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.

In recent years, RNA has reemerged as a versatile biological macromolecule capable of performing an astonishing number of biochemical activities. Initially described as the ubiquitous but transient carrier of genetic information in the Central Dogma, RNA has surprised scientists with its capacity to store genetic information, catalyze biochemical reactions, protect telomeres, guide proteins to their targets, help DNA replication and protein synthesis, scaffold ribonucleoprotein complexes, and transmit developmental and epigenetic information through mitotic and even meiotic cell divisions. The latest surprise came during the past decade with advances in deep sequencing technologies, which uncovered the pervasive world of noncoding RNAs (ncRNAs). Functional analysis of ncRNAs has revealed their wide-spread use in several biological pathways including the ones in the nucleus. We now know that nuclear ncRNAs of various sizes facilitate genome stability by inhibiting spurious recombination among repetitive DNA elements, repressing mobilization of transposable elements (TEs), templating or bridging DNA double-strand breaks (DSBs) during repair, and directing developmentally-regulated genome rearrangements in some ciliates. In this paper, we will survey the known mechanisms with which nuclear ncRNAs directly contribute to the maintenance of genome stability and outline the major advances in our understanding of the role of ncRNAs in the nucleus. These studies reveal an unexpected range of mechanisms by which ncRNAs contribute to genome stability and even potentially influence evolution by acting as templates for genome modification.

Summary Quiescence (G0) is a ubiquitous stress response through which cells enter reversible dormancy, acquiring distinct properties including reduced metabolism, resistance to stress and long life. G0 entry involves dramatic changes to chromatin and transcription of cells, but the mechanisms coordinating these processes remain poorly understood. Using the fission yeast, here we track G0-associated chromatin and transcriptional changes temporally and show that as cells enter G0, their survival and global gene expression programs become increasingly dependent on Clr4/SUV39H, the sole histone H3 lysine 9 (H3K9) methyltransferase, and RNA interference (RNAi) proteins. Notably, G0 entry results in RNAi-dependent H3K9 methylation of several euchromatic pockets, prior to which Argonaute1-associated small RNAs from these regions emerge. Overall our data reveal a function for constitutive heterochromatin proteins (the establishment of the global G0 transcriptional program) and suggest that stress-induced alterations in Argonaute-associated sRNAs can target the deployment of transcriptional regulatory proteins to specific sequences.

DNA helicases are present in all kingdoms of life and play crucial roles in processes of DNA metabolism such as replication, repair, recombination, and transcription. To date, however, the role of DNA helicases during homologous recombination in mycobacteria remains unknown. In this study, we show that Mycobacterium tuberculosis UvrD1 more efficiently inhibited the strand exchange promoted by its cognate RecA, compared to noncognate Mycobacterium smegmatis or Escherichia coli RecA proteins. The M. tuberculosis UvrD1(Q276R) mutant lacking the helicase and ATPase activities was able to block strand exchange promoted by mycobacterial RecA proteins but not of E. coli RecA. We observed that M. tuberculosis UvrA by itself has no discernible effect on strand exchange promoted by E. coli RecA but impedes the reaction catalyzed by the mycobacterial RecA proteins. Our data also show that M. tuberculosis UvrA and UvrD1 can act together to inhibit strand exchange promoted by mycobacterial RecA proteins. Taken together, these findings raise the possibility that UvrD1 and UvrA might act together in vivo to counter the deleterious effects of RecA nucleoprotein filaments and/or facilitate the dissolution of recombination intermediates. Finally, we provide direct experimental evidence for a physical interaction between M. tuberculosis UvrD1 and RecA on one hand and RecA and UvrA on the other hand. These observations are consistent with a molecular mechanism, whereby M. tuberculosis UvrA and UvrD1, acting together, block DNA strand exchange promoted by cognate and noncognate RecA proteins.

A central step in the process of homologous genetic recombination is the strand exchange between two homologous DNA molecules, leading to the formation of the Holliday junction intermediate. Several lines of evidence, both in vitro and in vivo, suggest a concerted role for the Escherichia coli RuvABC protein complex in the process of branch migration and the resolution of the Holliday junctions. A number of investigations have examined the role of RuvA protein in branch migration of the Holliday junction in conjunction with its natural cellular partner, RuvB. However, it remains unclear whether the RuvABC protein complex or its individual subunits function differently in the context of DNA repair and homologous recombination. In this study, we have specifically investigated the function of RuvA protein using Holliday junctions containing either homologous or heterologous arms. Our data show that Mycobacterium tuberculosis ruvA complements E. coli DeltaruvA mutants for survival to genotoxic stress caused by different DNA-damaging agents, and the purified RuvA protein binds HJ in preference to any other substrates. Strikingly, our analysis revealed two distinct types of structural distortions caused by M. tuberculosis RuvA between the homologous and heterologous Holliday junctions. We interpret these data as evidence that local distortion of base pairing in the arms of homologous Holliday junctions by RuvA might augment branch migration catalyzed by RuvB. The biological significance of two modes of structural distortion caused by M. tuberculosis RuvA and the implications for its role in DNA repair and homologous recombination are discussed.

Background: Helicases are implicated in fork remodeling. Results: Mycobacterium tuberculosis RecG helicase/translocase remodels stalled replication forks. Conclusion: M. tuberculosis RecG but not RuvAB or RecA is efficient in fork reversal activity. Significance: This study identifies a fork remodeling enzyme and provides insights into fork restart mechanisms in M. tuberculosis.

Background: Impediments to replication fork progression are an important source of genome instability and cell death. Results: We reveal the functional characteristics of MtRuvAB complex. Conclusion: MtRuvAB-catalyzed RFR is independent of symmetry at the fork junction, supercoiling and unimpeded by heterology. Significance: An understanding of MtRuvAB complex functions, whose expression is up-regulated following infection, has implications for drug discovery and development.

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