ADP-ribosyltransferases (ARTs) utilise NAD + to catalyse substrate ADP-ribosylation 1 , thereby regulating cellular pathways or contributing to toxin-mediated pathogenicity of bacteria [2][3][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][7][8][9] . Here, we present evidence that specific reversible DNA ADP-ribosylation 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, EPEC and Pseudomonas aeruginosa 10 . We report the first DarT structure which identifies this protein as a diverged member of the PARP family. Moreover, a set of high-resolution structures in ligand-free, pre-and post-reaction states reveals a specialised mechanism of catalysis that includes a key active-site arginine, extending the canonical ART toolkit. Comparison with the well-established DNA-repair protein ADP-ribosylation complex, PARP/HPF1, offers insights into how the DarT class of ARTs evolved into specific DNA-modifying enzymes. Together, the structural and mechanistic data provide unprecedented detail for a PARP family member and contribute to fundamental understanding of nucleic acid ADP-ribosylation. We furthermore show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin, functioning as non-canonical DNA-repair factor, are utilised 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 DarTG regulates growth by DNA ADP-ribosylation at the origin of chromosome replication.

ADP-ribosylation is a modification that targets a variety of macromolecules and regulates a diverse array of important cellular processes. ADP-ribosylation is catalysed by ADP-ribosyltransferases and reversed by ADP-ribosylhydrolases. Recently, an ADP-ribosyltransferase toxin termed ‘DarT’ from bacteria, which is distantly related to human PARPs, was shown to modify thymidine in single-stranded DNA in a sequence specific manner. The antitoxin of DarT is the macrodomain containing ADP-ribosylhydrolase DarG, which shares striking structural homology with the human ADP-ribosylhydrolase TARG1. Here, we show that TARG1, like DarG, can reverse thymidine-linked DNA ADP-ribosylation. We find that TARG1-deficient human cells are extremely sensitive to DNA ADP-ribosylation. Furthermore, we also demonstrate the first detection of reversible ADP-ribosylation on genomic DNA in vivo from human cells. Collectively, our results elucidate the impact of DNA ADP-ribosylation in human cells and provides a molecular toolkit for future studies into this largely unknown facet of ADP-ribosylation.

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Quantitative microscopy is a powerful method for performing phenotypic screens, from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments. Here, we examine the performance of the Cell Painting assay across multiple high-throughput microscope systems and find that all are compatible with this assay. Furthermore, we determine independently for each microscope system the best performing settings, providing those who wish to adopt this assay an ideal starting point for their own assays. We also explore the impact of microscopy setting changes in the Cell Painting assay and find that few dramatically reduce the quality of a Cell Painting profile, regardless of the microscope used.