Polymerase change raises mutation rates relevant to tuberculosis drug resistance

Drug resistance in Mycobacterium tuberculosis is a major challenge for treatment, and researchers are working to understand how bacteria pick up the mutations that make drugs fail. DNA replication is one of the key processes that influences how quickly new mutations arise, and previous work has linked expression of the DNA polymerase DnaE2 during stressful conditions with higher mutation rates. In the work led by Meindert H. Lamers, scientists set out to compare two polymerases: the high-fidelity, replicative DNA polymerase DnaE1 and the predicted error-prone DNA polymerase DnaE2. Their goal was to find which specific amino acid changes alter polymerase fidelity — in other words, which changes make the enzyme more likely to insert the wrong base and generate mutations. To do this they combined computational and laboratory approaches, using in silico analyses to flag candidate positions and following up with experimental tests to see whether altering those positions changed mutation rates in bacterial cells and in biochemical assays.

To identify positions likely to influence fidelity, the team applied a two-entropies sequence analysis to polymerase sequences and then performed experimental validation of the candidate sites. This combined in silico and in vivo approach pinpointed a specific double mutation in the palm domain of M. smegmatis DnaE1: D431S/R432D. Introducing D431S/R432D increased mutation frequencies both in vivo and in vitro, demonstrating that changes at these positions can reduce replication fidelity. Structural interpretation placed these two residues next to the DNA backbone of the template strand, and the authors propose that the amino acid swap loosens the polymerase’s grip on the template. A looser interaction with the template would make it easier for incorrect nucleotides to be incorporated, offering a mechanistic explanation for the observed rise in errors when D431S/R432D is present.

These findings provide a clearer picture of how specific changes in a replicative polymerase can shift it toward error-prone copying, a process that contributes to the emergence of drug resistance. By showing that particular residues in the palm domain of DnaE1 control fidelity, the work helps link molecular alterations in DNA replication machinery to population-level outcomes like faster accumulation of mutations. The study focuses on M. smegmatis as a model system, but the authors draw direct relevance to Mycobacterium tuberculosis because replication fidelity influences how drug resistance mutations arise during infection and treatment. Ultimately, the identification of fidelity-altering positions such as D431S/R432D offers a target list for further studies: researchers can monitor these sites, probe their effects in pathogenic strains, and consider how modulation of polymerase function might factor into future strategies to limit or manage drug resistance.