8-oxoguanine (8-oxoG) is a common oxidative DNA lesion that causes G > T substitutions. Determinants of local and regional differences in 8-oxoG-induced mutability across genomes are currently unknown. Here, we show DNA oxidation induces G > T substitutions and insertion/deletion (INDEL) mutations in human cells and cancers. Potassium bromate (KBrO₃)-induced 8-oxoGs occur with similar sequence preferences as their derived substitutions, indicating that the reactivity of specific oxidants dictates mutation sequence specificity. While 8-oxoG occurs uniformly across chromatin, 8-oxoG-induced mutations are elevated in compact genomic regions, within nucleosomes, and at inward facing guanines within strongly positioned nucleosomes. Cryo-electron microscopy structures of OGG1-nucleosome complexes indicate that these effects originate from OGG1’s ability to flip outward positioned 8-oxoG lesions into the catalytic pocket while inward facing lesions are occluded by the histone octamer. Mutation spectra from human cells with DNA repair deficiencies reveals contributions of a DNA repair network limiting 8-oxoG mutagenesis, where OGG1- and MUTYH-mediated base excision repair is supplemented by the replication-associated factors Pol η and HMCES. Transcriptional asymmetry of KBrO₃-induced mutations in OGG1- and Pol η-deficient cells also demonstrates transcription-coupled repair can prevent 8-oxoG-induced mutation. Thus, oxidant chemistry, chromatin structures, and DNA repair processes combine to dictate the oxidative mutational landscape in human genomes.

Eukaryotic genomic DNA is packaged into chromatin as nucleosomes, where it remains susceptible to reactive oxygen species (ROS) that generate the mutagenic lesion 8-oxo-7,8-dihydroguanine (8oxoG). While 8-oxoguanine DNA glycosylase 1 (OGG1) can initiate repair of 8oxoG base paired with C within the nucleosome core particle (NCP) in a position- dependent manner, it is unknown whether MutY homolog (MUTYH), the DNA glycosylase that excises misincorporated A opposite 8oxoG, can initiate repair of 8oxoG:A base pairs within NCPs. To address this, we combined cryo-EM, molecular dynamics (MD) simulations, and biochemical assays. We determined that MUTYH activity on nucleosomal 8oxoG:A is strongly suppressed, with detectable excision limited to the entry/exit region. Cryo-EM structures at four superhelical locations reveal that 8oxoG adopts the syn conformation and Hoogsteen base pairs with A, as in non-nucleosomal DNA, indicating that lesion presentation is not altered by the histone octamer. MD simulations further reveal that 8oxoG:A base pair dynamics and local DNA backbone perturbations are similar in nucleosomal and non-nucleosomal DNA. Together, these data establish that the NCP sterically excludes MUTYH from 8oxoG:A base pairs, making them largely inaccessible to MUTYH processing. This work ultimately provides mechanistic insight for the elevated G to T transversion rate observed in histone-bound DNA following oxidative stress.

Translesion synthesis is a major DNA damage bypass pathway in which specialized DNA polymerases, such as pol η and Rev1, are recruited to stalled replication forks where they catalyze nucleotide incorporation opposite DNA damage. Translesion synthesis is regulated by the Rad6-Rad18 complex, which catalyzes PCNA mono-ubiquitylation. Despite its central role in regulating translesion synthesis, its oligomeric state and molecular interactions are poorly understood. Here, we use mass photometry to show that Rad18 co-purifies with Rad6 and forms a series of larger oligomeric complexes. Using yeast two-hybrid studies, we showed that while the full-length Rad18 complex interacts with pol η, its interaction with Rev1 is controlled by auto-inhibition. The release of this auto-inhibition is associated with the dissociation of the Rad6-Rad18 complex, freeing Rad18 monomers and dimers. Because pol η participates in non-mutagenic translesion synthesis, while Rev1 participates in mutagenic translesion synthesis, this auto-inhibition likely allows pol η preferential access to stalled replication forks by delaying Rev1 recruitment. Such an ordered polymerase-recruitment mechanism would reduce the likelihood of mutations.

Eukaryotic genomic DNA is packaged into chromatin through a repeating unit known as the nucleosome. In this chromatin environment, DNA is constantly exposed to several sources of DNA damage, such as reactive oxygen species (ROS), which can lead to the formation of 8-oxo-7,8-dihydroguanine (8oxoG). 8oxoG can base pair with cytosine (8oxoG:C) or form a mutagenic base pair with adenine (8oxoG:A), which can lead to single base transversions if left unrepaired. To date, the structure and dynamics of these two possible 8oxoG base pairs in the nucleosome remain unclear. Furthermore, whether MutY homologue (MUTYH) excises 8oxoG:A base pairs in the nucleosome remains elusive. Here using a combination of cryogenic-electron microscopy, molecular dynamics simulations, and biochemistry we determined the structure and dynamics of 8oxoG:C and 8oxoG:A base pairs in the nucleosome and characterize MUTYH activity in nucleosomal DNA. We found that nucleosomal 8oxoG:C forms a stable base pair using its anti conformation, while nucleosomal 8oxoG:A forms a more dynamic base pair using its syn conformation that is unable to be processed by MUTYH. This work provides fundamental insight into the accommodation of oxidative damage in the nucleosome and how this damage contributes to increased mutagenic transversions in nucleosomal compared to linker DNA.