Mutagenesis induced by conflicts between replication and transcription machineries: control by the Mismatch Repair System in Bacillus subtilis

IBÁÑEZ BUSSETI MILAGROS; MARIELA R. MONTI

Congreso; LVIII Reunión Anual de SAIB; 2022

DNA replication and transcription use the same template and occur concurrently in bacteria. The lack of temporal and spatial separations of these two processes leads to conflicts between them. The 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), which are call to action by the Mfd factor. This protein recognizes RNA Pols stalled at a DNA template lesion, and subsequently displaces it from DNA. This exposes the offending lesion to the base and nucleotide excision repair systems (BER and NER) that generate a gap, which is fill in by LF-Pols. Our previous findings demonstrated that the Mismatch Repair protein, MutS, controls the mutagenesis produced when LF-Pols perform translesion synthesis by regulating their access to replication sites through the processivity beta clamp factor in Pseudomonas aeruginosa. In the present study, we analyzed if this novel MutS-dependent mechanisms 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 the mutator factor 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. Deletion of the gen encoding this NER protein had no effect on the mutagenesis levels of the highly transcribed rpoB and rpsL 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) and exogenous (thyp3) highly transcribed genes.