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.

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, whereaspolh-1(Polη) andrev-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/BRCA1andrad-51/RAD51 paralog mutants showed increased mutations across all mutation classes, 2)mus-81/MUS81andslx-1/SLX1nuclease, andhim-6/BLM,helq-1/HELQorrtel-1/RTEL1helicase 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 inhelq-1helicase mutants, and a unique pattern of ‘translocations’ involving homeologous sequences occurred inrip-1recombination mutants.atm-1/ATM checkpoint mutants harboured structural variants specifically enriched in subtelomeric regions. Interestingly, locally clustered mutagenesis was only observed for combinedbrc-1andcep-1/p53 deficiency. Our study provides a global view of how different DNA repair pathways contribute to prevent germ cell mutagenesis.

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.

Relatively little is known about the cross-talk between the spindle assembly checkpoint and the DNA damage response, especially in multicellular organisms. We performed a Caenorhabditis elegans forward genetic screen to uncover new genes involved in the repair of DNA damage induced by ionizing radiation. We isolated a mutation, gt2000, which confers hypersensitivity to ionizing radiation and showed that gt2000 introduces a premature stop in bub-3. BUB-3 is a key component of the spindle assembly checkpoint. We provide evidence that BUB-3 acts during development and in the germline; irradiated bub-3(gt2000) larvae are developmentally retarded and form abnormal vulvae. Moreover, bub-3(gt2000) embryos sired from irradiated worms show increased levels of lethality. Both bub-3 and san-1 (the C. elegans homolog of MAD3) deletion alleles confer hypersensitivity to ionizing radiation, consistent with the notion that the spindle assembly checkpoint pathway is required for the DNA damage response. bub-3(gt2000) is moderately sensitive to the cross-linking drug cisplatin but not to ultraviolet light or methyl methanesulfonate. This is consistent with a role in dealing with DNA double-strand breaks and not with base damage. Double mutant analysis revealed that bub-3 does not act within any of the three major pathways involved in the repair of double-strand breaks. Finally, the cdc-20 gain-of-function mutant cdc-20/fzy-1(av15), which is refractory to the cell cycle delay conferred by the spindle checkpoint, showed phenotypes similar to bub-3 and san-1 mutants. We speculate that BUB-3 is involved in the DNA damage response through regulation of cell cycle timing.