Involvement of transcription-coupled repair factor Mfd and DNA helicase UvrD in mutational processes in Pseudomonas putida.

Stalled RNA polymerases (RNAPs) pose an obstacle for the replicating complexes, which could lead to transcription-replication conflicts and result in genetic instability. Stalled RNAPs and DNA lesions blocking RNAP elongation are removed by transcription-coupled repair (TCR), the process which in bacteria is mediated by TCR factor Mfd and helicase UvrD. Although the mechanism of TCR has been extensively studied, its role in mutagenesis is still obscure. In the current study we have investigated the role of Mfd and UvrD in mutational processes in soil bacterium Pseudomonas putida. Our results revealed that UvrD helicase is essential to prevent the emergence of mutations, as the loss of uvrD resulted in elevated mutant frequency both in exponential- and stationary-phase bacterial cultures. UvrD was also found to be necessary to survive DNA damage, but NER or MMR pathways are not completely abolished in UvrD-deficient P. putida. Mfd-deficiency had a moderate impact on surviving DNA damage and did not influence the frequency of mutations occurred in exponentially growing bacteria. However, the absence of Mfd caused approximately a two-fold decline in stationary-phase mutant frequency compared to the P. putida wild-type strain and suppressed the elevated mutant frequency observed in the ΔuvrD strain. Remarkably, the Mfd-deficient strain also formed less UV-induced mutants. These results suggest that in P. putida the Mfd-mediated TCR could be associated with UV- and stationary-phase mutagenesis.