The postreplicative mismatch repair (MMR) is an almost ubiquitous DNA repair essential for maintaining genome stability. It has been suggested that Mycobacteria have an alternative MMR in which NucS, an endonuclease with no structural homology to the canonical MMR proteins (MutS/MutL), is the key factor. Here, we analyze the spontaneous mutations accumulated in a neutral manner over thousands of generations by Mycobacterium smegmatis and its MMR-deficient derivative (ΔnucS). The base pair substitution rates per genome per generation are 0.004 and 0.165 for wild type and ΔnucS, respectively. By comparing the activity of different bacterial MMR pathways, we demonstrate that both MutS/L- and NucS-based systems display similar specificity and mutagenesis bias, revealing a functional evolutionary convergence. However, NucS is not able to repair indels in vivo. Our results provide an unparalleled view of how this mycobacterial system works in vivo to maintain genome stability and how it may affect Mycobacterium evolution.

The DNA repair endonuclease EndoMS/NucS is highly conserved in Archaea and Actinobacteria. This enzyme is able to recognize and cleave dsDNA carrying a mismatched base pair, and its activity is enhanced by the interaction with the sliding clamp of the replisome. Today, EndoMS/NucS has been established as the key protein of a non-canonical mismatch repair (MMR) pathway, acting specifically in the repair of transitions and being essential for maintaining genome stability. Despite having some particularities, such as its lower activity on transversions and the inability to correct indels, EndoMS/NucS meets the main hallmarks of a MMR. Its absence leads to a hypermutator phenotype, a transition-biased mutational spectrum and an increase in homeologous recombination. Interestingly, polymorphic EndoMS/NucS variants with a possible effect in mutation rate have been detected in clinical isolates of the relevant actinobacterial pathogen Mycobacterium tuberculosis. Considering that MMR defects are often associated with the emergence of resistant bacteria, the existence of EndoMS/NucS-defective mutators could have an important role in the acquisition of antibiotic resistance in M. tuberculosis. Therefore, a further understanding of the EndoMS/NucS-mediated non-canonical MMR pathway may reveal new strategies to predict and fight drug resistance. This review is focused on the recent progress in NucS, with special emphasis on its effect on genome stability and evolvability in Actinobacteria.

Worldwide dissemination of pathogens resistant to almost all available antibiotics represent a real problem preventing efficient treatment of infectious diseases. Among antimicrobial used in therapy, β-lactam antibiotics represent 40% thus playing a crucial role in the management of infections treatment. We report a small series of phenylboronic acids derivatives (BAs) active against class A carbapenemases KPC-2 and GES-5, and class C cephalosporinases AmpC. The inhibitory profile of our BAs against class A and C was investigated by means of molecular docking, enzyme kinetics and X-ray crystallography. We were interested in the mechanism of recognition among class A and class C to direct the design of broad serine β-Lactamases (SBLs) inhibitors. Molecular modeling calculations vs GES-5 and crystallographic studies vs AmpC reasoned, respectively, the ortho derivative 2 and the meta derivative 3 binding affinity. The ability of our BAs to protect β-lactams from BLs hydrolysis was determined in biological assays conducted against clinical strains: Fractional inhibitory concentration index (FICI) tests confirmed their ability to be synergic with β-lactams thus restoring susceptibility to meropenem. Considering the obtained results and the lack of cytotoxicity, our derivatives represent validated probe for the design of SBLs inhibitors.

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.

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.