Oxidative stress drives mutagenesis through transcription- coupled repair in bacteria

In bacteria, mutations lead to the evolution of antibiotic resistance, which is one of the main public health problems of the twenty -first century. Therefore, determining which cellular processes most frequently contribute to mutagenesis, especially in cells that have not been exposed to exogenous DNA damage, is critical. Here, we show that endogenous oxidative stress is a key driver of mutagenesis and the subsequent development of antibiotic resistance. This is the case for all classes of antibiotics and highly divergent species tested, including patient-derived strains. We show that the transcription-coupled repair pathway, which uses the nucleotide excision repair pro-teins (TC- NER), is responsible for endogenous oxidative stress-dependent mutagenesis and subsequent evolution. This suggests that a majority of mutations arise through transcription-associated processes rather than the replication fork. In addition to determining that the NER proteins play a critical role in mutagenesis and evolution, we also identify the DNA polymerases responsible for this process. Our data strongly suggest that cooperation between three different mutagenic DNA polymerases, likely at the last step of TC- NER, is responsible for mutagenesis and evolution. Overall, our work identifies a highly conserved pathway that drives mutagenesis due to endogenous oxidative stress, which has broad implications for all diseases of evolution, including antibiotic resistance development.SignificanceA common way of becoming resistant to antibiotics is mutations in the bacterial genome. However, how these mutations arise remains poorly understood. Here, we determine that endogenous oxidative stress is the main driver of mutagenesis that leads to antibiotic resistance development. In addition, we find that a highly conserved DNA repair pathway, transcription-coupled repair, is responsible for this oxidative stress-driven evolution of antibiotic resistance. Our prior work showed that the Mfd protein, which contributes to TC- NER, accelerates the development of antibiotic resistance. The work presented here shows that, in fact, all of the proteins involved in TC- NER are contributing significantly to resistance development, expanding our knowledge of the fundamental mechanisms driving not only drug resistance, but also evolution in general.