FAM111A regulates replication origin activation and cell fitness

  1. Diana O. Rios-Szwed
  2. Elisa Garcia-Wilson
  3. Luis Sanchez-Pulido
  4. Vanesa Alvarez
  5. Hao Jiang
  6. Susanne Bandau
  7. Angus Lamond
  8. Chris P. Ponting
  9. Constance Alabert

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

FAM111A is a replisome associated protein and dominant mutations within its trypsin-like peptidase domain are linked to severe human developmental syndromes. However, FAM111A functions and its putative substrates remain largely unknown. Here, we showed that FAM111A promotes origin activation and interacts with the putative peptidase FAM111B, and we identified the first potential FAM111A substrate, the suicide enzyme HMCES. Moreover, unrestrained expression of FAM111A wild-type and patient mutants impaired DNA replication and caused cell death only when the peptidase domain remained intact. Altogether our data reveal how FAM111A promotes DNA replication in normal conditions and becomes harmful in a disease context.

Article activity feed

  1. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    The authors do not wish to provide a response at this time

  2. Review Commons

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

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Rios-Szwed and co-authors show that the depletion of FAM111A results in faster replication speed, longer intra-origin distances, and less chromatin-bound RPA even without induction of replication stress in U2OS cells. Induction of replication stress in FAM111A-depleted cells results in blunted response with less DNA damage, decreased checkpoint activation and resistance to the replication-stress inducing agent, HU. They show that cells without FAM111A display lower levels of single stranded DNA after treatment.

    In the second part, the authors show that FAM111A and FAM111B form a complex, although the similarities and differences of their functions are not explored in detail. From the little data shown, it looks like they might be working together in controlling amount of ssDNA. They find that both proteins are expected to have two conserved UBL domains, with one of them overlapping with ssDNA binding domain. Finally, the authors use overexpression of WT and mutant proteins to show that expression of WT and patient-derived mutant has increased level of DNA damage, increased levels of ssDNA, with and without DNA damage, and that the peptidase domain is necessary for the phenotypes.

    The data from the first two figures are consistent with FAM111A being involved in regulation of single stranded DNA formation during normal replication and during replication stress. Unfortunately, the work gives no indication of the mechanism of such regulation. I am not convinced that the function has much to do with controlling origin activation (see below). The data from the last two figures is also descriptive. Until the substrates of FAM111A are identified, there will be no understanding of its true function and the data will continue to be descriptive.

    Specific points:

    Figure 1: The siFAM11A-2 has a stronger phenotype in growth assay but has very little change in levels of cells in G1. No complementation of the phenotype is given.

    1D- there is no total RPA so it is unclear if there is no change in pRPA in relation to total RPA. Small differences will be missed without DNA damage and it would be helpful to use more sensitive assays to identify the reduction in ssDNA under unperturbed conditions.

    1E- what does the data look like if the lengths of IdU are plotted? This would be a measure of speed of the ongoing forks. Generally, this would be better than the CldU measurement.

    1F- the Inter-CldU distance increase could be secondary (indirect effect) of the increased replication speed

    1G- It looks like there are many more data points in the siFAM11A-1 and many fewer in the siFAM111A-2. The increase in the MCM quantified in H is bigger with si2 even though the G1 distribution has less change than with si1. Consequently, these data are incolclusive.

    1I- no plot is shown for si2 but it is quantified. It would be informative to see the plots for easy comparison.

    Figure 2: This is the most interesting part of the paper and generally is well done. As mentioned above, I believe that the phenotype the authors see in Figure 1 is the same phenotype as seen here- less production of ssDNA but it is hard to see this under unperturbed conditions, thus more data should be gathered to test that.

    Figure 3: shows novel findings but it is unclear how it relates to the rest of the paper except that it suggests that the paralogs may work together in the pathway that has been explored in Figure 1 and 2. The authors perform computational and predictive analysis that identifies two UBL domains in the FAM111A/B paralogs. The FAM111A UBL2 domain is known to bind ssDNA. The authors might test if the domain can also bind ssDNA in FAM111B and if FAM111B has similar ability to promote ssDNA formation

    Figure 4: The human mutations provide some insight as to the requirement for functional peptidase activity for the function of the protein. The work would also be strengthened if a ssDNA binding mutant was made and tested given the authors interest in defining the UBL domains.

    Not sure why they use a term "ssDNA exposure"? It implies a removal of something that was covering it which they certainly do not show. I would use ssDNA levels, maybe ssDNA production, formation?

    Other points:

    As QIBC is used throughout the paper, it would be nice to have a brief explanation of the technique when it is first introduced.

    The authors write that the function of FAM111A in promoting ssDNA formation is "distinct from overcoming protein-DNA complexes ahead of the replisome by Top1 or PARP1". It is not clear to this reader how they have determined that they are not the result of the same mechanism as the phenotypes seem very related. I would clarify this point.

    Since the authors are including patient mutations, more introduction to the diseases would be useful.

    Referee cross-commenting

    I have no further comments.

    Significance

    The findings add to the growing literature on the FAM111 proteins and will be of interest to scientists who are studying them and those interested in replication and replication stress response.

  3. Review Commons

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

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Rios-Szwed et al have investigated the role of FAM111A in DNA replication. Previous studies had identified that FAM111A suppresses DNA replication via an interaction with RFC and that hyperactive mutants induce apoptosis. Now, Rios-Szwed et al discovered that FAM111A knockdown affects inter origin distance without checkpoint induction. In particular, the firing of dormant origins when dNTPs are limiting is supressed and less ssDNA is produced. Although FAM111B is a strong interactor of FAM111A, no additive effect on DNA replication was detected when both proteins were depleted. On the other hand, overexpression or hyperactive mutants promote more gammaH2AX and ssDNA even in the presence of a caspase inhibitor, suggesting that the protease functions in ssDNA production prior to apoptosis.

    Major comments:

    Dormant origins are frequently inhibited by phosphatases - is there any evidence that phosphatases are the target of FAM111A. In this context I would suggest to blot for Treslin, as it is one of the first factors being recruited in a kinase dependent manner to the MCM2-7 complex.

    Minor comments:

    Abstract: Unclear why too much FAM111A causes cell death

    Introduction: the R569H point mutant needs to be better introduced - e.g. explain where the mutation is localised or what it affects e.g. it is localised in the predicted peptidase domain

    Figure 1A and 1D - are all the lanes shown originating from the same gel - if not please repeat.

    Page 3 - I am not sure that in FAM111A depleted cells the DNA synthesis rate is reduced. Could it be, that just fewer cells are in S-phase.

    Page 3 - It is stated: "In contrast, the inter-fork distance was slightly increased in FAM111A depleted cells (Fig. S1E)", however, the data but the data do not fully support this statement.

    Figure 4C - the quantification of the last lane looks wrong. Is the average or the median? Please find information in the figure and methods section.

    Question: If both FAM111A and FAM111B are overexpressed - is this better tolerated?

    Is there a homologue in other species?

    Referee cross-commenting

    I agree with the other reviewers that the study has a descriptive nature. I guess this could be acceptable dependent on the journal choice.

    Significance

    In general, I really like the study as it establishes how initiation of DNA replication is affected by inhibition and activation of FAM111A. The work is done well and deserves to be seen in a good journal.

    The study helps the field to move forward and will allow a more targeted search for specific protease targets. In this way it will help clinicians and also researchers.

    My expertise is in initiation of DNA replication.

  4. Review Commons

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

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Rios-Szwed and colleagues investigate functions of FAM111A, a protease that Dr. Alabert has previously shown to localize at nascent DNA and promote PCNA loading. In this manuscript, the authors first describe that FAM111A facilitates efficient activation of replication origins by using DNA combing experiments and by analyzing chromatin loading of DNA replication proteins. Next, they show that FAM111A KO cells show reduced levels of ssDNA exposure after replication stress. Then the authors move on to show that the major FAM111A interactor is FAM111B, which they show to localize at nascent DNA and is epistatic to FAM111A in promoting DNA replication as well as RPA loading after replication stress. Finally, the authors show that unregulated FAM111A activity, either by overexpression of WT FAM111A or disease-associated mutants, causes extensive exposure of ssDNA.

    Major comments

    1. Fig. S1G: Actual inter-origin distances (distance between replication tracks in which a CldU track is flanked by IdU tracks on both sides) should be plotted to estimate the changes in origin firing frequencies. The results should be presented as inter-origin distances, not ratios between UCN-01-treated and untreated. The revised experiment should be included in the main figures as this is central to the conclusion, and statistics should be included.
    2. The claim "FAM111A ... promotes DNA replication initiation of active and dormant origins" (page 4, line 4) is not fully supported by experiments. Does FAM111A localize at replication origins? Without direct evidence of FAM111A being present at replication origins, it remains possible that the changes in origin activity is secondary to the loss of FAM111A function at forks or something else.
    3. Fig. S1G: If FAM111A's function to promote activation of dormant origins in response to UCN-01 is unrelated to the function of FAM111A at forks, it is expected to be independent of the PIP motif. Is it the case?
    4. Fig. 2B: Increased survival after HU treatment might be secondary to reduced S-phase populations in FAM111A-depleted cells (Fig. 1C) as HU would affect only S-phase cells.
    5. Fig. 2B-I: Similarly, the blunted response to replication stress in FAM111A depleted cells could be simply explained by reduced number of forks per cell as indicated by increased inter-fork distance (Fig. 1F). Similarly, the authors' group has previously reported reduced PCNA levels on chromatin (Alabert et al, 2014), suggesting that there are reduced number of active forks per nucleus.
    6. Fig. 2H "FAM111A depletion reduced ssDNA exposure upon HU treatment (Fig. 2H, 2I)": The figure in Fig. 2H does not appear to be treated with FAM111A RNAi. If this is FAM111A RNAi cells, siControl cells need to be shown as a comparison.
    7. Fig. 3B,C: The interaction between FAM111A and FAM111B needs to be validated by coimmunoprecipitation-WB of endogenous proteins.
    8. Fig. 4A-C: Induction of DNA damage and apoptosis by FAM111A WT and disease mutants (including T338A that the authors claim unstudied) has been reported by Hoffman et al. and therefore not novel.
    9. Fig. 4E: The increase in ssDNA intensities is mild and might not be biologically significant.
    10. Fig. 4G: Cell cycle status needs to be assessed by FACS after treatment with each drug. Bleomycin might induce G1/S arrest if G1/S checkpoint is intact.
    11. ssDNA exposure after FAM111A OE might not be because FAM111A has a function in promoting ssDNA exposure, but could be simply explained by replication fork stalling, for example, due to degradation of essential proteins as proposed before (Hoffman et al, 2020).
    12. Page 8, line 17, "Altogether, these data revealed that unrestrained FAM111A peptidase activity leads to ssDNA exposure upstream of apoptosis.": Just because the caspase inhibitor did not block the ssDNA exposure, it does not mean ssDNA exposure is upstream of apoptosis - it could be happening in parallel and might be unrelated. A similar unsupported conclusion "ssDNA exposure is upstream of apoptosis" appears in other places: page 8, line 30; page 9, line 22.
    13. Whether protease activity is necessary for the FAM111A function in regulation of origin activation and in ssDNA exposure is not addressed. Can the phenotypes of FAM111A KO cells be rescued by FAM111A WT but not an active site mutant?
    14. Similarly, the authors need to test whether the PIP motif of FAM111A is required for the function of FAM111A at forks, such as promoting ssDNA exposure.

    Minor comments

    1. Page 2, Line 8, "FAM111A catalytic activity has not been shown in vitro": Protease activity of FAM111A has been shown using recombinant proteins in vitro by Hoffman et al, 2020.
    2. Page 7, line 26, "T338A is a previously unstudied GCLEB patient mutation.": The T338A mutant was studied by Hoffman et al. and shown to have hyperactivity in vitro and to cause DNA damage when overexpressed in cells.

    Referee cross-commenting

    I feel that this study has problems even as a descriptive study. As I mentioned in my review, there are alternative explanations for their observations that the authors have not ruled out. If the authors remove all unsupported claims, then there is not much to conclude from this study. I am not saying their conclusions are wrong - I think this study is just premature.

    Significance

    This study could be of interest to the audience in DNA replication/DNA repair field and could be unveiling a new function of FAM111A in DNA replication. However, in the current form, this study appears to be a collection of loosely connected observations of FAM111A-manipulated cells without a clear message of what FAM111A does at replication forks and origins. Each observation appears to be loosely tied together with a keyword of ssDNA exposure, but how FAM111A regulates or changes ssDNA exposure is not addressed. The described phenotypes are potentially interesting, but for each observation there is an alternative explanation that could affect authors' interpretation. As outlined in my comments, lack of mechanism, lack of clear conclusion, and misinterpretation of some of the data led to this less enthusiastic review.

  5. Version published to 10.1101/2020.04.22.055574v1 on bioRxiv