Abstract
The growing drug resistance of M. tuberculosis necessitates the search for new molecular targets. A promising candidate in this regard is the NucS endonuclease — a functional analog of the canonical MutS–MutL–MutH mismatch repair system, which is absent in mycobacteria. In the present study, the biochemical and functional characterization of NucS from M. tuberculosis was performed. Recombinant proteins MtbNucS and MtbDnaN were expressed in the E. coli Arctic Express (DE3) system. MtbNucS was shown to exhibit selective nuclease activity toward substrates containing G•T and U•G mismatches; optimal catalytic conditions were 25 mM Tris-HCl (pH 8.0), 40 mM MgCl₂, and 2.5 mM MnCl₂. The sliding clamp MtbDnaN exerts a biphasic, dose-dependent effect on enzyme activity. Site-directed mutagenesis confirmed the critical role of residues W52 and D137 in the formation of the NucS active site. The data obtained support the potential of MtbNucS as a target for the development of novel antituberculosis drugs.