Identification of determinants of high-fidelity DNA synthesis in <i>Mycobacterium smegmatis</i> DnaE1 through <i>in silico</i> and <i>in vivo</i> approaches

Drug resistance in Mycobacterium tuberculosis presents a major challenge in tuberculosis treatment, highlighting the need to understand the underlying mechanisms. DNA replication plays an important role in the acquisition of drug resistance, and the expression of the DNA polymerase DnaE2 during adverse conditions has been associated with increased mutation rates. Here, we investigate the functional differences between the high-fidelity replicative DNA polymerase DnaE1 and the predicted error-prone DNA polymerase DnaE2, focusing on which amino acid changes affect polymerase fidelity. For this, we identify potential fidelity-altering positions using a two-entropies sequence analysis combined with experimental validation to test whether changes of these positions affect the mutation rates. We find that a double mutation in the palm domain of Mycobacterium smegmatis DnaE1: D431S/R432D, increases mutation frequencies both in vivo and in vitro. The location of these two residues adjacent to the DNA backbone of the template strand suggests that the amino acid change results in a looser grip on the DNA, allowing for the incorporation of incorrect nucleotides. These insights improve our understanding of the mechanisms underlying drug resistance in M. tuberculosis and could help in the development of future strategies to combat it.