Hierarchical determinants of the oxidation-induced mutational landscape in human cells

8-oxoguanine (8-oxoG) is a common oxidative DNA lesion, which causes G>T substitutions that compose COSMIC single base substitution signature 18 (SBS18) in human cancers. Determinants of local and regional differences in 8-oxoG-induced mutability are currently unknown. To uncover factors influencing the topology of 8-oxoG-induced mutations, we assessed spontaneous and KBrO ₃ -induced 8-oxoG mutagenesis in human cell lines. KBrO ₃ exposure produced a SBS18-like substitution spectrum and a distinct never-before reported INDEL signature that we also observed in human cancers. KBrO ₃ -induced 8-oxoG lesions occurred with similar sequence preference as KBrO ₃ -induced substitutions, indicating that the reactivity of specific reactive oxygen species (ROS) dictates the trinucleotide motif specificity for 8-oxoG-induced mutagenesis. While 8-oxoG lesions occurred relatively uniformly across chromatin states and nucleosomes, 8-oxoG-induced mutations occurred more frequently in more compact regions of the genome, within nucleosomal DNA, and at inward facing guanines within strongly positioned nucleosomes. Cryo-EM structures of OGG1 bound to nucleosomes indicate that these effects originate from OGG1’s ability to flip outward positioned 8-oxoG lesions into the catalytic pocket with only minor alterations to nucleosome structure, while inward facing lesions occluded by the histone octamer are unrecognized. Mutation spectra from cells with DNA repair deficiencies revealed a hierarchical DNA repair network limiting 8-oxoG mutagenesis in human cells, where OGG1– and MUTY-mediated BER is supplemented by replication-associated factors participating in tolerance of 8-oxoG or derived repair intermediates (i.e. Pol η and HMCES). Surprisingly, analysis of transcriptional asymmetry of KBrO ₃ -induced mutations demonstrated transcription-coupled repair of 8-oxoG in Pol η-deficient cells. Thus, radical chemistry, chromatin structures, and DNA repair processes combine to dictate the oxidative mutational landscape in human genomes.