Abstract:
R-loops, three-stranded nucleic structures formed when RNA anneals to the DNA template strand forming an RNA/DNA hybrid and displaced non-template ssDNA, can lead to double strand breaks and genomic instability, and provide roadblocks to both DNA and RNA polymerases. R-loop accumulation can affect genomic stability even in non-replicating bacterial cells. This genotoxic stress can be prevented by the degradation of the RNA in the RNA/DNA hybrid by RNase HI. In M. tuberculosis, RNase HI activity is essential for the growth of the bacterium in vitro, which makes it a potential drug target, but not much is known about the cellular consequences of RNase HI inhibition in the mycobacteria. This study reports the phenotypic effect of depletion of RNase HI activity in M. smegmatis as a model for chemical inhibition of M. tuberculosis RNase HI. Depletion of RNase HI impaired the removal of R-loops in M. smegmatis, and induced markers of the SOS response and DNA topology modification. RNase HI depletion was either additive or synergistic with antibiotics currently used for anti-tubercular therapy. Inhibitors of HIV RNase HI were found to inhibit recombinant M. tuberculosis RNase HI and possessed whole-cell activity on both M. marinum and M. tuberculosis.
Structural analysis of M. tuberculosis RNase HI using small-angle X-ray scattering showed that this two domain protein exists in multiple conformations in solution, all of them compact. The C-terminal CobC domain increased the binding affinity of the RNase HI domain for RNA/DNA hybrid providing a rationale for the domain combination, and an explanation for the positive contribution of CobC towards the RNase HI catalytic activity of the full-length protein.
All of this offers further insight about the role of mycobacterial RNase HI in resolving R-loops in the genome, the evolutionary advantages of the fusion between RNase HI and CobC, and suggests that RNase HI is a vulnerable target in the mycobacteria that is suitable for drug development.