TARG1 protects against toxic DNA ADP-ribosylation

Abstract

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.

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]…”

“…As the toxin of the system, DarT induces strong bacteriostatic effects and activates the SOS-response since the thymidine-linked ADP-ribosylation modifications are perceived as severe DNA damage requiring two consecutive DNA-repair pathways (HR and NER) to be resolved in a DarG-independent manner [ 75 , 76 ]. DarT expression was also shown to exert highly toxic effects in human cells which can however be protected by the endogenous human TARG1 enzymatic activity [ 78 ]. TARG1 displays close structural homology to DarG sharing the same catalytic lysine residue in the active site [ 52 ] and was found, like DarG, to be able to reverse thymidine-linked DNA ADP-ribosylation.…”

“…TARG1 displays close structural homology to DarG sharing the same catalytic lysine residue in the active site [ 52 ] and was found, like DarG, to be able to reverse thymidine-linked DNA ADP-ribosylation. Consequently, the expression of bacterial DarT toxin in TARG1-deficient human cells causes extreme replication stress impacting replication fork progression and activating the DNA-damage response at DNA replication sites [ 78 ]. For bacteria, DarG — that is functioning as the antitoxin — is an essential gene for survival and relevant for enabling smooth replication processes in DarT-expressing cells by providing DarT control and regulation via the hydrolytic activity of its macrodomain as well as physical sequestration through complex formation [ 75 , 76 ].…”

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.…”