Oxidative stress drives mutagenesis through transcription coupled repair in bacteria

In bacteria, mutations lead to the evolution of antibiotic resistance, which is one the main public health problems of the 21st 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 tested and across highly divergent species, including patient-derived strains. We show that the transcription-coupled repair pathway, which uses the nucleotide excision repair proteins (TC-NER), is responsible for endogenous oxidative stress-dependent mutagenesis and subsequent evolution. This strongly 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 that a highly conserved pathway drives mutagenesis due to endogenous oxidative stress, which has broad implications for all diseases of evolution, including antibiotic resistance development.