The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Abstract

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.

Surviving the Sun: Repair and bypass of DNA UV lesions

“…34 ATP binding and hydrolysis likely results in domain movement in UvrA and may be correlated with DNA binding. 26,64,65,68 One possibility is that in the absence of ATP UvrA unwinds and stretches the DNA as observed in the crystal structure [ Fig. 4(B)]; upon binding and hydrolysis of ATP UvrA may continue to untwist the DNA but compress rather than stretching it (Fig.…”

“…23,24 Molecular mechanisms for coping with UV lesions by different repair pathways and translesion DNA synthesis have been extensively studied. In the last few years the structures of UvrA, UvrB, XPC, and XPE, which carry out the first step of UV-lesion recognition in bacteria and eukaryotes, have been determined alone or complexed with DNA substrates, [25][26][27][28][29][30][31][32][33][34][35][36] as have yeast and human DNA pol g complexed with CPDs. 37,38 DNA base lesions, which include mismatched basepairs, modified bases due to oxidation, deamination, or alkylation, losses of bases (abasic sites) and large base adducts like cisplatin and polyaromatic hydrocarbons, exhibit a general feature of reduced base stacking and reduced DNA persistence length (see the review 39 and references therein).…”

Beijing Sublineages of Mycobacterium tuberculosis Differ in Pathogenicity in the Guinea Pig

“…Rv1009 (rpfB) is one of the most immunogenic resuscitation-promoting factors (40), and deletion of this gene has been associated with delayed reactivation from chronic tuberculosis in mouse (50). Rv1638 (uvrA) is part of the nucleotide excision repair system which counteracts the deleterious effects of DNA lesions (41) and is essential for Mycobacterium smegmatis to survive under conditions of hypoxia and low carbon source (13). Rv2416c (eis) encodes a secretory protein that enhances intracellular survival of M. tuberculosis in monocytes and contributes to its pathogenicity (53).…”

Important Role for Mycobacterium tuberculosis UvrD1 in Pathogenesis and Persistence apart from Its Function in Nucleotide Excision Repair

“…To assess the susceptibilities of the mutant strains to both UV irradiation and in vitro stress, cultures were compared with that of the wild-type parental strain as previously described (40), but the cultures also included menadione (250 M) in addition to sodium nitrite (2 mM), mitomycin C (0.1 g/ml), and t-butyl hydroperoxide (0.1 mM). The OD of all cultures was determined at the start of the experiment, and approximately equal levels of inoculum were used for all strains.…”

“…We targeted uvrD1 for deletion in M. tuberculosis and assessed its phenotype along with that of a uvrA mutant described elsewhere (40). While UvrA functions solely within the NER pathway, in E. coli UvrD also plays a role in the mismatch repair pathway (23), which is absent in M. tuberculosis (49), and in recombination repair at blocked replication forks (19,30).…”