The goal of this project is to elucidate the mechanisms responsible for the increase in spontaneous mutagenesis, which is dependent on the Mfd protein in Escherichia coli cells growing without exogenous stressors. Our hypothesis posits that the most of Mfd-dependent mutations arise from the Mfd-mediated exposure of stretches of single-stranded DNA (ssDNA) situated between the elongating RNA polymerase (RNAP) and Mfd. We propose that this occurs after Mfd restarts RNAPs stalled due to obstacles other than DNA lesions, and while it remains connected to the elongating RNAP and the DNA. The rationale behind this hypothesis is that because ssDNA is significantly less chemically stable than double-stranded DNA, it is more susceptible to premutagenic chemical modifications, such as depurination, depyrimidination, deamination and oxidation. Importantly, these modifications are not well-recognized by the NER system, and they do not block the DNA replication process. Identifying the molecular mechanisms that regulate mutation rates carries significant implications for a wide array of scientific fields, including genetics, evolution, medicine, biotechnology, and environmental science. In this context, the contribution of Mfd, an evolutionarily conserved enzyme, to spontaneous mutation rates holds particular significance. This importance stems from the fact that the inactivation of the gene encoding Mfd can be categorized as an "antimutator mutation." In other words, it reduces the rate of mutations, which can be especially relevant in the context of drug resistance and disease prevention.