Publications

• The Toxin-Antitoxin System DarTG Catalyzes Reversible ADP-Ribosylation of DNA.

  Jankevicius G., Ariza A., Ahel M., Ahel I.

  The discovery and study of toxin-antitoxin (TA) systems helps us advance our understanding of the strategies prokaryotes employ to regulate cellular processes related to the general stress response, such as defense against phages, growth control, biofilm formation, persistence, and programmed cell ... >> More

  The discovery and study of toxin-antitoxin (TA) systems helps us advance our understanding of the strategies prokaryotes employ to regulate cellular processes related to the general stress response, such as defense against phages, growth control, biofilm formation, persistence, and programmed cell death. Here we identify and characterize a TA system found in various bacteria, including the global pathogen Mycobacterium tuberculosis. The toxin of the system (DarT) is a domain of unknown function (DUF) 4433, and the antitoxin (DarG) a macrodomain protein. We demonstrate that DarT is an enzyme that specifically modifies thymidines on single-stranded DNA in a sequence-specific manner by a nucleotide-type modification called ADP-ribosylation. We also show that this modification can be removed by DarG. Our results provide an example of reversible DNA ADP-ribosylation, and we anticipate potential therapeutic benefits by targeting this enzyme-enzyme TA system in bacterial pathogens such as M. tuberculosis. << Less

  Mol. Cell 64:1109-1116(2016) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• DNA ADP-Ribosylation Stalls Replication and Is Reversed by RecF-Mediated Homologous Recombination and Nucleotide Excision Repair.

  Lawaree E., Jankevicius G., Cooper C., Ahel I., Uphoff S., Tang C.M.

  ADP-ribosylation of proteins is crucial for fundamental cellular processes. Despite increasing examples of DNA ADP-ribosylation, the impact of this modification on DNA metabolism and cell physiology is unknown. Here, we show that the DarTG toxin-antitoxin system from enteropathogenic Escherichia c ... >> More

  ADP-ribosylation of proteins is crucial for fundamental cellular processes. Despite increasing examples of DNA ADP-ribosylation, the impact of this modification on DNA metabolism and cell physiology is unknown. Here, we show that the DarTG toxin-antitoxin system from enteropathogenic Escherichia coli (EPEC) catalyzes reversible ADP-ribosylation of single-stranded DNA (ssDNA). The DarT toxin recognizes specific sequence motifs. EPEC DarG abrogates DarT toxicity by two distinct mechanisms: removal of DNA ADP-ribose (ADPr) groups and DarT sequestration. Furthermore, we investigate how cells recognize and deal with DNA ADP-ribosylation. We demonstrate that DNA ADPr stalls replication and is perceived as DNA damage. Removal of ADPr from DNA requires the sequential activity of two DNA repair pathways, with RecF-mediated homologous recombination likely to transfer ADP-ribosylation from single-to double-stranded DNA (dsDNA) and subsequent nucleotide excision repair eliminating the lesion. Our work demonstrates that these DNA repair pathways prevent the genotoxic effects of DNA ADP-ribosylation. << Less

  Cell Rep. 30:1373-1384(2020) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Molecular basis for DarT ADP-ribosylation of a DNA base.

  Schuller M., Butler R.E., Ariza A., Tromans-Coia C., Jankevicius G., Claridge T.D.W., Kendall S.L., Goh S., Stewart G.R., Ahel I.

  ADP-ribosyltransferases use NAD⁺ to catalyse substrate ADP-ribosylation¹, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria^2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modificat ... >> More

  ADP-ribosyltransferases use NAD⁺ to catalyse substrate ADP-ribosylation¹, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria^2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification⁵, but recent in vitro studies have suggested nucleic acids as targets^6-9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT-DarG toxin-antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)¹⁰. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP-HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT-DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication. << Less

  Nature 596:597-602(2021) [PubMed] [EuropePMC]