DNA surveillance mechanisms are crucial for maintaining genome stability and minimizing mutation rates. Mismatch repair (MMR) corrects replication errors that escape DNA-polymerase proofreading. In most organisms, MMR is orchestrated by MutS and MutL proteins. However, certain Archaea and Actinobacteria, including the major human pathogen Mycobacterium tuberculosis , lack these components. Instead, they appear to rely on the nuclease EndoMS/NucS, a structurally distinct enzyme governing a non-canonical MMR pathway. Since M. tuberculosis acquires drug resistance exclusively through chromosomal mutations, understanding its mutation rate regulation is critical. Nevertheless, the role of NucS in drug resistance evolution remains largely unexplored. We investigated NucS function in M. tuberculosis and uncovered a unique resistance dynamic distinct from other Actinobacteria. Deleting nucS altered the mutational spectrum but had minimal impact on the emergence of antibiotic-resistant mutants, contrasting sharply with other Actinobacteria where nucS loss dramatically increases mutation rates. This atypical behavior cannot be attributed solely to the R144S NucS polymorphism present in the H37Rv reference strain. Introducing the consensus NucS sequence in H37Rv produced only subtle changes in mutational spectrum without major effects on mutation rates. Analysis of 44,921 M . tuberculosis genomes revealed that most R144S-containing strains belong to the Euro-American sub-lineage 4.9, with no significant association with antibiotic resistance. However, nucS is under strong purifying selection, and R144S changes arose independently during M. tuberculosis evolution (homoplasy). Overall, our findings challenge the view that nucS serves as a genome stability guardian in M. tuberculosis and suggest additional mismatch repair mechanism(s) beyond NucS in this pathogen. IMPORTANCE: DNA repair systems are crucial for maintaining the integrity of genetic information by scanning and correcting errors that arise during DNA replication. Most organisms use well-characterized proteins (MutS and MutL) for this task, but some bacteria and archaea, including most Actinobacteria, lack these conventional components. Instead, they employ an alternative enzyme, NucS, to fix replicative DNA errors. This alternative pathway is thought to limit harmful mutations and, in turn, reduce the likelihood of drug resistance development. In our study of Mycobacterium tuberculosis , the pathogen responsible for tuberculosis, we found that NucS plays only a minor role in preventing resistance-associated mutations. This unexpected result challenges current assumptions about DNA repair in this pathogen and points to the existence of other, yet unidentified mechanisms that safeguard its genome. Understanding these processes could open new avenues for therapeutic strategies against tuberculosis, a disease that remains a major global health threat.

Mismatch repair (MMR) system alterations can trigger transient hypermutation, promoting adaptive mutations under stress, such as antibiotic exposure. While most organisms use MutS and MutL protein families for MMR, many archaea and actinobacteria, including the major human pathogen Mycobacterium tuberculosis, lack these components and instead rely on NucS, a structurally distinct enzyme driving a non-canonical MMR pathway. Given the role of MMR in mutation control, understanding how nucS expression is regulated could be essential for uncovering the molecular basis of antibiotic resistance development in mycobacteria. In this study, we characterized the nucS promoter and transcription start site in Mycobacterium smegmatis. We found that nucS expression declines during the stationary phase in both M. smegmatis and M. tuberculosis, paralleling replication activity and canonical MMR downregulation. Our data suggest that the alternative sigma factor σ^B may negatively regulate nucS expression during this phase. Additionally, we identified candidate compounds that may modulate nucS expression, underscoring its responsiveness to environmental cues. These findings enhance our understanding of mycobacterial stress responses and lay the groundwork for exploring antibiotic resistance mechanisms. Strikingly, our work reveals a case of double convergent evolution: both canonical (MutS/MutL) and non-canonical (NucS) pathways have independently evolved not only the same DNA repair function, but also similar regulatory frameworks for genome integrity preservation under stress conditions.

DNA surveillance mechanisms play a vital role in maintaining genome stability and minimizing mutation rates. One such mechanism, post-replicative mismatch repair (MMR), corrects replication errors that escape DNA polymerase proofreading activity. In most bacteria and eukaryotes, MMR is orchestrated by MutS and MutL proteins. However, certain archaeal and actinobacterial species, including the major human pathogen Mycobacterium tuberculosis , lack these components. Instead, they rely on the nuclease EndoMS/NucS, a structurally distinct enzyme that governs a non-canonical MMR pathway. Given that M. tuberculosis acquires drug resistance exclusively through chromosomal mutations, understanding mutation rate regulation in this pathogen is critical. Nevertheless, despite its anticipated significance, the role of NucS in drug resistance evolution remains largely unexplored in this organism. This study investigates NucS function in M. tuberculosis and uncovers a unique resistance dynamic distinct from other Actinobacteria. While nucS deletion alters the mutational spectrum, it minimally affects the emergence of rifampicin-, isoniazid-, and ethambutol-resistant mutations, in stark contrast to its role in other Actinobacteria. We demonstrated that this atypical behaviour is not attributable to the presence of a single NucS polymorphism, R144S, in the NucS sequence of the M. tuberculosis reference strain H37Rv, which differs from the NucS consensus sequence. Constructing and analysing an H37Rv variant possessing the NucS consensus sequence revealed a subtly altered mutational spectrum but unchanged mutation rates. Notably, database analysis of the R144S polymorphism in clinical isolates revealed its prevalence and significant association with ethambutol resistance. These findings challenge the established view that nucS serves as a genome stability guardian that minimizes mutation rates in M. tuberculosis , suggesting additional mismatch repair mechanisms beyond NucS or a highly efficient replication system in this pathogen.