Peer review of: Ethanol exposure increases mutation rate through error-prone polymerases (2020)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
This is a very interesting study that aims to understand the mechanisms by which ethanol acts as 
a mutagen. Despite the long-established role of ethanol as a carcinogen, its role as a mutagen is 
still far from clear. In this work it is proposed that ethanol causes replication stress and so an 
increased mutation rate via the engagement of error-prone polymerases. The genetic experiments 
performed in yeast are strong and convincing: preventing the recruitment of the error-prone 
polymerases or inactivating them abolishes the mutagenic effect of ethanol. Recent analyses of 
cancer genomes have also pinpointed error-prone polymerases as crucial for the mutational impact 
of ethanol on cancer genomes. The conclusions of this study are therefore very relevant to cancer 
research. 
 
Minor points: 
 
55-57 ref missing 
 
64 ‘factors that cause stress’ – what kind of stress? Also in later sentences ‘stress’ should always 
be specified 
 
all figures. Show the data points instead of bar/dynamite plots. Bar plots hide data distributions. 
 
Fig 1 could be presented more clearly: Label with reporter used (CAN1, URA1), assign letter to 
each panel (separate a). 
 
195 This paragraph is confusing as the last sentence says that the statements from the previous 
ones are invalid? If the genetic approach does not alter acetaldehyde levels, it cannot inform about 
the effect of acetaldehyde. The same is true for the next paragraph. 
 
Fig 3 no error bars in bar charts to show variation between experiments? 
 
Fig 4a The GO terms describing up- and downregulated transcription were the most significant 
ones or picked by hand? 
 
The logic for studying Mrc1/Claspin is not well explained. 
 
437-453 info in paragraph partly repetitive with what was stated before. Then a new paragraph is 
started referring to info in the one before? It would be better to merge the two paragraphs in a 
concise way to avoid repeating information. 
 
Supplementary Figure 6: Why is this important (?) result shifted to the supplement 
 
The connection with cancer is very interesting. However this link to recent work elucidating the 
role of error prone polymerases in alcohol-associated mutations in tumors only comes in the final 
lines of the discussion whereas it would be more appropriate to introduce this v relevant work in 
the introduction. In the discussion it would be good to have a more direct comparison between the 
yeast and tumour data (e.g. PolZ suggested to be more important in yeast compared to polH in 
tumors (tho also likely some contribution of both in both systems e.g. a PolZ-like signature 
suggested in at least one analysis of cancer genomes: 
https://www.nature.com/articles/ncomms15290). 
 
 
 
 

Reviewer #2 (Remarks to the Author): 
 
Description: 
The current view is that ethanol metabolism leads to toxic derivatives that form DNA and protein 
adducts, which are a major cause of ethanol-induced mutagenesis and carcinogenesis. In this 
study the authors suggest an alternative mechanism, in which ethanol-induced mutations in S. 
cerevisiae are generated due to induction of replication stress by ethanol, which causes slow-down 
of replication, and generation of single-stranded DNA regions, to which TLS polymerases are 
recruited and perform error-prone DNA synthesis. 
 
Critique: 
This study is of general interest, aiming to gain insight into the mutagenic and carcinogenic 
activities of ethanol, and proposing a new mechanism of action. However, a key point is that the 
authors where unable to rule out the involvement of ethanol-induced DNA adducts in the effects 
that they have observed, most importantly mutagenesis. This is because they report that they did 
not see any change in acetaldehyde concentration under several manipulations, including 
knockdown of ADH2 gene (which may be insufficient) or chemical inhibition. In addition, the 
possibility of the formation of other ethanol derivatives was not examined. This means that the 
mutations that they observed could have been caused by DNA adducts, which makes the 
involvement of TLS expected, since it is the major mechanism which generates point mutations at 
DNA lesions. Also proteotoxic stress can be caused by ethanol-generated protein adducts. All of 
this means that some key results presented in this manuscript can be attributed to adducts 
generated by ethanol metabolism, a possibility that the authors do not seriously consider. 
The arguments described above do not rule out the possibility that ethanol acts also by the 
mechanism proposed by the authors, perhaps even in combination with adducts effects. However, 
the authors have not provided convincing evidence to support such a mechanism. Most 
importantly, they have not shown replication slow-down (they showed inhibition of the cell cycle), 
and they have not demonstrated the formation of ssDNA stretches in the cells, two key aspects in 
their model. The authors need more experimental evidence, and perhaps try to integrate the two 
models for ethanol-induced mutagenesis.