain why is EYFP in Fig. 5G is cytoplasmic compared to Fig 4C (nuclear). We consistently see this variability and it was there in our previous results (Okano-Uchida et al., 2018), where EYFP was cytoplasmic in tissues, but was nuclear (and some cytoplasmic) in hepatocytes in culture.

    We do not know the reason for this difference but consistently see this difference. We now say in the text: “We did not explore why the EYFP protein is mostly nuclear in hepatocytes in culture (Fig. 4C) and mostly cytoplasmic in hepatocytes in the liver tissue (Fig. 5G, 7G), but speculate that differences in signaling pathways or fixation techniques between the two conditions contribute to this difference.”

    Are authors using the same genotype of Alb-Cre mice as shown by Okano-Uchida et al., 2018 as I do not find the reference of Schuler et. al., 2004 (PMID:15282742).

    We have been using two independent Alb-Cre animals. This is now described in the Methods.

    Significance

    The article is exactly based on their previous published paper but instead of ORC1, they were interested in dissecting the role of ORC2. Although they have discussed that CDC6 may be involved in replacing ORC1 KO mice to rescue the extensive DNA replication in endoreduplication, but instead of going to hunt the role of CDC6 in endoreduplication they checked the effect of ORC2 which actually lower the overall impact of the paper.

    We studied ORC2 conditional KO mice in a similar manner to the previously published ORC1 conditional KO in order to ensure (1) that the lack of effect in the Orc1 KO was not because ORC1 can theoretically be substituted for by CDC6 and (2) to establish the double KO of Orc1 and Orc2. To the best of our knowledge this is the first description of removal of two subunits of ORC complex at once in a mouse model. Moreover, in the light of rising recognition of sex as biological variable, we report sex-dependent effects which are very intriguing.

    We have not attempted knocking out CDC6 to uncover novel mechanisms of DNA replication, because we first needed to make sure that the mice can truly endo-reduplicate without two of the six subunits of ORC. Note that our published results in cancer cell lines (Shibata, 2016) show that CDC6 is still essential in the ORC KO cell lines, so a future experiment will likely reveal that CDC6 is still essential for endoreduplication in the ORC KO mice in vivo.

    Reviewer #2

    __Evidence, reproducibility and clarity __

    It has been reported that in the absence of ORC1, liver cells can still endoreduplicate and it has been speculated that this might occur if CDC6 can replace, at least partially, the function of ORC1. Here, authors evaluate if this is also true in the absence of ORC2 and found that ORC2 is required for cell proliferation in mouse hepatocytes but not for endoreduplication. This is also the case after combining the conditional mutations of ORC1 and ORC2. They propose that a mechanism must exist to load sufficient MCM2-7 to support DNA replication in the absence of these two ORC subunits. Some of the conclusions need further experimental support. The rationale for testing the requirement of ORC2, with or without ORC1, for endoreduplication is valid. However, a key point is that the endoreduplication level seems to be higher in the absence of ORC2 or both ORC1 and ORC2, and this is not properly addressed. Also, mechanistic details on how this could be triggered are absent from this study. As indicated below almost every figure in this manuscript contains weak points (see below).

    We now discuss the following: “One possible explanation of the greater endoreduplication in both our papers is that mitosis may be arrested earlier in development by G2 DNA damage checkpoints activated by incomplete licensing and replication of the genome in the absence of ORC. As a result, endoreduplication cycles could begin earlier in development resulting in greater endoreduplication.”

    Major

    1. Fig 1G, needs a detailed comment and justification.

    We have added the following to the text: “The proliferation rate of the MEF were measured by MTT assays. Even in the Orc2+/+ MEF, the infection with adeno-Cre decreased proliferation a little (the orange line compared to the blue line in Fig. 1G). However, for Orc2f/f MEF infection with adeno-Cre impairs proliferation even further (yellow line compared to black line in Fig. 1G)..

    Note that Adeno-Cre has been reported to be toxic for cell proliferation (citations 1, 2, 3), and so we included Adeno-Cre expression in ORC2+/+ (WT) as a background control.

    Citation:

    1. Pfeifer A, Brandon EP, Kootstra N, Gage FH, Verma IM: Delivery of the Cre recombinase by a self deleting lentiviral vector: Efficient gene targeting in vivo. Proc Natl Acad Sci USA. 2001, 98: 11450-11455. 10.1073/pnas.201415498.
    2. Loonstra A, Vooijs M, Beverloo HB, Allak BA, Drunen EV, Kanaar R, Berns A, Jonkers J: Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci USA. 2001, 98: 9209-9214. 10.1073/pnas.161269798.
    3. Schmidt EE, Taylor DS, Prigge JR, Barnet S, Capecchi R: Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc Natl Acad Sci USA. 2000, 97: 13702-13707. 10.1073/pnas.240471297.

    Fig 2D-F. Is this conclusion applicable to other endoreplicating tissues? Have authors consider to analyze body weight and liver weight measurements after normalization with similar data from a non-affected organ? The conditional KO was performed specifically in the liver. ORC is intact in other tissues in these animals. As a future direction our lab plans to study cardiac-specific conditional KO of ORC subunits to test whether other endo-reduplicating tissues can also synthesize DNA in the absence of ORC subunits.

    Fig 3 shows inconsistent results or results that lack proper justification in the text. The 2C peak is missing in Fig 3E (yellow line, positive control). However, 2n nuclei appear in Fig 3F-H. Also, the blue and yellow peaks do not coincide in the flow cytometry profiles, in particular for 8C and 16C.

    There was an error in the plotting of the former Fig. 3E. The information is better presented in the former Fig. 3F-H (now Fig. 3E-G) and so have removed the former Fig. 3E from the paper.

    Fig 4. Shorter EdU pulses could be more informative of the actual amount of S-phase cells. Thus, the use of a 2h EdU pulse needs a clear justification.

    The half-life of EDU incorporation differs slightly between in vivo and in vitro conditions. In vivo, slower cell proliferation requires a longer time, approximately 4 hours. However, in vitro, liver cells grow faster, and a 2-hour EDU pulse with 20 µM is sufficient for detection compared to a 3-hour pulse with 10 µM BrdU (Okano-Uchida et al., 2018). Several publications also use a 2-hour EDU incubation time (https://doi.org/10.1098/rsob.150172).

    Fig 5. EYFP is cytoplasmic, in contrast with results shown in Fig 4C

    We consistently see this variability and it was there in our previous results (Okano-Uchida et al., 2018), where EYFP was cytoplasmic in tissues, but was nuclear (and some cytoplasmic) in hepatocytes in culture.

    We do not know the reason for this difference but consistently see this difference. We now say in the text: “We did not explore why the EYFP protein is mostly nuclear in hepatocytes in culture (Fig. 4C) and mostly cytoplasmic in hepatocytes in the liver tissue (Fig. 5G, 7G), but speculate that differences in signaling pathways or fixation techniques between the two conditions contribute to this difference.”

    Fig 6. Results obtained with the double mutant are poorly described.

    We have split the figure into two figures (New Fig. 6 and 7) edited the results section to ensure that they are easily comprehended by the readers. We have also included Westerns from hepatocyte cell lysates of four DKO mice to show that ORC1 and ORC2 proteins are reproducible decreased (New Fig. 6D).

    What are the level of other pre-RC components in the mutants used in this study. This could be easily evaluated by Western blotting

    Despite the technical difficulty of not having antibodies that recognize all the mouse initiation proteins, we have now measured mouse ORC1, ORC2, ORC3, ORC5, ORC6, CDC6 and the MCM2 and MCM3 subunits of MCM2-7. The results do not show a consistent decrease or increase of any of these proteins in individual mice of the two genotypes, Orc2-/- or DKO (New Fig. 2D and 6E)

    How do authors justify their claim that a very limited amount of ORC are sufficient to load a vast excess of MCM2-7 hexamers?

    The rationale is stated in the introduction from data from cancer cell lines: “Given that WT cells have about 150,000 molecules of ORC2, even if this truncated protein is functional ORC2, ~150 molecules of the protein would be expected to load MCM2-7 double hexamers on at least 50,000 origins of replication. Experimentally, we show in Shibata, 2020 (Fig. 7C), that although ORC subunits are undetectable on Westerns, MCM2-7 association with the chromatin is unchanged. By the way, we do not say “vast excess” of MCM2-7, just sufficient MCM2-7 to fire 50,000 origins.

    Minor

    1. The titles of the Results section could be more informative of the main conclusion rather than simply descriptive

    We updated our Results titles to be more informative.

    The Discussion is too long

    We have shortened the discussion by removing our calculations to the Results section and abbreviating some of the discussion on endoreduplication. However we had to insert new items brough forth by the reviewers. Due to the controversy of this topic in our field, we had to include extensive discussion of current literature and put our results in their proper context.

    Significance

    The topic is relevant and the hypothesis tested is reasonable, although the conceptual advance is limited (see also below). The major limitation is the absence of mechanistic details addressing the occurrence of extra endoreduplication cycles (compared to controls) in the ORC1 and ORC2 mutants.

    Reviewer #3

    __Evidence, reproducibility and clarity: __

    The origin recognition complex (ORC) is an essential loading factor for the replicative Mcm2-7 helicase complex. Despite ORC's critical role in DNA replication, there have been instances where the loss of specific ORC subunits has still seemingly supported DNA replication in cancer cells, endocycling hepatocytes, and Drosophila polyploid cells. Critically, all tested ORC subunits are essential for development and proliferation in normal cells. This presents a challenge, as conditional knockouts need to be generated, and a skeptic can always claim that there were limiting but sufficient ORC levels for helicase loading and replication in polyploid or transformed cells. That being said, the authors have consistently pushed the system to demonstrate replication in the absence or extreme depletion of ORC subunits.

    Here, the authors generate conditional ORC2 mutants to counter a potential argument with prior conditional ORC1 mutants that Cdc6 may substitute for ORC1 function based on homology. They also generate a double ORC1 and ORC2 mutant, which is still capable of DNA replication in polyploid hepatocytes. While this manuscript provides significantly more support for the ability of select cells to replicate in the absence or near absence of select ORC subunits, it does not shed light on a potential mechanism. While a mechanistic understanding of how these cells proliferate in the absence or extreme depletion of ORC subunits is outside the scope of the current manuscript, it would have been beneficial to see more functional analyses to help guide the field. For example, is there a delay or impairment in Mcm2-7 loading in G1 (FACs-based loading assay from the Cook Lab (Matson et al., eLife. 2017)) in primary hepatocytes with the ORC2 conditional deletion? Is copy number maintained as cells increase polyploidy in the absence of ORC subunits, or are some regions of the genome more sensitive to ORC depletion (CGH arrays or sequencing of the flow-sorted polyploid cells)?

    We thank the reviewer for recognizing the main point of these experiments: to dispel the argument that CDC6 can substitute for ORC1 in the six-subunit ORC (although no one has demonstrated this, the argument is made on the basis of close sequence homology between CDC6 and ORC1). The second point, also appreciated by the reviewer is to show that it is possible to find cells that replicate in the absence or near absence of two ORC subunits.

    The mechanistic questions raised are important, and we will address them here:

    Is there a delay or impairment of MCM2-7 loading in G1? The hepatocytes in culture are fragile and not immortalized and thus, this issue can be much more easily addressed in the cancer cell lines we have made that are missing several ORC subunits and will do that in a later paper. Note however, the surprising lack of change in MCM2-7 association in cell lines where both ORC2 and ORC5 are deleted (Shibata, 2020, Fig. 7C).

    Are some regions of the genome more sensitive to ORC deletion during the polyploidization? We could not find any paper where people have investigated whether the whole genome is uniformly polyploidized in livers. In other words, the baseline conditions in WT livers have not been established. We therefore have postponed experiments to answer this question for a later paper. Note that in unpublished data from mapping SNS-seq origins in WT and ORC deletion cell lines there does not appear to be selective firing of certain origins over others in the deletion cell lines.

    Additional points: I didn't understand how the numbers were derived in Table 2. Was there really a 20-fold decrease in nuclear density for female ORC1 and ORC2 double-deletion hepatocytes? The differences in Figure S2 are dramatic, but not 20-fold dramatic.

    We measure the relative nuclear density by counting the number of plump nuclei (hepatocytes) per field as described for Fig. 5F and 7F now in the Methods section. The reviewer is correct in that we overestimated the decrease of nuclear density in the female DKO mice by two-fold. The revised calculations suggest that 6 cell divisions occur in the female DKO mice after the ORC proteins have decreased to at least __Significance: __

    The strengths of this manuscript are the mouse genetics and the generation of conditional alleles of Orc2 and the rigorous assessment of phenotypes resulting from limiting amounts of specific ORC subunits. It also builds on prior work with ORC1 to rule out Cdc6 complementing the loss of ORC1. The weakness is that it is a very hard task to resolve the fundamental question of how much ORC is enough for replication in cancer cells or hepatocytes. Clearly, there is a marked reduction in specific ORC subunits that is sufficient to impact replication during development and in fibroblasts, but the devil's advocate can always claim limiting levels of ORC remaining in these specialized cells. The significance of the work is that the authors keep improving their conditional alleles (and combining them), thus making it harder and harder (but not impossible) to invoke limiting but sufficient levels of ORC. At this point, the investigators and the field are well-positioned to attempt future functional CRISPR screens to identify other factors that may modulate the response to the loss of ORC subunits. This work will be of interest to the DNA replication, polyploidy, and genome stability communities.

    We thank the reviewer for getting the important point of this paper: “making it harder and harder (but not impossible) to invoke limiting but sufficient levels of ORC….” In other words, either ORC is completely dispensable for loading MCM2-7 in certain cancer cell lines and hepatocytes or it is highly catalytic and one molecule of ORC can load a few hundred MCM2-7 doublets so that most origins in the genome are licensed and capable of firing. We are trying the CRISPR screens in cancer cell lines that the reviewer envisages

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    Referee #3

    Evidence, reproducibility and clarity

    The origin recognition complex (ORC) is an essential loading factor for the replicative Mcm2-7 helicase complex. Despite ORC's critical role in DNA replication, there have been instances where the loss of specific ORC subunits has still seemingly supported DNA replication in cancer cells, endocycling hepatocytes, and Drosophila polyploid cells. Critically, all tested ORC subunits are essential for development and proliferation in normal cells. This presents a challenge, as conditional knockouts need to be generated, and a skeptic can always claim that there were limiting but sufficient ORC levels for helicase loading and replication in polyploid or transformed cells. That being said, the authors have consistently pushed the system to demonstrate replication in the absence or extreme depletion of ORC subunits.

    Here, the authors generate conditional ORC2 mutants to counter a potential argument with prior conditional ORC1 mutants that Cdc6 may substitute for ORC1 function based on homology. They also generate a double ORC1 and ORC2 mutant, which is still capable of DNA replication in polyploid hepatocytes. While this manuscript provides significantly more support for the ability of select cells to replicate in the absence or near absence of select ORC subunits, it does not shed light on a potential mechanism. While a mechanistic understanding of how these cells proliferate in the absence or extreme depletion of ORC subunits is outside the scope of the current manuscript, it would have been beneficial to see more functional analyses to help guide the field. For example, is there a delay or impairment in Mcm2-7 loading in G1 (FACs-based loading assay from the Cook Lab (Matson et al., eLife. 2017)) in primary hepatocytes with the ORC2 conditional deletion? Is copy number maintained as cells increase polyploidy in the absence of ORC subunits, or are some regions of the genome more sensitive to ORC depletion (CGH arrays or sequencing of the flow-sorted polyploid cells)?

    Additional points: I didn't understand how the numbers were derived in Table 2. Was there really a 20-fold decrease in nuclear density for female ORC1 and ORC2 double-deletion hepatocytes? The differences in Figure S2 are dramatic, but not 20-fold dramatic.

    Significance

    The strengths of this manuscript are the mouse genetics and the generation of conditional alleles of Orc2 and the rigorous assessment of phenotypes resulting from limiting amounts of specific ORC subunits. It also builds on prior work with ORC1 to rule out Cdc6 complementing the loss of ORC1. The weakness is that it is a very hard task to resolve the fundamental question of how much ORC is enough for replication in cancer cells or hepatocytes. Clearly, there is a marked reduction in specific ORC subunits that is sufficient to impact replication during development and in fibroblasts, but the devil's advocate can always claim limiting levels of ORC remaining in these specialized cells. The significance of the work is that the authors keep improving their conditional alleles (and combining them), thus making it harder and harder (but not impossible) to invoke limiting but sufficient levels of ORC. At this point, the investigators and the field are well-positioned to attempt future functional CRISPR screens to identify other factors that may modulate the response to the loss of ORC subunits. This work will be of interest to the DNA replication, polyploidy, and genome stability communities.

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    Referee #2

    Evidence, reproducibility and clarity

    It has been reported that in the absence of ORC1, liver cells can still endoreduplicate and it has been speculated that this might occur if CDC6 can replace, at least partially, the function of ORC1. Here, authors evaluate if this is also true in the absence of ORC2 and found that ORC2 is required for cell proliferation in mouse hepatocytes but not for endoreduplication. This is also the case after combining the conditional mutations of ORC1 and ORC2. They propose that a mechanism must exist to load sufficient MCM2-7 to support DNA replication in the absence of these two ORC subunits.

    Some of the conclusions need further experimental support. The rationale for testing the requirement of ORC2, with or without ORC1, for endoreduplication is valid. However, a key point is that the endoreduplication level seems to be higher in the absence of ORC2 or both ORC1 and ORC2, and this is not properly addressed. Also, mechanistic details on how this could be triggered are absent from this study. As indicated below almost every figure in this manuscript contains weak points (see below).

    Major

    1. Fig 1G, needs a detailed comment and justification.
    2. Fig 2D-F. Is this conclusion applicable to other endoreplicating tissues? Have authors consider to analyze body weight and liver weight measurements after normalization with similar data from a non-affected organ?
    3. Fig 3 shows inconsistent results or results that lack proper justification in the text. The 2C peak is missing in Fig 3E (yellow line, positive control). However, 2n nuclei appear in Fig 3F-H. Also, the blue and yellow peaks do not coincide in the flow cytometry profiles, in particular for 8C and 16C.
    4. Fig 4. Shorter EdU pulses could be more informative of the actual amount of S-phase cells. Thus, the use of a 2h EdU pulse needs a clear justification.
    5. Fig 5. EYFP is cytoplasmic, in contrast with results shown in Fig 4C
    6. Fig 6. Results obtained with the double mutant are poorly described.
    7. What are the level of other pre-RC components in the mutants used in this study. This could be easily evaluated by Western blotting
    8. How do authors justify their claim that a very limited amount of ORC are sufficient to load a vast excess of MCM2-7 hexamers?

    Minor

    1. The titles of the Results section could be more informative of the main conclusion rather than simply descriptive
    2. The Discussion is too long

    Significance

    The topic is relevant and the hypothesis tested is reasonable, although the conceptual advance is limited (see also below). The major limitation is the absence of mechanistic details addressing the occurrence of extra endoreduplication cycles (compared to controls) in the ORC1 and ORC2 mutants

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    The work by Przanowska et al., sought to understand the role of ORC2 in murine development and further wanted to discover its role in liver endo-reduplication. The overall methods used is sufficient enough to address its role but is not very conclusive based on their overall results and data provided as elaborated in below comments.

    Major Comments:

    1. The major issue of the paper is how well is ORC2 depleted in perinatal liver (Fig. 2C) and is not very clear from the data as all the western blots are at very low exposure levels and bands are very weak (still weak bands seen). There are good antibodies of ORC2 which can be used for IHC staining and can be used to address the extent of ORC2 depletion.
    2. Why in Fig 2C, the M2 mice is showing an equivalent level of ORC2 protein compared to mice M1 with NO CRE expression (compare lane1 and lane5). So, the results are based on one mouse which I do not think is significant enough to come to the conclusion. The authors need to add more data from different mice for statistical significance. Please use IHC to show the depletion of ORC2 protein in the liver sections.
    3. As nicely demonstrated in the previous paper by Okano-Uchida et al., 2018 that ORC1 depletion in the liver shows an DNA ploidy effect from 6-week onwards. The authors need to demonstrate in this paper also when the 16N phenotype is observed starting from week1 to 12 months.
    4. In the double knockout experiments (ORC1 and ORC2) the authors are not even bothered to demonstrate that how much are both the proteins are actually depleted from the cells, so on the results obtained from these mice experiments are not conclusive or explanatory.

    Minor points:

    1. Why are scale bars missing in right panel of Fig. 2G, Fig. 6D Supp Fig. 2B KO studies. The authors need to confirm that that all the large nuclei have NO or less significant ORC2 protein through IHC H&E staining.
    2. Please explain why is EYFP in Fig. 5G is cytoplasmic compared to Fig 4C (nuclear).
    3. Are authors using the same genotype of Alb-Cre mice as shown by Okano-Uchida et al., 2018 as I do not find the reference of Schuler et. al., 2004 (PMID:15282742).

    Significance

    The article is exactly based on their previous published paper but instead of ORC1, they were interested in dissecting the role of ORC2. Although they have discussed that CDC6 may be involved in replacing ORC1 KO mice to rescue the extensive DNA replication in endoreduplication, but instead of going to hunt the role of CDC6 in endoreduplication they checked the effect of ORC2 which actually lower the overall impact of the paper.

  6. Version published to 10.1101/2024.04.04.588006v1 on bioRxiv