Mutagenesis induced by replication-transcription conflicts: control by the Mismatch Repair System in Bacillus subtilis

IBÁÑEZ MILAGROS; MARIELA R. MONTI

Congreso; XVIII Reunión Anual de SAMIGE; 2023

DNA replication and transcription machineries use the same DNA template and occur concurrently in bacteria without temporal and spatial separations. Depending on the orientation of a given gene, the replication machinery can face RNA polymerases (RNAPs) in either a head-on (HO) or a co-directional (CD) manner. These transcription-replication conflicts (TRCs) have detrimental consequences on replication and cell viability, as well as they promote mutagenesis in highly transcribed genomic regions. The TRCs-induced mutagenesis is produced by the DNA synthesis catalyzed by low fidelity DNA polymerases (LF-Pols) in the nucleotide excision repair (NER). Briefly, Mfd protein recognizes stalled RNAPs at a DNA template lesion, and subsequently displaces it from DNA. The exposed lesion is excised by the NER proteins leaving a single nucleotide gap, which is fill in by LF-Pols. Our previous results have demonstrated that the Mismatch Repair protein, MutS, modulates the access of LF-Pols to replication sites by regulating their interaction with the processivity beta clamp factor. In the present study, we analyzed if this novel MutS-dependent mechanism modulates the TRCs-mutagenesis induced by LF-Pols in Bacillus subtilis. With this aim, mutation rates in endogenous genes with low (thyA) and high (rpoB and rpsL) transcription levels were estimated in a mutSβ strain, which expressed a MutS mutant that does not bind to β clamp and therefore does not control LF-Pols, compared to the wild type (WT) strain. We found a significant increase in the mutation rates to resistance to rifampicin (target gene: rpoB, rifR) and streptomicine (target gene: rpsL, smR) in the mutSβ strain relative to the WT strain. In contrast, both strains showed similar mutation rates to trimetoprim resistance (target gene: thyA, tmpR). Then, we tested if Mfd and the LF-Pols, Pol I, PolY1 and PolY2, are involved in the increased mutation exhibited by the highly transcribed genes in the mutSβ strain. Inactivation of Mfd and Pol I specifically decreased mutation rates to rifR and smR but not to tmpR in the mutSβ genetic background. We also analyzed if the UvrA factor, which initiates the NER pathway, is implicated in the increased mutagenesis observed in mutSβ. Inactivation of the uvrA gene diminished mutations levels of the highly transcribed genes. Similar results were obtained with the exogenous thyP3 reporter gen, which was placed under an IPTG-inducible promoter. In conclusion, these results suggest that MutS regulates the action of the low fidelity Pol I in the Mfd-dependent mutagenesis resulting from TRCs. Currently, we are analyzing the molecular signatures of this process, by determining the mutation spectra of the endogenous (rpoB) highly transcribed gene, and lesions triggering the TC-NER.