PONE-D-22-17741Using canavanine resistance to measure mutation rates in Schizosaccharomyces pombePLOS ONE

Dear Dr. Kearsey,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The second reviewer has many concerns, both experimental and related to the presentation.  Many of the issues could be resolved by further analysis of the data and changes to the manuscript, but a few experiments may be necessary.  A revised manuscript should provide a full rebuttal to all points, concentrating on the points below:

[1] The reviewer considers that not enough recognition is given to previous work, and provides numerous references that should be cited (see suggestions throughout the report).

[2] The hypothesis of the authors regarding the cdc25 effect should be easily checked by WB to determine the protein level of Cdc25 in Any1+ and Any1-R175C.

[3] Fluctuation test data should be repeated using independent colonies.

Please also address the minor points raised by both reviewers.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Pai et al., ” Using canavanine resistance to measure mutation rates in Schizosaccharomyces

pombe”

In this paper, the authors investigate canavanine-resistant mutants of S. pombe as a tool to quantitate mutation rates. In S. cerevisiae, this is a main way to measure the rate of forward mutations. They find that, in contrast to S. cerevisiae where canavanine resistance largely arises from mutations in the CAN1 arginine transporter gene, in S. pombe all genome-sequenced resistant strains carry mutations in the any1+ gene, encoding a beta-arrestin and ubiquitin ligase adapter (homologous to S. cerevisiae ART1). Some isolates in addition show mutations in membrane transporter genes, but the authors conclude that the contribution to canavanine resistance from those are marginal. The authors then go on to show that in any1 mutants, the Cat1 membrane-bound nutrient transporter is absent from the plasma membrane and internalized. Specifically, the any1 point mutation (R175C) that is found in canavanine resistant strains, hits an evolutionarily conserved position that is close to a ubiquitylation site, necessary for regulation of the intracellular localization of the transporter.

A main conclusion is that the mutational spectrum in the any1+ gene isolated from canavanine-resistant mutants is limited to one single allele, and that this method therefore is not suitable for analyzing mutagenesis by DNA damaging agents or mutator alleles. As a measure of mutation rates to e.g. compare DNA damaging agents, canavanine resistance may work, although less efficiently than 5-FOA resistance.

The paper is focused and clearly written, some exceptions are listed below. The authors use adequate methods to discover the main mechanism for canavanine resistance in S. pombe, and describe the consequences of this for its use to measure mutation rates. The results are useful to the fission yeast community. I have only a few points that should be addressed:

In Fig. 1B, how are mutation rates actually represented? Taken literally, the Y axis would show rates relative to wt on a linear scale. The relative mutation rates for the mutants would then range from maybe 100 % (V412L on Ade) to 7000 % (S298F on 5-FOA) – is this correct? If so, all alleles would show hypermutation. Yet, the authors say that “V412L shows a low mutation rate, while the equivalent human mutation V411L causes hypermutation” (lines 154 -156). Please clarify what is meant by “low mutation rate” (= lower than wt or something else?), and explain the scale on the Y-axis.

Minor comments:

Line 148 - 150: The three measures of mutation rates show similar trends – why not show this by correlation coefficients? Or are the underlying numbers of mutant colonies counted too low to allow a statistical analysis?

The use of standard annotation for S. pombe should be used throughout the manuscript: lower case italics for mutants and mutations, same but followed by superscript “+” for genes and wt RNA; non-italics with initial upper case for proteins.

It would be interesting to hear the authors’ views on why the corresponding situation does not occur in S. cerevisiae: why are mutation found in the arginine transporter gene CAN1 in canavanine-resistant mutants, and not in ART1/LDB19, the S. cerevisiae ortholog of any1+?

Reviewer #2: Manuscript Number: PONE-D-22-17741 “Using canavanine resistance to measure mutation rates in Schizosaccharomyces pombe" by Chen-Chun Pai; Ellen Heitzer; Sibyl Bertrand; Sophia Toumazou; Timothy Humphrey; Stephen Kearsey.

In this study, Pai et al investigate the effect of mutant alleles of polymerase epsilon associated with cancers on mutagenesis using three assays available in S. pombe. In general, the paper consists of two parts: a small part describing the effect of the mutant alleles and a substantial part yet mostly reproducing other labs results dedicated to canavanine resistance in S. pombe. Although the impact of mutant alleles is potentially interesting, this study has multiple problems outlined below.

An effort should be made to substantiate certain findings (cdc25) and integrate accurately previous findings made by other labs. Specifically, most of the Any1R175C characterization by Pai et al. has already been reported. Cat1 internalization in Any1R175C was reported by Ait Saada et al. Canavanine resistance of vhc1, cat1, aat1 and cat1 aat1 deletions was reported by Aspuria and Tamanoi (PMID: 18219492), Ma et al. (PMID:23934889) and Ait Saada et al (PMID: 35639710). Two of those papers (PDIM 18219492, PDIM 35639710) are allegedly cited by Pai et al without giving credit to their findings. Instead of ignoring or overlooking other researchers' findings, the authors should make an effort to give more value to their real findings by giving more substance to their observations.

The only “new” results are figures 1B and 5. The rest is a reproduction of what is already known or simple alignments.

Abstract: “The any1R175C strain showed internalization of the Cat1 arginine transporter, explaining the canavanine resistance phenotype.” This has already been reported in Ait Saada et al, 2022 PlosOne and should not be presented as the main finding of this paper.

“it is likely that Cdc25 ubiquitylation and downregulation by Any1R175C-Pub1 delays mitotic entry.” This is only speculative, especially since the nature of the interaction between Any1-R175C and Pub1 is not known. Besides, the hypothesis of the authors regarding the cdc25 effect can be and should be easily checked by WB to determine the protein level of Cdc25 in Any1+ and Any1-R175C. In addition, Pub1-mediated Cdc25 ubiquitination can be assessed by WB in mts2ts mutant (Nefsky and Beach 1996 PMID: 8635463).

Fluctuation test data. The data are shown only relative to WT. What are the absolute rates? It is not clear what the error bars correspond to (CI, SD, SEM). In addition, the fluctuation test was performed from a single culture that was aliquoted into 12 wells of a 96-well plate, which means that the fluctuation test data are not obtained from independent clones. The reference cited by the authors (Lang and Murray 2008) reports using 10 independent clones. How many colonies were on average did grow on canavanine plates? It is also not clear whether the canavanine-resistant colonies obtained from the fluctuation test exhibit the any1-R175C mutation since it is not those clones that have been sequenced. Since POLE mutants are mutators by nature, there is no reason to expect only Any1-R175C mutation.

Section “Mechanism of canavanine resistance conferred by Any1R175C” There are no new findings in this result section. Cat1 internalization in Any1R175C has already been reported by Ait Saada et al. This reference is not cited even once in this result section.

Figure S1: The authors found that decreasing the pH in PMG media allows faster growth of canavanine-resistant cells. Is the effect of the pH specific to strain 4355? An appropriate strain control should be added (WT and can1-1).

Whole-genome sequencing. The strains sequenced are 2299 and 3938, WT and clr4Δ, respectively. It is not clear why the authors performed sequencing in clr4Δ background. Besides, none of the POLE mutants was included. Do the POLE mutants resistant to canavanine show any1-523C>T mutation and is C>T an expected mutational signature for the POLE mutants? Also, why POLE P286R was not include? In addition, the medium used to select for canavanine-resistant clones contains glutamate as a source of nitrogen, which is known to confer a stricter resistance compared to ammonium chloride. It is known that POLE mutants exhibit a mutator phenotype and that canavanine resistance in S. pombe can be very strong in certain double mutants. Since canavanine-resistant clones derived from POLE mutants have not been sequenced, it is not clear if the whole genome sequencing performed here provides a holistic view of the use of canavanine resistance to measure mutation rates.

Figure S2, vhc1 is not an ortholog of any arginine permease. It is not clear why it is part of the alignment. It is not even clear what is the purpose of this alignment per se. The secondary mutations identified in aat1 and cat1 are not shown to induce any phenotype related to canavanine resistance. The relevance of these mutations would be appreciated if the authors reproduce the single mutations and show that they behave like the corresponding single gene deletion. Using the appropriate medium, deletion of cat1 and aat1 (although to a lesser extent) have been shown to be resistant to canavanine (Aspuria and Tamanoi 2008).

Line 197. And figure 2. What is the relevance of the any1-R175 mutation being close to any2-K163? This later site is not very conserved between Any1 and Any2. Evidence shows that Any1 might be ubiquitinylated at K263 (Nakashima et al., 2014 PMID: 24876389), which corresponds to K486 in Art1 that has been shown to be ubiquitinated (Lin et al, 2008 PMID: 18976803).

Figure 3. and line 204 “To compare the canavanine resistance of an any-R175C strain with mutants defective in amino-acid transporters,” Vhc1 is not an amino acid transporter. The media used is PMG. It is known that glutamate leads to a higher uptake of arginine and explains why certain mutants are less resistant to canavanine in the presence of glutamate compared to ammonium chloride (Fantes and Creanor, 1984). In addition, all the deletion mutants used in this experiment have already been characterized for their canavanine resistance/sensitivity (Ma et al., 2013; Ait Saada et al., 2022; Aspuria and Tamanoi 2008).

Section “Implications for use of canavanine in mutation rate assays in fission yeast”. The authors conclude that only one kind of mutation can be selected for using their protocol which (i) makes the title of their paper misleading and (ii) the sensitivity of the assay questionable.

Minor comments

Line 97. no Δ should be in vhc1::hphMX6. The same applies to the other strain deletions in Table S1. Wild type should be written WT.

Line 104. Determination OF mutation rates by fluctuation analysis

Line 182. Replace “can1 gene” by “vhc1 gene previously called can1”.

Line 235. WT

Line 251. PMG

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Reviewer #1: No

Reviewer #2: No

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Using canavanine resistance to measure mutation rates in Schizosaccharomyces pombe

PONE-D-22-17741R1

Dear Dr. Kearsey,

We’re pleased to inform you that your manuscript has now been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have adquately addressed my concerns, mainly about Fig. 1. They have also made clear how the difference between fission and budding yeast in the number of arginine transporters can explain why canavanine resistance more often occurs through mutation in the CAN1 gene in S. cerevisiae, but in the any1+ gene in S. pombe.

Reviewer #2: The authors removed the cdc25 section and now introduced appropriate references. They also answered almost all my comments. As a result, the paper is significantly improved.

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Reviewer #1: Yes: Per Sunnerhagen

Reviewer #2: No

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