Mycobacterium tuberculosis UvrD1 and UvrD2 helicases unwind G-quadruplex DNA.

Unresolved G-quadruplex (G4) DNA secondary structures impede DNA replication and can lead to DNA breaks and to genome instability. Helicases are known to unwind G4 structures and thereby facilitate genome duplication. Escherichia coli UvrD is a multifunctional helicase that participates in DNA repair, recombination and replication. Previously, we had demonstrated a novel role of E. coli UvrD helicase in resolving G4 structures. Mycobacterium tuberculosis genome encodes two orthologs of E. coli UvrD helicase, UvrD1 and UvrD2. It is unclear whether UvrD1 or UvrD2 or both helicases unwind G4 DNA structures. Here, we demonstrate that M. tuberculosis UvrD1 and UvrD2 unwind G4 tetraplexes. Both helicases were proficient in resolving previously characterized tetramolecular G4 structures in an ATP hydrolysis and single-stranded 3'-tail-dependent manner. Notably, M. tuberculosis UvrD1 and UvrD2 were efficient in unwinding G4 structures derived from the potential G4 forming sequences present in the M. tuberculosis genome. These data suggest an extended role for M. tuberculosis UvrD1 and UvrD2 helicases in resolving G4 DNA structures and provide insights into the maintenance of genome integrity via G4 DNA resolution.

Mycobacterium tuberculosis DinG is a structure-specific helicase that unwinds G4 DNA: implications for targeting G4 DNA as a novel therapeutic approach.

The significance of G-quadruplexes and the helicases that resolve G4 structures in prokaryotes is poorly understood. The Mycobacterium tuberculosis genome is GC-rich and contains >10,000 sequences that have the potential to form G4 structures. In Escherichia coli, RecQ helicase unwinds G4 structures. However, RecQ is absent in M. tuberculosis, and the helicase that participates in G4 resolution in M. tuberculosis is obscure. Here, we show that M. tuberculosis DinG (MtDinG) exhibits high affinity for ssDNA and ssDNA translocation with a 5' → 3' polarity. Interestingly, MtDinG unwinds overhangs, flap structures, and forked duplexes but fails to unwind linear duplex DNA. Our data with DNase I footprinting provide mechanistic insights and suggest that MtDinG is a 5' → 3' polarity helicase. Notably, in contrast to E. coli DinG, MtDinG catalyzes unwinding of replication fork and Holliday junction structures. Strikingly, we find that MtDinG resolves intermolecular G4 structures. These data suggest that MtDinG is a multifunctional structure-specific helicase that unwinds model structures of DNA replication, repair, and recombination as well as G4 structures. We finally demonstrate that promoter sequences of M. tuberculosis PE_PGRS2, mce1R, and moeB1 genes contain G4 structures, implying that G4 structures may regulate gene expression in M. tuberculosis. We discuss these data and implicate targeting G4 structures and DinG helicase in M. tuberculosis could be a novel therapeutic strategy for culminating the infection with this pathogen.