Molecular basis for DarT ADP-ribosylation of a DNA base

Abstract

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.

Beyond protein modification: the rise of non-canonical ADP-ribosylation

“…The heavy focus on ADP-ribosylation as protein modification may have just overshadowed the richness of ADP-ribosylation as general ( protein-unrelated) signalling event. Its study will not only uncover exciting facets of life and evolution but also comes with great potential for the development of new antimicrobials and anticancer agents as well as biotechnological tools [73][74][75]77,78]…”

“…2.9 revealed that DarT links ADP-ribose with the anomeric carbon of the adenine-distal ribose to the in-ring nitrogen of the thymidine base. The reaction requires an additional catalytic arginine residue in the active site that is assumed to be particularly essential for proton abstraction from the thymine nitrogen and that extends the canonical set of so far known ART catalytic residues [ 77 ]. It is indicative for the evolving diversity of ARTs with their ability to adapt to versatile specialised functions unrelated to the classical protein-targeting activity.…”

“…It is indicative for the evolving diversity of ARTs with their ability to adapt to versatile specialised functions unrelated to the classical protein-targeting activity. The DarT-catalysed reaction can be reversed by the macrodomain-containing hydrolase DarG (DNA ADP-ribosylglycohydrolase) acting, compared with DarT, in a motif sequence-independent manner, thus is also able to reverse DarT reactions from non-cognate species [ 75 , 77 ]. By being downstream-coded in the same operon, DarG is genetically linked to DarT and together, DarT and DarG (DarTG) form a toxin-antitoxin (TA) pair.…”

Assay technologies facilitating drug discovery for ADP‐ribosyl writers, readers and erasers

“…[14] Initially, PARP activity was measured through incorporation of a radioactive ATP into an acid-insoluble material, although the nature of this product of the DNA-dependent enzyme PARP1 was not known at this stage. [3] Later, an assay method to detect PARP protein and PARP activity directly from nitrocellulose blots using radiolabeled substrate [ 32 P]NAD + was developed by Simonin et al [15] The low-throughput Western blot or dot blot methods have been used with other detection methods including biotinylated NAD + analogs [16] and streptavidin coupled HRP, PAR H10, or MAR/PAR antibodies [17][18][19] and various ADP-ribosylation affinity reagents developed recently. [20][21][22][23] It is noteworthy that when using purified proteins, it is possible to see the disappearance of a protein band upon addition of NAD + with a simultaneous appearance of a high molecular weight smear on a coomassie stained SDS-PAGE.…”

“…This modification is readily detectable on a gel when small oligonucleotides are used as targets. [18,19,25,26] Interpretation of the modification by mass spectrometry (MS) proves difficult due to the lack of sequence specificity and presence of multiple modification sites per target protein. Toxins however are sequence specific and modify typically only certain residues in the target proteins and therefore MS spectra is easy to interpret.…”