A multilayered repair system protects the mycobacterial chromosome from endogenous and antibiotic-induced oxidative damage.

Oxidative damage to DNA is a threat to the genomic integrity and coding accuracy of the chromosomes of all living organisms. Guanine is particularly susceptible to oxidation, and 8-oxo-dG (OG), when produced in situ or incorporated by DNA polymerases, is highly mutagenic due to mispairing with adenine. In many bacteria, defense against OG depends on MutT enzymes, which sanitize OG in the nucleotide pool, and the MutM/Y system, which counteracts OG in chromosomal DNA. In , antibiotic lethality has been linked to oxidative stress and the downstream consequences of OG processing. However, in mycobacteria, the role of these systems in genomic integrity and antibiotic lethality is not understood, in part because mycobacteria encode four MutT enzymes and two MutMs, suggesting substantial redundancy. Here, we definitively probe the role of OG handling systems in mycobacteria. We find that, although MutT4 is the only MutT enzyme required for resistance to oxidative stress, this effect is not due to OG processing. We find that the dominant system that defends against OG-mediated mutagenesis is MutY/MutM1, and this system is dedicated to in situ chromosomal oxidation rather than correcting OG incorporated by accessory polymerases (DinB1/DinB2/DinB3/DnaE2). In addition, we uncover that mycobacteria resist antibiotic lethality through nucleotide sanitization by MutTs, and in the absence of this system, accessory DNA polymerases and MutY/M contribute to antibiotic-induced lethality. These results reveal a complex, multitiered system of OG handling in mycobacteria with roles in oxidative stress resistance, mutagenesis, and antibiotic lethality.