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ADP-ribosylation--the transfer of ADP-ribose (ADPr) from NAD+ onto target molecules--is catalyzed by members of the ADP-ribosyltransferase (ART) superfamily of proteins, found in all kingdoms of life. Modification of amino acids in protein targets by ADPr regulates critical cellular pathways in eukaryotes and underlies the pathogenicity of certain bacteria. Several members of the ART superfamily are highly relevant for disease; these include the poly(ADP-ribose) polymerases (PARPs), recently shown to be important cancer targets, and the bacterial toxins diphtheria toxin and cholera toxin, long known to be responsible for the symptoms of diphtheria and cholera that result in morbidity. In this Review, we discuss the functions of amino acid ADPr modifications and the ART proteins that make them, the nature of the chemical linkage between ADPr and its targets and how this impacts function and stability, and the way that ARTs select specific amino acids in targets to modify.

The poly(ADP-ribose) polymerase (PARP) family comprises 17 enzymes, which generate poly(ADP-ribose) and/or mono(ADP-ribose) (MAR) that can modify target protein function and can function as a signalling scaffold. These modifications may have various roles in cancer and, as discussed in this Opinion article, inhibitors of MARylation in particular may warrant investigation as anticancer drugs. Poly(ADP-ribose) polymerases (PARPs) modify target proteins post-translationally with poly(ADP-ribose) (PAR) or mono(ADP-ribose) (MAR) using NAD + as substrate. The best-studied PARPs generate PAR modifications and include PARP1 and the tankyrase PARP5A, both of which are targets for cancer therapy with inhibitors in either clinical trials or preclinical development. There are 15 additional PARPs, most of which modify proteins with MAR, and their biology is less well understood. Recent data identify potentially cancer-relevant functions for these PARPs, which indicates that we need to understand more about these PARPs to effectively target them.

Taking PAR apart Proteins can be reversibly modified through the addition of repeating, polymerized ADP-ribose (PAR) subunits catalysed by poly(ADP-ribose) polymerase (PARP). Removal of PAR requires a glycohydrolase (PARG), which cleaves the ribose–ribose bond between subunits. Ivan Ahel and colleagues report that bacteria and fungi have a divergent PARG, which is unrelated to other enzymes that cleave PAR. Its structure, in complex with ADP-ribose and with a PARG inhibitor, and the results of mutational analysis suggest that the mechanism used in mammals and bacteria may be conserved. PARP inhibitors are being developed as pharmaceuticals for diseases including cancer, and this work suggests that small, cell-permeable PARG inhibitors might also be possible drug candidates. Post-translational modification of proteins by poly(ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis 1 . Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose–ribose bond, and is synthesized from NAD by PAR polymerases 1 , 2 . PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose–ribose bonds present in PAR chains; its deficiency leads to cell death 3 , 4 . Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata ), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family 5 , 6 . High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl)ation and potentially of how the defects in this regulation are linked to human disease.

Structural and biochemical approaches are used to show how RNF146 activity is allosterically regulated by the binding of poly(ADP-ribose) ligand, and how substrate specificity is achieved with protein poly(ADP-ribosyl)ation and ubiquitination occurring in the same protein complex. PARylation-dependent ubiquitination mechanism PARylation is a post-translational modification in which ADP-ribose polymers are covalently attached to protein targets. One of its many cellular functions is to control the ubiquitination and degradation of cell regulators such as Axin and PTEN. Wenqing Xu and colleagues use structural and biochemical approaches to show how the activity of RNF146, an E3 ligase responsible for PARylation-dependent ubiquitination, is regulated by the binding of PAR ligand and how substrate specificity is achieved with PARylation and ubiquitination occurring in the same protein complex. RNF146 represents a new class of RING E3 ligases, the activity of which can be regulated by ligand binding. Protein poly(ADP-ribosyl)ation (PARylation) has a role in diverse cellular processes such as DNA repair, transcription, Wnt signalling, and cell death 1 , 2 , 3 , 4 , 5 , 6 . Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several crucial regulatory proteins, including Axin and 3BP2 (refs 7 , 8 , 9 ). The RING-type E3 ubiquitin ligase RNF146 (also known as Iduna) is responsible for PARylation-dependent ubiquitination (PARdU) 10 , 11 , 12 . Here we provide a structural basis for RNF146-catalysed PARdU and how PARdU specificity is achieved. First, we show that iso -ADP-ribose ( iso -ADPr), the smallest internal poly(ADP-ribose) (PAR) structural unit, binds between the WWE and RING domains of RNF146 and functions as an allosteric signal that switches the RING domain from a catalytically inactive state to an active one. In the absence of PAR, the RING domain is unable to bind and activate a ubiquitin-conjugating enzyme (E2) efficiently. Binding of PAR or iso -ADPr induces a major conformational change that creates a functional RING structure. Thus, RNF146 represents a new mechanistic class of RING E3 ligases, the activities of which are regulated by non-covalent ligand binding, and that may provide a template for designing inducible protein-degradation systems. Second, we find that RNF146 directly interacts with the PAR polymerase tankyrase (TNKS). Disruption of the RNF146–TNKS interaction inhibits turnover of the substrate Axin in cells. Thus, both substrate PARylation and PARdU are catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate–TNKS interaction. We propose that the maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146–TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell.

A proteomic method to identify human proteins post-translationally modified by poly(ADP-ribosyl)ation is reported, which will help yield further insights into the biological role of this modification. Poly(ADP-ribosyl)ation is catalyzed by a family of enzymes known as PARPs. We describe a method to characterize the human aspartic acid– and glutamic acid–ADP-ribosylated proteome. We identified 1,048 ADP-ribosylation sites on 340 proteins involved in a wide array of nuclear functions; among these were many previously unknown PARP downstream targets whose ADP-ribosylation was sensitive to PARP inhibitor treatment. We also confirmed that iniparib had a negligible effect on PARP activity in intact cells.

Poly(ADP-ribose) (PAR) is a homopolymer of adenosine diphosphate ribose that is added to proteins as a posttranslational modification to regulate numerous cellular processes. PAR also serves as a scaffold for protein binding in macromolecular complexes, including biomolecular condensates. It remains unclear how PAR achieves specific molecular recognition. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) to evaluate PAR flexibility under different cation conditions. We demonstrate that, compared to RNA and DNA, PAR has a longer persistence length and undergoes a sharper transition from extended to compact states in physiologically relevant concentrations of various cations (Na⁺, Mg2+, Ca2+, and spermine4+). We show that the degree of PAR compaction depends on the concentration and valency of cations. Furthermore, the intrinsically disordered protein FUS also served as a macromolecular cation to compact PAR. Taken together, our study reveals the inherent stiffness of PAR molecules, which undergo switch-like compaction in response to cation binding. This study indicates that a cationic environment may drive recognition specificity of PAR.

Selective inhibition of poly(ADP-ribose) polymerase 1 (PARP1) may reduce the hematologic toxicity associated with dual PARP1/PARP2 inhibition. We performed molecular dynamics simulations for five selective inhibitors in complexes with PARP1 and PARP2, using three independent 50 ns runs per complex after docking and equilibration, followed by protein–ligand interaction fingerprint and statistical analyses. All complexes remained dynamically stable, with ligand root-mean-square deviation values generally within 0.3 nm. Comparative analysis identified three αF-helix residue pairs with nominally reduced interaction frequencies in PARP2: Asn767/Ala336, Leu769/Gly338, and Asp770/Asp339 (p < 0.05). After Benjamini–Hochberg correction for multiple comparisons, Leu769/Gly338 remained significant (q < 0.05), indicating that this pair represents the most statistically robust interaction difference within this region. Using palacaparib as the most selective inhibitor, these differences were associated with weakened or lost hydrophobic, van der Waals, and cation–π interactions in PARP2. Selective binding of modern PARP1 inhibitors appears to be associated with αF-helix-dependent interaction patterns, providing a mechanistic basis for the rational design of next-generation selective inhibitors with improved selectivity and potentially reduced toxicity.

The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells 1 , 2 . After binding to genomic lesions, these enzymes use NAD + to modify numerous proteins with mono- and poly(ADP-ribose) signals that are important for the subsequent decompaction of chromatin and the recruitment of repair factors 3 , 4 . These post-translational modifications are predominantly serine-linked and require the accessory factor HPF1, which is specific for the DNA damage response and switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine residues 5 – 10 . Here we report a co-structure of HPF1 bound to the catalytic domain of PARP2 that, in combination with NMR and biochemical data, reveals a composite active site formed by residues from HPF1 and PARP1 or PARP2 . The assembly of this catalytic centre is essential for the addition of ADP-ribose moieties after DNA damage in human cells. In response to DNA damage and occupancy of the NAD + -binding site, the interaction of HPF1 with PARP1 or PARP2 is enhanced by allosteric networks that operate within the PARP proteins, providing an additional level of regulation in the induction of the DNA damage response. As HPF1 forms a joint active site with PARP1 or PARP2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors. Assembly of a catalytic centre formed by HPF1 bound to PARP1 or PARP2 is essential for protein ADP-ribosylation after DNA damage in human cells.

Key Points Poly(ADP-ribose) (PAR) is synthesized from NAD + by PAR polymerases (PARPs) and regulates many physiological processes such as the maintenance of DNA integrity, gene expression and cell division. PARPs form a superfamily of 17 members in humans, and display diverse subcellular distributions and functions. Some members might function together and possess overlapping properties. PAR that is synthesized in response to DNA-strand breaks is a DNA-damage signalling molecule that allows a rapid and efficient cellular evaluation of the damage range. It is also an essential recruiting molecule that, in a few seconds, concentrates key factors of the single-strand break repair pathway at the site of the lesion. The poly(ADP-ribosyl)ation of histones that are associated with open chromatin conformation at the DNA-damage site provided the first clue to the roles of PAR as an epigenetic modification. Recent evidence revealed an important role of PAR in the epigenetic regulation of chromatin structure and in gene expression under physiological conditions in which the integrity of the DNA is not affected. The dogma that the DNA-damage-dependent PARP-1 is activated by DNA-strand breaks has to be reconsidered now due to recent studies that showed the activation of PARP-1 in the absence of DNA interruptions. Elucidating the triggers is currently one of our most exciting challenges. PARP-1 and PAR play key roles in various acute and chronic inflammatory disorders as well as in a number of degenerative diseases by contributing to the caspase-independent, apoptosis-inducing factor (AIF)-dependent cell death. PARP inhibition confers protection to these pathologies. PARP inhibitors have promising pharmacological applications in potentializing the effect of antitumour drugs in cancer therapy as well as in the treatment of inflammatory, neurological and cardiac disorders. Emerging evidence indicates a possible functional interplay between the PAR metabolic pathway and the SIRT1-mediated deacetylation pathway in the regulation of chromatin structure and function that is associated with broad biological activities. The transfer of poly(ADP-ribose) (PAR) to proteins is mediated by the growing family of PAR polymerases. This post-translational modification regulates many important cellular processes, including maintenance of genome integrity, gene expression and cell division, and is emerging as an important epigenetic mark. The addition to proteins of the negatively charged polymer of ADP-ribose (PAR), which is synthesized by PAR polymerases (PARPs) from NAD + , is a unique post-translational modification. It regulates not only cell survival and cell-death programmes, but also an increasing number of other biological functions with which novel members of the PARP family have been associated. These functions include transcriptional regulation, telomere cohesion and mitotic spindle formation during cell division, intracellular trafficking and energy metabolism.

Poly-ADP-ribosylation is a post-translational modification catalyzed by enzymes such as PARP1, which responds to metabolic and genotoxic stress. Now macrodomain-containing proteins are shown to rapidly move to PARP1 activation sites, and recruitment of the macrodomain-containing histone macroH2A1.1 results in local chromatin changes. Poly-ADP-ribosylation is a post-translational modification catalyzed by PARP enzymes with roles in transcription and chromatin biology. Here we show that distinct macrodomains, including those of histone macroH2A1.1, are recruited to sites of PARP1 activation induced by laser-generated DNA damage. Chemical PARP1 inhibitors, PARP1 knockdown and mutation of ADP-ribose–binding residues in macroH2A1.1 abrogate macrodomain recruitment. Notably, histone macroH2A1.1 senses PARP1 activation, transiently compacts chromatin, reduces the recruitment of DNA damage factor Ku70–Ku80 and alters γ-H2AX patterns, whereas the splice variant macroH2A1.2, which is deficient in poly-ADP-ribose binding, does not mediate chromatin rearrangements upon PARP1 activation. The structure of the macroH2A1.1 macrodomain in complex with ADP-ribose establishes a poly-ADP-ribose cap-binding function and reveals conformational changes in the macrodomain upon ligand binding. We thus identify macrodomains as modules that directly sense PARP activation in vivo and establish macroH2A histones as dynamic regulators of chromatin plasticity.