C. elegans genome-wide analysis reveals DNA repair pathways that act cooperatively to preserve genome integrity upon ionizing radiation.

Ionizing radiation (IR) is widely used in cancer therapy and accidental or environmental exposure is a major concern. However, little is known about the genome-wide effects IR exerts on germ cells and the relative contribution of DNA repair pathways for mending IR-induced lesions. Here, using C. elegans as a model system and using primary sequencing data from our recent high-level overview of the mutagenic consequences of 11 genotoxic agents, we investigate in detail the genome-wide mutagenic consequences of exposing wild-type and 43 DNA repair and damage response defective C. elegans strains to a Caesium (Cs-137) source, emitting γ-rays. Cs-137 radiation induced single nucleotide variants (SNVs) at a rate of ~1 base substitution per 3 Gy, affecting all nucleotides equally. In nucleotide excision repair mutants, this frequency increased 2-fold concurrently with increased dinucleotide substitutions. As observed for DNA damage induced by bulky DNA adducts, small deletions were increased in translesion polymerase mutants, while base changes decreased. Structural variants (SVs) were augmented with dose, but did not arise with significantly higher frequency in any DNA repair mutants tested. Moreover, 6% of all mutations occurred in clusters, but clustering was not significantly altered in any DNA repair mutant background. Our data is relevant for better understanding how DNA repair pathways modulate IR-induced lesions.

Protection of the C. elegans germ cell genome depends on diverse DNA repair pathways during normal proliferation.

Maintaining genome integrity is particularly important in germ cells to ensure faithful transmission of genetic information across generations. Here we systematically describe germ cell mutagenesis in wild-type and 61 DNA repair mutants cultivated over multiple generations. ~44% of the DNA repair mutants analysed showed a >2-fold increased mutagenesis with a broad spectrum of mutational outcomes. Nucleotide excision repair deficiency led to higher base substitution rates, whereas polh-1(Polη) and rev-3(Polζ) translesion synthesis polymerase mutants resulted in 50-400 bp deletions. Signatures associated with defective homologous recombination fall into two classes: 1) brc-1/BRCA1 and rad-51/RAD51 paralog mutants showed increased mutations across all mutation classes, 2) mus-81/MUS81 and slx-1/SLX1 nuclease, and him-6/BLM, helq-1/HELQ or rtel-1/RTEL1 helicase mutants primarily accumulated structural variants. Repetitive and G-quadruplex sequence-containing loci were more frequently mutated in specific DNA repair backgrounds. Tandem duplications embedded in inverted repeats were observed in helq-1 helicase mutants, and a unique pattern of 'translocations' involving homeologous sequences occurred in rip-1 recombination mutants. atm-1/ATM checkpoint mutants harboured structural variants specifically enriched in subtelomeric regions. Interestingly, locally clustered mutagenesis was only observed for combined brc-1 and cep-1/p53 deficiency. Our study provides a global view of how different DNA repair pathways contribute to prevent germ cell mutagenesis.

Analysis of mutational signatures in C. elegans: Implications for cancer genome analysis.

Genome integrity is constantly challenged by exogenous and endogenous insults, and mutations are associated with inherited disease and cancer. Here we summarize recent studies that utilized C. elegans whole genome next generation sequencing to experimentally determine mutational signatures associated with mutagen exposure, DNA repair deficiency or a combination of both and discuss the implications of these results for the understanding of cancer genome evolution. The experimental analysis of wild-type and DNA repair deficient nematodes propagated under unchallenged conditions over many generations revealed increased mutagenesis in approximately half of all DNA repair deficient strains, its rate, except for DNA mismatch repair, only being moderately increased. The exposure of wild-type and DNA repair defective strains to selected genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin-B1, and cisplatin enabled the systematic analysis of the relative contributions of redundant repair modalities that mend DNA damage. Combining genotoxin exposure with DNA repair deficiency can manifest as altered mutation rates and/or as a change in mutational profiles, and reveals how different DNA alterations induced by one genotoxin are repaired by separate DNA repair pathways, often in a highly redundant way. Cancer genomes provide a snapshot of all mutational events that happened prior to cancer detection and sequencing, necessitating computational models to deduce mutational signatures using mathematical best fit approaches. While computationally deducing signatures from cancer genomes has been tremendously successful in associating some signatures to known mutagenic causes, many inferred signatures lack a clear link to a known mutagenic process. Moreover, analytical signatures might not reflect any distinct mutagenic processes. Nonetheless, combined effects of mutagen exposure and DNA damage-repair deficiency are also present in cancer genomes, but cannot be as easily detected owing to the unknown histories of genotoxic exposures and because biallelic in contrast to monoallelic DNA repair deficiency is rare. The impact of damage-repair interactions also manifests through selective pressure for DNA repair gene inactivation during cancer evolution. Using these considerations, we discuss a theoretical framework that explains why minute mutagenic changes, possibly too small to manifest as change in a signature, can have major effects in oncogenesis. Overall, the experimental analysis of mutational processes underscores that the interpretation of mutational signatures requires considering both the primary DNA lesion and repair status and imply that mutational signatures derived from cancer genomes may be more variable than currently anticipated.

Mutational signatures are jointly shaped by DNA damage and repair.

Cells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes.

Mutational signatures of DNA mismatch repair deficiency in C. elegans and human cancers.

Throughout their lifetime, cells are subject to extrinsic and intrinsic mutational processes leaving behind characteristic signatures in the genome. DNA mismatch repair (MMR) deficiency leads to hypermutation and is found in different cancer types. Although it is possible to associate mutational signatures extracted from human cancers with possible mutational processes, the exact causation is often unknown. Here, we use C. elegans genome sequencing of pms-2 and mlh-1 knockouts to reveal the mutational patterns linked to C. elegans MMR deficiency and their dependency on endogenous replication errors and errors caused by deletion of the polymerase ε subunit pole-4 Signature extraction from 215 human colorectal and 289 gastric adenocarcinomas revealed three MMR-associated signatures, one of which closely resembles the C. elegans MMR spectrum and strongly discriminates microsatellite stable and unstable tumors (AUC = 98%). A characteristic difference between human and C. elegans MMR deficiency is the lack of elevated levels of NCG > NTG mutations in C. elegans, likely caused by the absence of cytosine (CpG) methylation in worms. The other two human MMR signatures may reflect the interaction between MMR deficiency and other mutagenic processes, but their exact cause remains unknown. In summary, combining information from genetically defined models and cancer samples allows for better aligning mutational signatures to causal mutagenic processes.