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Mutational signatures are jointly shaped by DNA damage and repair

Nadezda V Volkova, Bettina Meier, Víctor González-Huici, Simone Bertolini, Santiago Gonzalez, Harald Voeringer, Federico Abascal, Iñigo Martincorena, Peter J Campbell, Anton Gartner, Moritz Gerstung

Posted on: 24 February 2020 , updated on: 25 February 2020

Preprint posted on 11 February 2020

Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-020-15912-7

A combinatory mutational screen in C. elegans, combining 54 DNA repair mutants and 12 genotoxins, demonstrates that mutations are a result of both DNA damage and failed repair.

Selected by Kerryn Elliott

Categories: cancer biology, cell biology, genetics, genomics

Background

The DNA of a given cell is constantly altered by DNA replication errors and genotoxic stresses. An assemblage of repair pathways specifically designed to keep mutations at bay usually efficiently repairs these alterations. Occasionally this process fails, and DNA lesions escape detection, or are repaired by error-prone pathways, leading to diseases such as cancer.

With the recent advances in whole genome sequencing, the mutational spectra of human cancers have been dissected into individual mutagenic processes using computational pattern recognition programs that correlate genotoxic exposures, such as UV light, or DNA repair deficiencies, such as defects in mismatch repair (MMR), with certain mutational signatures. There are now more than 50 mutational signatures identified computationally, and approximately one third of these have no known etiology (Alexandrov et. al., 2020). It is possible that these unknown signatures represent combinations of mutagens and repair pathway defects. In this paper the authors perform a combinatorial mutagenesis screen with 12 genotoxic agents on 53 different DNA repair mutants in C. elegans to show that mutations are a result of both DNA damage AND failed repair.

Figure 1. Experimental setup and representative mutational spectra in the study made available under a CC-BY-NC-ND 4.0 International license

 

Main findings

The combination of using genotoxin treatment in backgrounds of DNA repair deficiency was a clever approach to understand whether the genotoxins themselves, or the failed repair contributed more to the mutational signature. The authors found that over 40% of the combination treatments demonstrated unique mutational signatures or altered mutation rates, but unexpectedly there were also combinations where the loss of translesion synthesis enzymes (TLS) led to a decrease in mutagenesis. This suggests that the majority of mutations induced by genotoxins are caused by error-prone translesion synthesis. By comparing to the genotoxin treatments on a wild type background, the authors were able to attribute the mutations to either the genotoxin itself (54%), DNA repair deficiencies themselves (26%), and to positive (23%) and negative (3%) genotoxin-repair interactions. Notably, nucleotide excision repair (NER) was found to be responsible for the majority of the repair following genotoxin exposure, as the loss of NER proteins increased the mutational load substantially.

Interestingly, while the interactions between genotoxins and DNA repair deficiencies usually retained a similar mutation spectrum with an increased mutation rate, around 10% of the interactions resulted in a change in the mutational spectrum. The authors found lesion-specific mutations for alkylating agents under different DNA repair deficiency backgrounds. The alkylating agent MMS was highlighted, as MMS can alkylate A and G nucleotides to generate different modifications, mainly N3-methyladenine and O6-methylguanine. These modifications are processed by different repair pathways, and therefore blocking one or the other pathway led to a different mutational outcome. Using the specific mutants, the authors were able to link the translesion synthesis enzyme Pol κ to the repair of specific adenine lesions (N3-methyladenine), and the alkyl-transferase AGT-1 to the repair of guanine modifications (O6-methylguanine). They also proposed that the error-prone TLS enzyme Pol ζ was responsible for a large proportion of the additional mutations occurring in the knockouts, as the Pol ζ deficient strain showed a decrease in single base mutations, and instead showed an increase in large deletions, possibly due to fork-stalling. It is this combination of DNA repair deficiency alongside genotoxin exposure which makes this paper a very interesting read!

Why I chose this paper:

Mutational signatures, particularly those driven by the combination of damage and repair are a main research interest of mine. The use of C. elegans as a model organism allowed the combination of treatments to be studied in detail, leading to the ability to dissect out the cause of the mutational signature, be it the mutagen itself or the associated repair process.

This work is important for the field to understand the heterogeneity of mutagenesis and the importance of the combination of damage and failed repair in generating mutations.

Questions to the authors:

Are these results directly relatable to human cancers, given the treatment was on C. elegans? You mention an organism-specific spectrum in Cisplatin, but are there additional complexities (transcription factor families, orthologs, levels of mutagen exposure in human life etc.) that are missing in the model organism that may influence mutations in human cells?

It is interesting that some of these “created” signatures can be detected in human cancers. How do you propose we move forward to understand “combinatory” signatures in humans? How confident are you in the current mutational signature spectra, particularly in assigning etiology?

The increase in large deletions and decrease in SNVs in some TLS mutants was quite interesting to me. Could you expand on your reasoning as to why this might happen, as you suggested this was likely due to replication stalling and fork collapse? Has this been detected in humans, or are there other TLS enzymes that take over this role and prevent large deletions from occurring?

References:

Alexandrov, L.B., Kim, J., Haradhvala, N.J. et al. The repertoire of mutational signatures in human cancer. Nature 578, 94–101 (2020).

 

Tags: cancer, mutagen

doi: https://doi.org/10.1242/prelights.16938

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Author's response

Nadezda Volkova, Bettina Meier, Anton Gartner and Moritz Gerstung shared

Are these results directly relatable to human cancers, given the treatment was on C. elegans? You mention an organism-specific spectrum in Cisplatin, but are there additional complexities (transcription factor families, orthologs, levels of mutagen exposure in human life etc.) that are missing in the model organism that may influence mutations in human cells?

 

While DNA repair pathways are highly conserved across eukaryotes some differences exist. For cisplatin, the precise reason for discrepancies in the spectrum is yet to discover, but for e.g. UV-induced damage there is evidence that orthologs of the same repair enzymes act at different efficiency leading to differences between organisms and experimental systems (Hartman et al. 1989). Endogenous sources of mutations can also contribute to the divergence of signatures: as we have shown previously, 5-methylcytosines abundant in human cells but absent in C. elegans can undergo deamination and turn into thymines, which contributes the mutational spectra of ageing, but also mismatch repair deficiency in cancer (Meier et al. 2018). Further differences include humans having a more diverse set of translesion synthesis polymerases. Lastly, some compounds are only genotoxic upon metabolic activation, which can explain additional differences between organisms and even experimental systems derived from the same organism.

 

It is interesting that some of these “created” signatures can be detected in human cancers. How do you propose we move forward to understand “combinatory” signatures in humans? How confident are you in the current mutational signature spectra, particularly in assigning etiology?

 

Our data show that the same genotoxin and also the same DNA repair deficiency can produce a multitude of different mutational signatures. This might challenge a unique attribution of observed patterns to underlying mechanisms and more factors need to be accounted for establishing a signature’s etiology. The situation is especially challenging when a repair deficiency removes mutations in which case the breakdown of mutational spectra into positive signature contributions may not provide a correct answer.

 

The increase in large deletions and decrease in SNVs in some TLS mutants was quite interesting to me. Could you expand on your reasoning as to why this might happen, as you suggested this was likely due to replication stalling and fork collapse? Has this been detected in humans, or are there other TLS enzymes that take over this role and prevent large deletions from occurring?

 

It was indeed one of the most fascinating insights derived from our study that the majority of base substitutions appears to be contributed by error-prone TLS seemingly to avoid fewer and possibly more deleterious deletions. This phenomenon has been described in other model systems such as yeast (Lawrence and Hinkle 1996). In mammalian cells, polymerase ζ was shown to prevent replication-dependent DNA breaks (Lange et al. 2012), and also indicated as having a major role in replication of human cells under UV exposure (Gibbs et al. 1998), but its contribution was never assessed at a genome scale across multiple genotoxins. To our knowledge, no human mutational signature of TLS deficiency is known, and TLS deficiency does not appear to be selected for in human cancers, but germline variants in POLH underlie the disorder Xeroderma Pigmentosum Variant that predisposes to skin cancers (Masutani et al. 1999).

 

References:

Alexandrov, L.B., Kim, J., Haradhvala, N.J. et al. The repertoire of mutational signatures in human cancer. Nature 578, 94–101 (2020).

Hartman, P. S. et al. Excision repair of UV radiation-induced DNA damage in Caenorhabditis elegans. Genetics 122, 379–385 (1989).

Meier, B., Volkova, N.V., et al. Mutational signatures of DNA mismatch repair deficiency in C. elegans and human cancers. Genome Res. 28, 666–675 (2018).

Lawrence, C. W. & Hinkle, D. C. DNA polymerase zeta and the control of DNA damage induced mutagenesis in eukaryotes. Cancer Surv. 28, 21–31 (1996).

Lange, S.S, Wittschieben, J.P., and Wood, R.D. DNA polymerase zeta is required for proliferation of normal mammalian cells. Nucleic Acids Res. 40(10), 4473–4482 (2012).

Gibbs, P.E.M. et al. A human homolog of the Saccharomyces cerevisiae REV3 gene, which encodes the catalytic subunit of DNA polymerase ζ. PNAS 95(12), 6876–6880 (1998).

Masutani, C., et al. The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase η. Nature 399(6737), 700–704 (1999).

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