The unique role of <i>nucS</i> -mediated noncanonical mismatch repair in <i>Mycobacterium tuberculosis</i> resistance evolution

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 <i>Mycobacterium tuberculosis</i>, lack these components. Instead, they appear to rely on the nuclease EndoMS/NucS, a structurally distinct enzyme governing a non-canonical MMR pathway. Since <i>M. tuberculosis</i> 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 <i>M. tuberculosis</i> and uncovered a unique resistance dynamic distinct from other Actinobacteria. Deleting <i>nucS</i> altered the mutational spectrum but had minimal impact on the emergence of antibiotic-resistant mutants, contrasting sharply with other Actinobacteria where <i>nucS</i> 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 <i>M</i>. <i>tuberculosis</i> genomes revealed that most R144S-containing strains belong to the Euro-American sub-lineage 4.9, with no significant association with antibiotic resistance. However, <i>nucS</i> is under strong purifying selection, and R144S changes arose independently during <i>M. tuberculosis</i> evolution (homoplasy). Overall, our findings challenge the view that <i>nucS</i> serves as a genome stability guardian in <i>M. tuberculosis</i> and suggest additional mismatch repair mechanism(s) beyond NucS in this pathogen.IMPORTANCEDNA 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 <i>Mycobacterium tuberculosis</i>, 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.