Functional Characterization of the Transcriptional response to DNA damage in mycobacteria Authors: Oyindamola Adefisayo, Michael Glickman Affiliations: Weill Cornell Medicine, Memorial Sloan Kettering Cancer Center Abstract: It is critical to understand the mycobacterial DNA damage response (DDR) due to its role in mutagenesis, the ultimate cause of antibiotic resistance, and in resisting DNA damage inflicted by host immunity. Mycobacteria encode two transcriptional DDR pathways that control the expression of multiple genes essential for DNA repair and mutagenesis. The two pathways are controlled by the activator PafBC and the LexA repressor. Although the regulons controlled by these systems are defined, the division of labor between them for repair and mutagenesis, and the activating signal for the PafBC pathway, are unknown. To address these questions, we ablated the PafBC pathway (, the SOS pathway (through an uncleavable lexA S167A point mutation), or both, along with ablation of recA, which has been used in literature as a proxy for the SOS pathway in Mycobacterium smegmatis (Msmeg). We used these strains to analyze the division of labor between these pathways in response to multiple DNA damaging agents, including quinolones, UV light and mitomycin C. Transcriptional profiling by RNA sequencing in response to both UV and ciprofloxacin induced DNA damage confirmed prior reports that PafBC controls a larger transcriptional program than LexA. However, ablation of the SOS response reveals a dominant role for this system in sensitivity to killing by DNA damage and mutagenesis. In addition, although SOS deficient bacteria are more sensitive to UV damage than PafBC deficient bacteria, only double PafBC/SOS deficiency phenocopies loss of RecA, indicating functional redundancy between the pathways. In contrast to prior studies that equated ablation of RecA with loss of the SOS pathway, we find that the ablation of recA actually closely phenocopies the double pafBC/lexA S167A strain, particularly when comparing DNA damage induced gene expression. Additionally, transcriptional profiling and RecA protein expression in both M. smegmatis and M. tuberculosis reveal that quinolone stress may be a preferential inducer of the PafBC pathway. Taken together, our results show that although the PafBC pathway controls a larger transcriptional regulon, the SOS pathway plays a dominant role in DNA damage survival and mutagenesis. Our studies identify quinolone stress as an inducer of PafBC dependent gene expression, potentially providing a window into the activating signal of the PafBC pathway. Finally, our data indicate that intact RecA function is required for the full expression of both the SOS and PafBC pathways.