The team built a diploid yeast strain in which both copies of the SOD1 gene had been deleted, creating a hybrid background containing roughly 50,000 sequence differences between its two parental genomes. This design allowed the researchers to map, with extraordinary resolution, every loss of heterozygosity event that accumulated as cells divided. After subculturing 32 independent isolates for hundreds of generations, whole-genome sequencing revealed a staggering catalogue of damage. Single nucleotide variations accumulated at 13 times the wild-type rate, loss of heterozygosity rose 12-fold, large chromosomal rearrangements increased 4-fold, and aneuploidy—the gain or loss of entire chromosomes—surged an astonishing 48-fold above normal levels.

The scale and diversity of these lesions underscored that Sod1 deficiency does not merely nudge the genome toward instability; it fundamentally rewrites the rules of genome maintenance. The researchers detected 322 interstitial and 134 terminal loss-of-heterozygosity events across their isolates, occurring at rates 15- and 16-fold higher than in wild-type cells, respectively. Gene conversion tracts—the stretches of DNA copied from one homologous chromosome to repair damage on the other—were also markedly longer in the mutant, averaging 10.8 kilobases compared with 5.9 kilobases in healthy cells. Most structural rearrangements were flanked by Ty retrotransposon sequences, pointing to unequal recombination between repetitive elements as a major driver of large-scale chromosome changes.

Perhaps the most striking result came when the researchers repeated the experiment under anaerobic conditions. Deprived of oxygen, the sod1 mutant’s genomic chaos vanished almost entirely: mutation rates fell to wild-type levels and recombination rates dropped dramatically. Supplementing the growth medium with glutathione, a potent cellular antioxidant, similarly cut mutation and recombination frequencies by more than half. Together, these experiments identified reactive oxygen species as the sole executioner behind the genomic storm, confirming that the instability arises from direct chemical attack on DNA rather than from indirect disruption of repair machinery or metabolic collapse.

The mutational fingerprint left by Sod1 loss proved highly distinctive. All six classes of base substitutions increased, but transversions dominated, particularly C:G to A:T changes concentrated within 5′-CCA-3′ sequence motifs. This pattern is the classic signature of 8-oxoguanine, an oxidized form of guanine that mispairs with adenine during replication, producing the characteristic transversion after a second round of DNA copying. The finding directly implicates 8-oxoguanine overproduction as the molecular engine of Sod1-deficient mutagenesis, and it links the enzyme’s antioxidant function to a specific, measurable chemical lesion on DNA.

Dissecting how cells cope with these lesions revealed a sophisticated hierarchy of repair and bypass. The base excision repair glycosylase Ogg1 emerged as the frontline defense: deleting OGG1 alone caused a 19-fold rise in C:G to A:T transversions, and combining OGG1 deletion with SOD1 deletion produced a synergistic 7.5-fold increase over the ogg1 mutant alone. By these calculations, Ogg1-mediated excision corrects roughly 90 percent of the 8-oxoguanine lesions generated in the absence of Sod1. When repair is overwhelmed, translesion synthesis polymerases take over, and here the outcomes diverge sharply. DNA polymerase eta, encoded by RAD30, performs relatively accurate bypass by inserting cytosine opposite the oxidized base, preventing about a third of the characteristic transversions. In contrast, the error-prone polymerase zeta and its partner Rev1 account for approximately two-thirds of all Sod1-induced point mutations, illustrating the costly trade-off between completing replication and preserving sequence fidelity.

Timing analysis of recombination events added another layer of mechanistic insight. Using a colony-sectoring assay on chromosome IV, the team showed that about 65 percent of mitotic crossovers in sod1 mutants were initiated by double-strand breaks arising during the G1 phase of the cell cycle, closely mirroring the pattern in wild-type cells. Fluorescence microscopy of Rad52 repair foci confirmed a four-fold increase in spontaneous DNA breaks. Intriguingly, the distribution of crossover breakpoints was not random: 33 of 35 events on chromosome IV clustered distal to the SSD1 locus, a bias the researchers traced to post-mutational selection rather than intrinsic recombination hotspots. Restoring a functional SSD1 allele, which improved fitness in the sod1 background, redistributed breakpoints uniformly, demonstrating that observed genomic landscapes reflect both where damage occurs and which recombinant cells survive to form colonies.

The study then turned to human disease, testing eight ALS-associated SOD1 variants in the yeast system. Wild-type human SOD1 fully rescued the growth defects and genomic instability of yeast lacking endogenous Sod1, confirming deep functional conservation. But the clinical variants were not created equal. The H48Q mutation, which disrupts a copper-binding residue essential for catalytic activity, shattered genome stability, producing 8- and 10-fold increases in mutation and recombination rates alongside severe growth impairment. The G85R and S133N variants showed moderate effects, while the well-known A4V, G36R, D90A, and G93A variants remained essentially benign in this context. These results suggest that for a subset of ALS patients, loss of genomic protection—driven by failure to quench nuclear reactive oxygen species—may constitute an additional, mutation-specific pathogenic mechanism beyond the prevailing toxic gain-of-function model of protein aggregation.

Notably, the researchers found that human Sod1 is far more tolerant of amino acid substitutions than its yeast counterpart, a difference they attribute to the extraordinary structural robustness of the human enzyme, which remains stable even in metal-deficient states. This resilience likely reflects evolutionary pressure in long-lived organisms to protect the proteome against decades of somatic mutation. The work also carries a cautionary message for cancer therapy: because tumor cells often depend on elevated Sod1 to manage their oxidative burden, pharmacological inhibition of the enzyme, while potentially cytotoxic, risks triggering a mutational burst that could accelerate the emergence of drug-resistant clones. By mapping the full genomic consequences of antioxidant failure—from single nucleotides to whole chromosomes—this study establishes a mechanistic framework for understanding how metabolic defense and genome integrity are inseparably coupled in eukaryotic life.

Subject of Research: The role of superoxide dismutase 1 in protecting eukaryotic genome stability against oxidative DNA damage

Article Title: Decoupling metabolic defense: Mutational and recombinational chaos in Sod1-deficient cells

Article References: Sui, Y., Zheng, D.-Q., Wang, Y., Zhao, S.-S., Li, K.-J., Ye, C., & Zhang, K. (2026). Decoupling metabolic defense: Mutational and recombinational chaos in Sod1-deficient cells. PLOS Genetics, 22(10), e1012326. https://doi.org/10.1371/journal.pgen.1012326

Image Credits: AI Generated

DOI: 10.1371/journal.pgen.1012326

Keywords: Sod1, genome instability, reactive oxygen species, 8-oxoguanine, yeast, loss of heterozygosity, aneuploidy, base excision repair, translesion synthesis, ALS, SOD1 mutations, oxidative stress

The team, led by Hidehiko Kawai and Hiroyuki Kamiya, set out to dissect the mutational consequences of reactive oxygen species generated under chronic irradiation conditions that mimic low-dose, low-dose-rate exposures. This regime is biologically important because cancers arising after such exposures are largely indistinguishable from spontaneous tumors, making it extremely hard to define the mutational mechanisms at work. Under these conditions, radiation-generated reactive oxygen species are thought to inflict persistent low-level oxidative DNA damage rather than the dramatic double-strand breaks associated with high-dose irradiation. The central challenge has been to connect that diffuse chemical damage to specific, quantifiable mutational outcomes at defined DNA positions, something conventional mutagenesis assays have struggled to achieve with single-molecule resolution.

To overcome this limitation, the researchers employed a next-generation sequencing version of the classical supF shuttle vector assay, enhanced with a random twelve-nucleotide barcode incorporated into each vector molecule. This nucleotide-barcoding strategy is the technical heart of the study. Because every vector molecule carries a unique barcode, each independent mutational event can be identified and counted individually, allowing the team to distinguish true single-molecule mutation events from sequencing artifacts or clonal amplification. The assay was run under both standard supF-selection conditions and without selection, and mutation frequencies and spectra were quantified across the entire supF reporter gene. In parallel, the team prepared shuttle vector libraries carrying a single, site-specific 8-oxo-G lesion at defined positions, providing a controlled benchmark against which the irradiation-induced mutational patterns could be compared.

The results from chronic gamma irradiation were striking. Irradiation significantly increased mutations at C:G base pairs located within 5′-TCN-3′:5′-NGA-3′ sequence contexts, where N denotes any nucleotide. This trinucleotide-level specificity echoes the kind of sequence-context dependence that has become familiar from large-scale cancer genomics, in which different mutational processes leave characteristic footprints in particular sequence neighborhoods. But the more unexpected finding was positional: many of the positions that responded to irradiation were already spontaneous mutation hotspots, and they also coincided with distant C:G sites that were preferentially mutated in response to a single 8-oxo-G lesion placed elsewhere in the vector.

That coincidence points to the study’s most conceptually provocative result, a phenomenon the authors describe as strand-biased action-at-a-distance mutagenesis. When a single 8-oxo-G lesion was introduced at one defined site, mutations did not simply accumulate at or immediately adjacent to the lesion. Instead, the lesion induced extensive mutations at distant C:G sites, and these mutations showed a pronounced strand bias, preferentially affecting one strand of the duplex over the other. Chronic gamma irradiation further enhanced these distant mutations without substantially altering their positional distribution. In other words, irradiation did not create a new and distinct set of mutation sites; it amplified a pre-existing pattern of susceptible positions that was already latent in the molecule.

This amplification model has significant implications for how scientists interpret mutational signatures in cancer genomes. If radiation-induced mutagenesis largely reinforces spontaneous hotspots rather than generating an independent radiation-specific landscape, then the mutational signature of chronic low-dose radiation may be subtle and entangled with background processes, which is consistent with the clinical observation that radiation-associated cancers are so hard to distinguish from spontaneous ones. The finding also suggests that the identity of the damaged base may matter less than the structural and sequence context in which it sits, since a lesion at one position can shape mutational outcomes many bases away.

What could mechanistically explain action-at-a-distance mutagenesis and its strand bias? The authors found important clues in the local secondary structures that single-stranded DNA can adopt. During replication, transcription, or repair, stretches of DNA transiently become single-stranded, and these single-stranded regions are chemically vulnerable and can fold into hairpins and other structures. The study’s hotspot and coldspot behaviors, as well as position-dependent substitution patterns, were associated with predicted local secondary structures of single-stranded DNA. This suggests that structural context contributes to mutagenesis beyond the trinucleotide sequence context alone, adding a layer of physical geometry to the sequence-based models that currently dominate the field of mutational signature analysis.

Strand bias itself is a well-recognized feature of mutational processes in cancer genomics, often reflecting whether a lesion sits on the leading or lagging strand during replication, or on the transcribed versus non-transcribed strand during transcription-coupled repair. The strand-biased mutations observed here, driven by a single oxidative lesion and modulated by chronic irradiation, provide a controlled experimental system in which such biases can be studied mechanistically rather than inferred statistically from tumor sequencing data. The authors note that processes such as translesion DNA synthesis, in which specialized polymerases copy across damaged bases, and base excision repair, in which glycosylases such as OGG1 remove oxidized guanine, are natural candidates for the enzymatic pathways that convert a localized lesion into distant, strand-biased mutations, although the present study defines the patterns rather than fully resolving the responsible mechanisms.

Methodologically, the barcoded supF NGS platform represents a versatile new framework for mutagenesis research. Classical shuttle vector assays, which date back decades, allowed mutated plasmids recovered from cells to be sequenced, but they could not easily distinguish independent events or achieve the throughput needed to map mutation positions comprehensively. The addition of random twelve-nucleotide barcodes and next-generation sequencing changes that calculus, enabling precise quantification of mutation frequencies and spectra at single-molecule resolution. The authors position the platform as a general tool for mechanistic studies of oxidative, radiation-associated, and cancer-related mutagenesis, and it is easy to imagine its application to other lesions, other exposure regimes, and cells deficient in specific repair pathways.

The broader significance of the work lies in reframing how low-dose radiation risk might be understood at the molecular level. Rather than radiation writing a wholly new mutational script into the genome, the study suggests it turns up the volume on a script that oxidative metabolism has already written, preferentially at pre-existing susceptible sites whose locations are governed by sequence context and single-stranded DNA structure. For cancer epidemiology, this offers a potential explanation for why chronic low-dose exposures are so difficult to trace in tumor genomes, and for mutagenesis biology, it elevates DNA secondary structure from a curiosity to a first-class determinant of where mutations strike. As the authors conclude, chronic low-dose-rate gamma irradiation and a single 8-oxo-G lesion promote closely related mutational processes that act preferentially at pre-existing susceptible sites and generate strand-biased action-at-a-distance mutations, a set of strand-bias laws that may ultimately sharpen both radiation risk assessment and the interpretation of mutational signatures across human cancers.

Subject of Research: Strand-biased mutagenesis induced by oxidative DNA damage and chronic low-dose-rate gamma irradiation

Article Title: Systematic mutagenesis assay promotes comprehension of the strand-bias laws for mutations induced by oxidative DNA damage

Article References: Kawai, H., Ebi, S., Sugihara, R., Fujiwara, C., Fujikawa, Y., Kimura, S., & Kamiya, H. (2026). Systematic mutagenesis assay promotes comprehension of the strand-bias laws for mutations induced by oxidative DNA damage. BMC Cancer. https://doi.org/10.1186/s12885-026-17081-0

Image Credits: AI Generated

DOI: 10.1186/s12885-026-17081-0

Keywords: 8-oxo-G, oxidative DNA damage, ionizing radiation, mutagenesis, strand bias, mutation hotspots, supF shuttle vector, next-generation sequencing, reactive oxygen species, DNA secondary structure, cancer, base excision repair