Introduction of cytosine-5 DNA methylation sensitizes cells to oxidative damage

Reviewer #1 (Public review):

Summary:

The manuscript titled "Introduction of cytosine-5 DNA methylation sensitizes cells to oxidative damage" proposes that 5mC modifications to DNA, despite being ancient and wide-spread throughout life, represent a vulnerability, making cells more susceptible to both chemical alkylation and, of more general importance, reactive oxygen species. Sarkies et al take the innovative approach of introducing enzymatic genome-wide cytosine methylation system (DNA methyltransferases, DNMTs) into E. coli, which normally lacks such a system. They provide compelling evidence that the introduction of DNMTs increases the sensitivity of E. coli to chemical alkylation damage. Surprisingly they also show DNMTs increase the sensitivity to reactive oxygen species and propose that the DNMT generated 5mC presents a target for the reactive oxygen species that is especially damaging to cells. Evidence is presented that DNMT activity directly or indirectly produces reactive oxygen species in vivo, which is an important discovery if correct, though the mechanism for this remains obscure.

I am satisfied that the points #2, #3 and #4 relating to non-addativity, transcriptional changes and ROS generation have been appropriately addressed in this revised manuscript. The most important point (previously #1) has not been addressed beyond the acknowledgement in the results section that: "Alternatively, 3mC induction by DNMT may lead to increased levels of ssDNA, particularly in alkB mutants, which could increase the risk of further DNA damage by MMS exposure and heighten sensitivity." This slightly miss-represents the original point that 5mC the main enzymatic product of DNMTs rather or in addition to 3mC is likely to lead to transient damage susceptible ssDNA, especially in an alkB deficient background. And more centrally to the main claims of this manuscript, the authors have not resolved whether methylated cytosine introduced into bacteria is deleterious in the context of genotoxic stress because of the oxidative modification to 5mC and 3mC, or because of oxidative/chemical attack to ssDNA that is transiently exposed in the repair processing of 5mC and 3mC, especially in an alkB deficient background. This is a crucial distinction because chemical vulnerability of 5mC would likely be a universal property of cytosine methylation across life, but the wide-spread exposure of ssDNA is expected to be peculiarity of introducing cytosine methylation into a system not evolved with that modification as a standard component of its genome.

These two models make different predictions about the predominant mutation types generated, in the authors system using M.SssI that targets C in a CG context - if oxidative damage to 5mC dominates then mutations are expected to be predominantly in a CG context, if ssDNA exposure effects dominate then the mutations are expected to be more widely distributed - sequencing post exposure clones could resolve this.

Strengths:

This work is based on an interesting initial premise, it is well motivated in the introduction and the manuscript is clearly written. The results themselves are compelling.

Weaknesses:

I am not currently convinced by the principal interpretations and think that other explanations based on known phenomena could account for key results. Specifically the authors have not resolved whether oxidative modification to 5mC and 3mC, or chemical attack to ssDNA that is transiently exposed in the repair processing of 5mC and 3mC is the principal source of the observed genotoxicity.

(1) Original query which still stands: As noted in the manuscript, AlkB repairs alkylation damage by direct reversal (DNA strands are not cut). In the absence of AlkB, repair of alklylation damage/modification is likely through BER or other processes involving strand excision and resulting in single stranded DNA. It has previously been shown that 3mC modification from MMS exposure is highly specific to single stranded DNA (PMID:20663718) occurring at ~20,000 times the rate as double stranded DNA. Consequently the introduction of DNMTs is expected to introduce many methylation adducts genome-wide that will generate single stranded DNA tracts when repaired in an AlkB deficient background (but not in an AlkB WT background), which are then hyper-susceptible to attack by MMS. Such ssDNA tracts are also vulnerable to generating double strand breaks, especially when they contain DNA polymerase stalling adducts such as 3mC. The generation of ssDNA during repair is similarly expected follow the H2O2 or TET based conversion of 5mC to 5hmC or 5fC neither of which can be directly repaired and depend on single strand excision for their removal. The potential importance of ssDNA generation in the experiments has not been [adequately] considered.

Reviewer #2 (Public review):

5-methylcytosine (5mC) is a key epigenetic mark in DNA and plays a crucial role in regulating gene expression in many eukaryotes including humans. The DNA methyltransferases (DNMTs) that establish and maintain 5mC, are conserved in many species across eukaryotes, including animals, plants, and fungi, mainly in a CpG context. Interestingly, 5mC levels and distributions are quite variable across phylogenies with some species even appearing to have no such DNA methylation.

This interesting and well-written paper discusses continuation of some of the authors' work published several years ago. In that previous paper, the laboratory demonstrated that DNA methylation pathways coevolved with DNA repair mechanisms, specifically with the alkylation repair system. Specifically, they discovered that DNMTs can introduce alkylation damage into DNA, specifically in the form of 3-methylcytosine (3mC). (This appears to be an error in the DNMT enzymatic mechanism where the generation 3mC as opposed to its preferred product 5-methylcytosine (5mC), is caused by the flipped target cytosine binding to the active site pocket of the DNMT in an inverted orientation.) The presence of 3mC is potentially toxic and can cause replication stress, which this paper suggests may explain the loss of DNA methylation in different species. They further showed that the ALKB2 enzyme plays a crucial role in repairing this alkylation damage, further emphasizing the link between DNA methylation and DNA repair.

The co-evolution of DNMTs with DNA repair mechanisms suggest there can be distinct advantages and disadvantages of DNA methylation to different species which might depend on their environmental niche. In environments that expose species to high levels of DNA damage, high levels of 5mC in their genome may be disadvantageous. This present paper sets out to examine the sensitivity of an organism to genotoxic stresses such as alkylation and oxidation agents as the consequence of DNMT activity. Since such a study in eukaryotes would be complicated by DNA methylation controlling gene regulation, these authors cleverly utilize Escherichia coli (E.coli) and incorporate into it the DNMTs from other bacteria that methylate the cytosines of DNA in a CpG context like that observed in eukaryotes; the active sites of these enzymes are very similar to eukaryotic DNMTs and basically utilize the same catalytic mechanism (also this strain of E.coli does not specifically degrade this methylated DNA) .

The experiments in this paper more than adequately show that E. coli expression of these DNMTs (comparing to the same strain without the DNMTS) do indeed show increased sensitivity to alkylating agents and this sensitivity was even greater than expected when a DNA repair mechanism was inactivated. Moreover, they show that this E. coli expressing this DNMT is more sensitive to oxidizing agents such as H2O2 and has exacerbated sensitivity when a DNA repair glycosylase is inactivated. Both propensities suggest that DNMT activity itself may generate additional genotoxic stress. Intrigued that DNMT expression itself might induce sensitivity to oxidative stress, the experimenters used a fluorescent sensor to show that H2O2 induced reactive oxygen species (ROS) are markedly enhanced with DNMT expression. Importantly, they show that DNMT expression alone gave rise to increased ROS amounts and both H2O2 addition and DNMT expression has greater effect that the linear combination of the two separately. They also carefully checked that the increased sensitivity to H2O2 was not potentially caused by some effect on gene expression of detoxification genes by DNMT expression and activity. Finally, by using mass spectroscopy, they show that DNMT expression led to production of the 5mC oxidation derivatives 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) in DNA. 5fC is a substrate for base excision repair while 5hmC is not; more 5fC was observed. Introduction of non-bacterial enzymes that produce 5hmC and 5fC into the DNMT expressing bacteria again showed a greater sensitivity than expected. Remarkedly, in their assay with addition of H2O2, bacteria showed no growth with this dual expression of DNMT and these enzymes.

Overall, the authors conduct well thought-out and simple experiments to show that a disadvantageous consequence of DNMT expression leading to 5mC in DNA is increased sensitivity to oxidative stress as well as alkylating agents.

Again, the paper is well-written and organized. The hypotheses are well-examined by simple experiments. The results are interesting and can impact many scientific areas such as our understanding of evolutionary pressures on an organism by environment to impacting our understanding about how environment of a malignant cell in the human body may lead to cancer.

In a new revised version of the paper, the authors have adequately addressed issues put forth by other reviewers. The result is even a better manuscript. Additions to the Results and Discussion sections and a new Supplemental Figure 2 give further credence to their conclusions.

Reviewer #3 (Public review):

Summary:

Krwawicz et al., present evidence that expression of DNMTs in E. coli results in (1) introduction of alkylation damage that is repaired by AlkB; (2) confers hypersensitivity to alkylating agents such as MMS (and exacerbated by loss of AlkB); (3) confers hypersensitivity to oxidative stress (H2O2 exposure); (4) results in a modest increase in ROS in the absence of exogenous H2O2 exposure; and (5) results in the production of oxidation products of 5mC, namely 5hmC and 5fC, leading to cellular toxicity. The findings reported here have interesting implications for the concept that such genotoxic and potentially mutagenic consequences of DNMT expression (resulting in 5mC) could be selectively disadvantageous for certain organisms. The other aspect of this work which is important for understanding the biological endpoints of genotoxic stress is the notion that DNA damage per se somehow induces elevated levels of ROS.

Strengths:

The manuscript is well-written, and the experiments have been carefully executed providing data that support the authors' proposed model presented in Fig. 7 (Discussion, sources of DNA damage due to DNMT expression).

Weaknesses:

(1) The authors have established an informative system relying on expression of DNMTs to gauge the effects of such expression and subsequent induction of 3mC and 5mC on cell survival and sensitivity to an alkylating agent (MMS) and exogenous oxidative stress (H2O2 exposure). The authors state (p4) that Fig. 2 shows that "Cells expressing either M.SssI or M.MpeI showed increased sensitivity to MMS treatment compared to WT C2523, supporting the conclusion that the expression of DNMTs increased the levels of alkylation damage." This is a confusing statement and requires revision as Fig. 2 does ALL cells shown in Fig. 2 are expressing DNMTs and have been treated with MMS. It is the absence of AlkB and the expression of DNMTs that that causes the MMS sensitivity.

(2) It would be important to know whether the increased sensitivity (toxicity) to DNMT expression and MMS is also accompanied by substantial increases in mutagenicity. The authors should explain in the text why mutation frequencies were not also measured in these experiments.

(3) Materials and Methods. ROS production monitoring. The "Total Reactive Oxygen Species (ROS) Assay Kit" has not been adequately described. Who is the Vendor? What is the nature of the ROS probes employed in this assay? Which specific ROS correspond to "total ROS"?

(4) The demonstration (Fig. 4) that DNMT expression results in elevated ROS and its further synergistic increase when cells are also exposed to H2O2 is the basis for the authors' discussion of DNA damage-induced increases in cellular ROS. S. cerevisiae does not possess DNMTs/5mC, yet exposure to MMS also results in substantial increases in intracellular ROS (Rowe et al, (2008) Free Rad. Biol. Med. 45:1167-1177. PMC2643028). The authors should be aware of previous studies that have linked DNA damage to intracellular increases in ROS in other organisms and should comment on this in the text.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Summary:

The manuscript proposes that 5mC modifications to DNA, despite being ancient and widespread throughout life, represent a vulnerability, making cells more susceptible to both chemical alkylation and, of more general importance, reactive oxygen species. Sarkies et al take the innovative approach of introducing enzymatic genome-wide cytosine methylation system (DNA methyltransferases, DNMTs) into E. coli, which normally lacks such a system. They provide compelling evidence that the introduction of DNMTs increases the sensitivity of E. coli to chemical alkylation damage. Surprisingly they also show DNMTs increase the sensitivity to reactive oxygen species and propose that the DNMT generated 5mC presents a target for the reactive oxygen species that is especially damaging to cells. Evidence is presented that DNMT activity directly or indirectly produces reactive oxygen species in vivo, which is an important discovery if correct, though the mechanism for this remains obscure.

Strengths:

This work is based on an interesting initial premise, it is well-motivated in the introduction and the manuscript is clearly written. The results themselves are compelling.

We thank the reviewer for their positive response to our study. We also really appreciate the thoughtful comments raised. We have addressed the comments raised as detailed below.

Weaknesses:

I am not currently convinced by the principal interpretations and think that other explanations based on known phenomena could account for key results. Specific points below.

(1) As noted in the manuscript, AlkB repairs alkylation damage by direct reversal (DNA strands are not cut). In the absence of AlkB, repair of alklylation damage/modification is likely through BER or other processes involving strand excision and resulting in single stranded DNA. It has previously been shown that 3mC modification from MMS exposure is highly specific to single stranded DNA (PMID:20663718) occurring at ~20,000 times the rate as double stranded DNA. Consequently, the introduction of DNMTs is expected to introduce many methylation adducts genome-wide that will generate single stranded DNA tracts when repaired in an AlkB deficient background (but not in an AlkB WT background), which are then hyper-susceptible to attack by MMS. Such ssDNA tracts are also vulnerable to generating double strand breaks, especially when they contain DNA polymerase stalling adducts such as 3mC. The generation of ssDNA during repair is similarly expected follow the H2O2 or TET based conversion of 5mC to 5hmC or 5fC neither of which can be directly repaired and depend on single strand excision for their removal. The potential importance of ssDNA generation in the experiments has not been considered.

We thank the reviewer for this interesting and insightful suggestion. Our interpretation of our findings is that a subset of MMS-induced DNA damage, specifically 3mC, overlaps with the damage introduced by DNMTs and this accounts for increased sensitivity to MMS when DNMTs are expressed. However, the idea that the introduction of 3mC by DNMT actually makes the DNA more liable to damage by MMS, potentially through increasing the level of ssDNA, is also a potential explanation, which could operate in addition to the mechanism that we propose.

(2) The authors emphasise the non-additivity of the MMS + DNMT + alkB experiment but the interpretation of the result is essentially an additive one: that both MMS and DNMT are introducing similar/same damage and AlkB acts to remove it. The non-additivity noted would seem to be more consistent with the ssDNA model proposed in #1. More generally non-additivity would also be seen if the survival to DNA methylation rate is non-linear over the range of the experiment, for example if there is a threshold effect where some repair process is overwhelmed. The linearity of MMS (and H2O2) exposure to survival could be directly tested with a dilution series of MMS (H2O2).

We thank the reviewer for this point. As in the response to point #1, the reviewer’s hypothesis of increased potency of MMS, potentially through increased ssDNA, downstream of 3mC induction by DNMT, is a good one. We have added a dose-response curve for DNMT-expressing cells to MMS to the revised version of the manuscript. This shows that there is a non-linear response to MMS in the WT background. Sensitivity is exacerbated by expression of DNMT and alkB mutation individually but there is also a strong non-additive effect that is particularly marked at low MMS concentrations where sensitivity is much higher in the double mutant than predicted from the two single mutants. This is consistent with induction of DNA damage by DNMT that is repaired by alkB because alkB can be ‘overwhelmed’ even in WT backgrounds as the reviewer suggests. However, it is also perfectly possible that the effect is due to increased levels of DNA damage induction in DNMT-expressing cells. Both these results are compatible with our central hypothesis, namely that DNMT expression induces 3mC. We have included these results along with discussion of them in the revised text in the results section:

In order to investigate the non-additivity between DNMT expression and alkB mutation further, we investigated the effect of MMS over a range of concentrations for the different strains (Supplemental Figure 1A). We quantified the non-additivity by comparing between the survival of alkB expressing DNMT to the predicted combined effect of either alkB mutation alone or DNMT expression alone(Supplemental Figure 1B). Significantly reduced survival than expected was observed, most notably at low concentrations of MMS, which could be due to the saturation of the effect at high concentrations of MMS for alkB mutants expressing DNMT, where extremely high levels of sensitivity were observed. The non-linear shape of the graph observed for WT cells expressing DNMTs further suggests that the ability of AlkB to repair the DNA is overwhelmed at high MMS concentrations even in the WT background. These results are consistent with the idea that AlkB repairs a form of DNA damage from MMS that is more prevalent when DNMT is expressed. This could be because DNMT induces 3mC, repaired by AlkB, and further 3mC is induced by MMS leading to much higher 3mC levels in the absence of AlkB activity. Alternatively, 3mC induction by DNMT may lead to increased levels of ssDNA, particularly in alkB mutants, which could increase the risk of further DNA damage by MMS exposure and heighten sensitivity. Either of these mechanisms are consistent with induction of 3mC by DNMT, and indicate that the induction of DNA damage by DNMT expression has a fitness cost for cells when exposed to genotoxic stress in their environment.

(3) The substantial transcriptional changes induced by DNMT expression (Supplemental Figure 4) are a cause for concern and highlight that the ectopic introduction of methylation into a complex system is potentially more confounded than it may at first seem. Though the expression analysis shows bulk transcription properties, my concern is that the disruptive influence of methylation in a system not evolved with it adds not just consistent transcriptional changes but transcriptional heterogeneity between cells which could influence net survival in a stressed environment. In practice I don't think this can be controlled for, possibly quantified by single-cell RNA-seq but that is beyond the reasonable scope of this paper.

We fully agree with the reviewer and, indeed, we are very interested in what is driving the transcriptional changes that we observed. Work is currently underway in the lab to investigate this further but, as the reviewer suggests, is beyond the scope of this paper. Importantly, we have used the transcriptional data to determine that the effect of DNMTs on ROS is unlikely to be due to failure of ROS-induced detoxification mechanisms by investigating the expression of oxyR regulated genes. Nevertheless we have explicitly mentioned the concern raised by the reviewer in the revised manuscript as follows:

“The substantial transcriptional responses could potentially affect how individual cells respond to genotoxic stress and thus could be contributing to some of the excess sensitivity to MMS and H2O2 in cells expressing DNMTs. However, the induction of oxyR regulated genes such as catalase was unaffected by 5mC (Supplementary Figure 4B). Thus, the increased sensitivity to H2O2 is unlikely to be caused by failure of detoxification gene induction by DNMT expression.”

(4) Figure 4 represents a striking result. From its current presentation it could be inferred that DNMTs are actively promoting ROS generation from H2O2 and also to a lesser extent in the absence of exogenous H2O2. That would be very surprising and a major finding with far-reaching implications. It would need to be further validated, for example by in vitro reconstitution of the reaction and monitoring ROS production. Rather, I think the authors are proposing that some currently undefined, indirect consequence of DNMT activity promotes ROS generation, especially when exogenous H2O2 is available. It would help if this were clarified.

We thank the reviewer for picking this up. In the discussion, we raise two possible explanations for why DNMT (even without H2O2) increases the ROS levels. One idea is direct activity of DNMT, and one is through the product of DNMT activity (5mC) acting as a platform to generate more ROS from endogenous or exogenous sources. Whilst we attempted to measure ROS from mSSSI activity in vitro, this experiment gave inconsistent results and therefore we cannot distinguish between these two possibilities. However, we argued that direct activity is less likely, exactly as the reviewer points out. We have clarified our discussion in the revised version, rewriting the entire section titled

Oxidative stress as a new source of DNA damage induction by DNMT expression to more clearly set out these possibilities.

Reviewer #2 (Public review):

5-methylcytosine (5mC) is a key epigenetic mark in DNA and plays a crucial role in regulating gene expression in many eukaryotes including humans. The DNA methyltransferases (DNMTs) that establish and maintain 5mC, are conserved in many species across eukaryotes, including animals, plants, and fungi, mainly in a CpG context. Interestingly, 5mC levels and distributions are quite variable across phylogenies with some species even appearing to have no such DNA methylation.

This interesting and well-written paper discusses the continuation of some of the authors' work published several years ago. In that previous paper, the laboratory demonstrated that DNA methylation pathways coevolved with DNA repair mechanisms, specifically with the alkylation repair system. Specifically, they discovered that DNMTs can introduce alkylation damage into DNA, specifically in the form of 3-methylcytosine (3mC). (This appears to be an error in the DNMT enzymatic mechanism where the generation 3mC as opposed to its preferred product 5-methylcytosine (5mC), is caused by the flipped target cytosine binding to the active site pocket of the DNMT in an inverted orientation.) The presence of 3mC is potentially toxic and can cause replication stress, which this paper suggests may explain the loss of DNA methylation in different species. They further showed that the ALKB2 enzyme plays a crucial role in repairing this alkylation damage, further emphasizing the link between DNA methylation and DNA repair.

The co-evolution of DNMTs with DNA repair mechanisms suggests there can be distinct advantages and disadvantages of DNA methylation to different species which might depend on their environmental niche. In environments that expose species to high levels of DNA damage, high levels of 5mC in their genome may be disadvantageous. This present paper sets out to examine the sensitivity of an organism to genotoxic stresses such as alkylation and oxidation agents as the consequence of DNMT activity. Since such a study in eukaryotes would be complicated by DNA methylation controlling gene regulation, these authors cleverly utilize Escherichia coli (E.coli) and incorporate into it the DNMTs from other bacteria that methylate the cytosines of DNA in a CpG context like that observed in eukaryotes; the active sites of these enzymes are very similar to eukaryotic DNMTs and basically utilize the same catalytic mechanism (also this strain of E.coli does not specifically degrade this methylated DNA) .

The experiments in this paper more than adequately show that E. coli expression of these DNMTs (comparing to the same strain without the DNMTS) do indeed show increased sensitivity to alkylating agents and this sensitivity was even greater than expected when a DNA repair mechanism was inactivated. Moreover, they show that this E. coli expressing this DNMT is more sensitive to oxidizing agents such as H2O2 and has exacerbated sensitivity when a DNA repair glycosylase is inactivated. Both propensities suggest that DNMT activity itself may generate additional genotoxic stress. Intrigued that DNMT expression itself might induce sensitivity to oxidative stress, the experimenters used a fluorescent sensor to show that H2O2 induced reactive oxygen species (ROS) are markedly enhanced with DNMT expression. Importantly, they show that DNMT expression alone gave rise to increased ROS amounts and both H2O2 addition and DNMT expression has greater effect that the linear combination of the two separately. They also carefully checked that the increased sensitivity to H2O2 was not potentially caused by some effect on gene expression of detoxification genes by DNMT expression and activity. Finally, by using mass spectroscopy, they show that DNMT expression led to production of the 5mC oxidation derivatives 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) in DNA. 5fC is a substrate for base excision repair while 5hmC is not; more 5fC was observed. Introduction of non-bacterial enzymes that produce 5hmC and 5fC into the DNMT expressing bacteria again showed a greater sensitivity than expected. Remarkedly, in their assay with addition of H2O2, bacteria showed no growth with this dual expression of DNMT and these enzymes.

Overall, the authors conduct well thought-out and simple experiments to show that a disadvantageous consequence of DNMT expression leading to 5mC in DNA is increased sensitivity to oxidative stress as well as alkylating agents.

Again, the paper is well-written and organized. The hypotheses are well-examined by simple experiments. The results are interesting and can impact many scientific areas such as our understanding of evolutionary pressures on an organism by environment to impacting our understanding about how environment of a malignant cell in the human body may lead to cancer.

We thank the reviewer for their response to our study, and value the time taken to produce a public review that will aid readers in understanding the key results of our study.

Reviewer #3 (Public review):

Summary:

Krwawicz et al., present evidence that expression of DNMTs in E. coli results in (1) introduction of alkylation damage that is repaired by AlkB; (2) confers hypersensitivity to alkylating agents such as MMS (and exacerbated by loss of AlkB); (3) confers hypersensitivity to oxidative stress (H2O2 exposure); (4) results in a modest increase in ROS in the absence of exogenous H2O2 exposure; and (5) results in the production of oxidation products of 5mC, namely 5hmC and 5fC, leading to cellular toxicity. The findings reported here have interesting implications for the concept that such genotoxic and potentially mutagenic consequences of DNMT expression (resulting in 5mC) could be selectively disadvantageous for certain organisms. The other aspect of this work which is important for understanding the biological endpoints of genotoxic stress is the notion that DNA damage per se somehow induces elevated levels of ROS.

Strengths:

The manuscript is well-written, and the experiments have been carefully executed providing data that support the authors' proposed model presented in Fig. 7 (Discussion, sources of DNA damage due to DNMT expression).

Weaknesses:

(1) The authors have established an informative system relying on expression of DNMTs to gauge the effects of such expression and subsequent induction of 3mC and 5mC on cell survival and sensitivity to an alkylating agent (MMS) and exogenous oxidative stress (H2O2 exposure). The authors state (p4) that Fig. 2 shows that "Cells expressing either M.SssI or M.MpeI showed increased sensitivity to MMS treatment compared to WT C2523, supporting the conclusion that the expression of DNMTs increased the levels of alkylation damage." This is a confusing statement and requires revision as Fig. 2 does ALL cells shown in Fig. 2 are expressing DNMTs and have been treated with MMS. It is the absence of AlkB and the expression of DNMTs that that causes the MMS sensitivity.

We thank the reviewer for this and agree that this needs to be clarified with regards to the figure presented and will do so in the revised manuscript. The key comparison is between the active and inactive mSSSI which shows increased sensitivity when active methyltransferases are expressed. We have clarified this in the revised version of the manuscript as follows:

“Cells expressing either M.SssI or M.MpeI showed increased sensitivity to MMS treatment compared to cells expressing inactive M.SssI”

(2) It would be important to know whether the increased sensitivity (toxicity) to DNMT expression and MMS is also accompanied by substantial increases in mutagenicity. The authors should explain in the text why mutation frequencies were not also measured in these experiments.

This is an important point because it is not immediately obvious that increased sensitivity would be associated with increased mutagenicity (if, for example, 3mC was never a cause of innacurate DNA repair even in the absence of AlkB). We have now added a Rif resistance assay which demonstrates increased mutagenesis in the presence of DNMT, and that this is exacerbated by loss of AlkB. This is now added as supplemental figure 2 and described in the manuscript as follows:

“One potential consequence of DNMT activity in inducing DNA damage might be increased mutagenesis. To test this we performed a rifampicin resistance mutagenesis assay, in the absence of MMS, to test whether DNMT induced damage was sufficient to lead to mutation rate increase. Mutation rate was increased by DNMT expression (p=1.6e-12; two way anova; Supplemental Figure 2) and alkB mutation (two way anova) separately (p<1e-16). Moreover, there was a significant interaction such that combined alkB mutation and DNMT expression led to a further increased mutation rate compared to the expectation from alkB mutation and DNMT expression separately (p = 7.9e-10; Supplemental Figure 2). Importantly, DNMT induction alone would be expected to lead to increased mutations due to cytosine deamination(Sarkies, 2022a); however, there is a synergistic effect on mutations when this is combined with loss of AlkB function in alkB mutants. This is consistent with 3mC induction by DNMTs which is repaired by AlkB in WT cells but leads to mutations in alkB mutant cells.

(3) Materials and Methods. ROS production monitoring. The "Total Reactive Oxygen Species (ROS) Assay Kit" has not been adequately described. Who is the Vendor? What is the nature of the ROS probes employed in this assay? Which specific ROS correspond to "total ROS"?

The ROS measurement was with a kit from ThermoFisher: https://www.thermofisher.com/order/catalog/product/88-5930-74. The probe is DCFH-DA. This is a general ROS sensor that is oxidised by a large number of cellular reactive oxygen species hence we cannot attribute the signal to a single species. Use of a technique with the potential to more precisely identify the species involved is something we plan to do in future, but is beyond what we can do as part of this study. We have added a comment as to the specificity of the ROS sensor in the revised version as follows:

“The ROS detection reagent in this system is DCFH-DA, a generalised ROS sensor that is not specific to any particular ROS molecule.”

(4) The demonstration (Fig. 4) that DNMT expression results in elevated ROS and its further synergistic increase when cells are also exposed to H2O2 is the basis for the authors' discussion of DNA damage-induced increases in cellular ROS. S. cerevisiae does not possess DNMTs/5mC, yet exposure to MMS also results in substantial increases in intracellular ROS (Rowe et al, (2008) Free Rad. Biol. Med. 45:1167-1177. PMC2643028). The authors should be aware of previous studies that have linked DNA damage to intracellular increases in ROS in other organisms and should comment on this in the text.

We thank the reviewer for this point. We note that the increased ROS that we observed occur in the presence of DNMTs alone and in the presence of H2O2, not in the presence of MMS; however, the point that DNA damage in general can promote increased ROS in some circumstances is well taken. We have included a comment on this in the revised version as follows:

“We believe this is a plausible mechanism to explain both increased ROS and increased sensitivity to oxidative stress when DNMT is expressed. However, other explanations are possible, and it is notable that DNA damaging agents such as MMS can lead to ROS generation(Rowe et al., 2008). A more detailed chemical and kinetic study of the ROS formation in DNMT-expressing cells would be needed to resolve these questions.”