The intrinsic ATPase activity of Mycobacterium tuberculosis UvrC is crucial for its damage-specific DNA incision function

To ensure genome stability, bacteria have evolved a network of DNA repair mechanisms; among them, the UvrABC-dependent nucleotide excision repair (NER) pathway is essential for the incision of a variety of bulky adducts generated by exogenous chemicals, UV radiation and by-products of cellular metabolism. However, very little is known about the enzymatic properties ofMycobacterium tuberculosisUvrABC excinuclease complex. Furthermore, the biochemical properties ofEscherichia coliUvrC (EcUvrC) are not well understood (compared to UvrA and UvrB), perhaps due to its limited availability and/or activity instabilityin vitro. In addition, homology modelling ofM. tuberculosisUvrC (MtUvrC) revealed the presence of a putative ATP-binding pocket, although its function remains unknown. To elucidate the biochemical properties of UvrC, we constructed and purified wild-type MtUvrC and its eight variants harbouring mutations within the ATP-binding pocket. The data from DNA-binding studies suggest that MtUvrC exhibits high-affinity for duplex DNA containing a bubble or fluorescein-dT moiety, over fluorescein-adducted single-stranded DNA. Most notably, MtUvrC has an intrinsic UvrB-independent ATPase activity, which drives dual incision of the damaged DNA strand. In contrast, EcUvrC is devoid of ATPase activity; however, it retains the ability to bind ATP at levels comparable to that of MtUvrC. The ATPase-deficient variants map to residues lining the MtUvrC ATP-binding pocket. Further analysis of these variants revealed separation of function between ATPase and DNA-binding activities in MtUvrC. Altogether, these findings reveal functional diversity of the bacterial NER machinery and a paradigm for the evolution of a catalytic scaffold in UvrC.

Automated summary

The study explores the enzymatic properties of the Mycobacterium tuberculosis UvrABC excinuclease complex, revealing that MtUvrC has UvrB-independent ATPase activity which drives dual incision of damaged DNA strands. In contrast, EcUvrC lacks ATPase activity but retains the ability to bind ATP.