4.8 Article

Molecular basis for DarT ADP-ribosylation of a DNA base

Journal

NATURE
Volume 596, Issue 7873, Pages 597-+

Publisher

NATURE PORTFOLIO
DOI: 10.1038/s41586-021-03825-4

Keywords

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Funding

  1. Wellcome Trust [101794, 210634]
  2. Biotechnology and Biological Sciences Research Council [BB/R007195/1, BB/R006393/1, BB/N004590/1]
  3. Cancer Research United Kingdom [C35050/A22284]

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ADP-ribosyltransferases use NAD(+) to catalyze substrate ADP-ribosylation and regulate cellular pathways or contribute to bacterial pathogenicity. Recent studies have suggested nucleic acids as targets of reversible ADP-ribosylation. This research provides evidence of specific ADP-ribosylation of DNA in cells through the DarT-DarG system in bacteria, including global pathogens. The study reveals the structure and mechanism of the DarT enzyme, showing its evolution into specific DNA-modifying enzymes and its role in regulating growth through DNA ADP-ribosylation.
ADP-ribosyltransferases use NAD(+) to catalyse substrate ADP-ribosylation(1), 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 modification(5), 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.

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