Genome dynamics in Mycobacterium tuberculosis – new candidates in the hunt for novel drug targets

Mycobacterium tuberculosis (Mtb), the etiologic agent of the re-emerging disease tuberculosis, continues to cause mortality and morbidity worldwide claiming 1.5-2 million people’s lives annually. Mtb is an intracellular pathogen that survives and replicates inside macrophages. Intracellularly, Mtb is exposed to abundant oxidative and nitrosative stress, inducing macro-molecular damage. The ability to repair DNA damage caused by exposure to reactive oxygen and nitrogen intermediates produced by the phagocytic host cell is likely to play an important role in this species. Genome maintenance functions such as DNA repair, replication and recombination (3R) play important roles in the pathogenicity and survival of this pathogen. Hence, characterization of 3R components will enlighten Mtb genome dynamics and pathogenesis. Helicases, motor proteins that catalyze separation of double stranded helices into single strands, are involved in almost all aspects of nucleic acid transactions. In E. coli, Ercc3, DinG and RecG homologs have been found to unwind a variety of branched DNA substrates in vitro, including holliday junction, D-loops and R-loops. We have cloned, overexpressed and purified the Mtb Ercc3, DinG and RecG helicases. The purified proteins were assessed with regard to their ability to bind and unwind a range of DNA substrates mimicking replication and recombination intermediates and for ATPase activity. Mtb Ercc3, DinG and RecG were found to exhibit ATP dependent helicase activities with various substrate specificities, were able to unwind full and partial replication fork substrates and converted holliday junctions to flayed duplexes. This is the first characterization of Mtb helicase homologs. The results indicate that the Mtb Ercc3, DinG and RecG are active helicases and ATPases that most probably serves as general guardians of the Mtb genome. On-going structure-function analysis at the atomic level and screening for inhibitory components indicate that in addition to their roles in mycobacterial pathogenicity and survival in the host, helicases represent attractive potential drug targets.