Ionizing radiation-induced cancer is largely indistinguishable from spontaneous cancer, particularly following low-dose and low-dose-rate exposures, making the underlying mutational mechanisms difficult to define. Under such exposure conditions, radiation-generated reactive oxygen species (ROS) can trigger persistent low-level DNA damage via the induction of oxidative DNA lesions such as 8-oxo-7,8-dihydroguanine (8-oxo-G). However, how this DNA damage is converted into mutational outcomes remains poorly understood. To address this question, we applied a nucleotide-barcoded (N₁₂-BC) supF shuttle vector next-generation sequencing (supF NGS) assay.

Mutagenesis induced by chronic gamma irradiation was analyzed using supF NGS assay under both supF-selection and w/o-supF-selection conditions. By incorporating a random N₁₂ barcode, the assay enables analysis of independent mutational events at the single-molecule level, allowing precise quantification of mutation frequencies and spectra. In parallel, shuttle vector libraries containing a single site-specific 8-oxo-G lesion were used to compare irradiation-associated mutagenesis with mutations induced by a defined oxidative DNA lesion.

Chronic gamma irradiation significantly increased mutations at C:G base pairs within 5′-TCN-3′:5′-NGA-3′ sequence contexts. Unexpectedly, many irradiation-responsive positions coincided with spontaneous mutation hotspots and with distant C:G sites preferentially mutated in response to a single 8-oxo-G lesion. A single 8-oxo-G lesion induced extensive strand-biased “action-at-a-distance” mutations at these distant sites, and chronic irradiation further enhanced these mutations without substantially altering their positional distributions. Thus, irradiation amplified pre-existing mutation patterns rather than generating a distinct set of mutation sites. In addition, hotspot and coldspot behaviors, as well as position-dependent substitution patterns, were associated with predicted local secondary structures of single-stranded DNA, suggesting that structural context contributes to mutagenesis beyond trinucleotide sequence context alone.

Chronic low-dose-rate gamma irradiation and a single 8-oxo-G lesion promoted closely related mutational processes that preferentially acted at pre-existing susceptible sites and generated strand-biased action-at-a-distance mutations. Furthermore, our data suggest that local secondary-structure features contribute to hotspot and coldspot formation as well as position-dependent mutation spectra. The supF shuttle vector NGS platform provides a versatile framework for mechanistic studies of oxidative, radiation-associated, and cancer-related mutagenesis.