Molecular basis for DarT ADP-ribosylation of a DNA base

Nature. 2021 Aug;596(7873):597-602. doi: 10.1038/s41586-021-03825-4. Epub 2021 Aug 18.


ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6-9. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP-Ribosylation*
  • Adenosine Diphosphate Ribose / metabolism
  • Antitoxins
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Bacterial Toxins
  • Base Sequence
  • Biocatalysis
  • DNA / chemistry*
  • DNA / genetics
  • DNA / metabolism*
  • DNA Adducts / chemistry
  • DNA Adducts / metabolism
  • DNA Damage
  • DNA Repair
  • DNA Transposable Elements / genetics
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism
  • Models, Molecular
  • Mycobacterium / enzymology
  • Mycobacterium / genetics
  • Nitrogen / chemistry
  • Nitrogen / metabolism
  • Poly(ADP-ribose) Polymerases / chemistry
  • Replication Origin / genetics
  • Substrate Specificity
  • Thermus / enzymology
  • Thymidine / chemistry
  • Thymidine / metabolism
  • Thymine / chemistry*
  • Thymine / metabolism*


  • Antitoxins
  • Bacterial Proteins
  • Bacterial Toxins
  • DNA Adducts
  • DNA Transposable Elements
  • DNA, Bacterial
  • Adenosine Diphosphate Ribose
  • DNA
  • Poly(ADP-ribose) Polymerases
  • Nitrogen
  • Thymine
  • Thymidine