The unique role of nucS-mediated non-canonical mismatch repair in Mycobacterium tuberculosis resistance evolution

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.