Poly(ADP-ribose) or PAR regulates multiple aspects of cell biology, both as an independent signaling molecule and as a modification on biomolecules. As a posttranslational modification, PAR can modulate the biochemical properties of target proteins. Isolated free PAR molecules function in cellular signaling. This chapter describes two methods to isolate and purify free PAR and protein-linked PAR from biochemical reactions, one using chemical fractionation and another using physical separation. A method to isolate free PAR and protein-linked PAR from human cells is also presented. These methods allow monitoring of free PAR and protein-linked PAR levels under different biochemical conditions or in response to different cellular stimuli.

Adenosine diphosphate (ADP) ribosylation (ADPr) regulates multiple stress responses, yet substrates in the apoptotic machinery remain elusive. We show that a single, DNA damage-induced ADPr event controls proapoptotic PIDDosome (PIDD1/RAIDD/caspase-2) formation in response to unresolved interstrand DNA cross-links (ICL). ADPr targets conserved E783 in the PIDD1 death domain (DD); is catalyzed by poly(ADP-ribose) polymerase 4 (PARP4), a phylogenetically orphan PARP of previously unknown function; is reversed by the ribosylhydrolase activity of PARP14; and is triggered by Ataxia Telangiectasia and RAD3-related (ATR) phosphorylation-induced, PIAS1-mediated SUMOylation of the PIDD1 DD, which enables PARP4 docking. PIDD1 ADPr is dispensable for the recruitments of RAIDD and caspase-2 but essential for the dimerization of the caspase. Hence, denying E783 ADPr spares the onset of PIDDosome assembly but blocks its completion, thus eliminating caspase-2 activation and ensuing apoptosis. Conversely, removal of PARP14 forces apoptosis, even in cells with tolerable damage. The data identify PARP4 as an ICL response effector and illuminate a three-step modification sequence of the PIDD1 DD that conducts PIDDosome assembly from initiation to completion.

PARP1 detection of DNA strand breaks allosterically leads to PARP1 synthesis of poly(ADP-ribose) modifications that signal DNA damage. HPF1 engages activated PARP1 to control modification site selection. Understanding of the mechanism of DNA break detection and catalytic activation is incomplete, due largely to limited structural information for full-length PARP1. Here, single-particle cryo-EM provides views of the full complement of PARP1 domains engaging a DNA single-strand break in the presence of HPF1 and a fragment of binding partner Timeless. Cryo-EM, single-molecule DNA dynamics, and small-angle X-ray scattering analysis indicate that PARP1 remains dynamic even when the multi-domain structure is organized on a DNA break, with the minimal catalytic region displaying high mobility relative to domains engaging damage. We propose that the organization of PARP1 domains on a DNA break releases a tethered, constitutively active catalytic region to modify molecules in a radius surrounding the DNA break site.

RNA editing in mitochondria is vital for many eukaryotes. In plants, mitochondrial C-to-U deamination editing is catalyzed by tens to hundreds of PPR-DYW proteins, each typically dedicated to a specific site. Where retained, PPR-DYW family expanded independently in all eukaryotic groups examined here -- except in marine microeukaryotes diplonemids, which encode a single homolog despite deaminating their mitochondrial RNA at more than 100 sites. Here we characterize this unconventional deaminase, PPRD1, from Diplonema papillatum. Native affinity pulldown of the protein identified 20 predominantly sub-stoichiometric partners, including potential RNA-binding helical-repeat proteins. Among them, the divergent PolX-like protein DAPX1 consistently and reciprocally co-purified with PPRD1 in near-equal proportions. Structural modelling suggests that DAPX1 may stabilize the deaminase catalytic domain and expand its interaction interface. Silencing either PPRD1 or DAPX1 inhibited cell growth and reduced in vivo not only C-to-U, but also A-to-I deamination across five mitochondrial RNA-editing clusters encompassing 110 sites. Together, these results support a model of PPRD1 and DAPX1 forming the core of the Diplonema deamination-editing machinery, with sub-stoichiometric partners acting as specificity factors that guide accurate RNA editing with minimal off-target effects.

Transcription-coupled nucleotide excision repair (TC-NER or TCR) is initiated when the ATPase Cockayne syndrome protein B (CSB) recognizes a DNA lesion stalled RNA polymerase II (RNAPII) and forms a stable complex. Here, we report that poly(ADP-ribose) polymerase-1 (PARP1), that plays a key role in the lesion recognition step of global genomic NER, also facilitates the earliest step of TCR. PARP1, which is associated with RNAPII during normal transcription, interacts with and stabilizes CSB on the lesion-stalled RNAPII. CSB stimulates PARP1's activity to form PAR, and in turn CSB is PARylated mainly at its N-terminal PAR-binding motif (PBM) to promote its stabilization with RNAPII, whereas its minor PARylation at the C-terminal domain suppresses its ATPase function, thus limiting the window of time for ATP-dependent lesion recognition by CSB. The loss of PARP1, treatment with inhibitors of PARP or poly(ADP-ribose) glycohydrolase (PARG) to prevent PAR synthesis or its catabolism to generate free PAR or engineering N-terminal PARylation-resistant CSB decrease the efficiency of cells for TCR. PARP1 mutant Caenorhabditis elegans larvae exhibit a pronounced TCR-deficient phenotype. Our findings uncover an evolutionarily conserved role of PARP1 and PAR metabolism in the initiation of TCR.

PARP4 is an ADP-ribosyltransferase typically associated with the cytoplasmic vault organelle. PARP4 has a distinct domain composition relative to other PARP enzymes; however, the N-terminal region of PARP4 is homologous to a collection of domains found in PARP1, a regulator of multiple nuclear processes including the cellular response to DNA damage. The N-terminal region of PARP4 interacts in vitro with nucleic acid, in particular a noncoding RNA associated with vault particles, and a BRCT domain is implicated in this interaction. Here, we report the X-ray structure of the BRCT domain of PARP4 and structure-based mutagenesis that interrogates the nucleic acid-binding activity using vault RNA. The isolated BRCT domain is capable of mediating interaction with vault RNA, and we identified four BRCT mutants that disrupt vault RNA interaction to varying degrees. X-ray structures of the BRCT mutants indicate that perturbations to an electropositive region of the BRCT surface underlie the loss of nucleic acid binding. Comparison to other nucleic acid-binding BRCT domains highlights distinct features of the PARP4 BRCT structure. The study presents the experimental structure of the PARP4 BRCT domain, establishes this domain as nucleic acid-binding module, and provides PARP4 BRCT mutants that can be used to investigate PARP4 cellular functions.

Non-covalent interactions of poly(ADP-ribose) (PAR) facilitate condensate formation, yet the impact of these interactions on condensate properties remains unclear. Here, we demonstrate that PAR-mediated interactions through PARP13, specifically the PARP13.2 isoform, are essential for modulating the dynamics of stress granules—a class of cytoplasmic condensates that form upon stress, including types frequently observed in cancers. Single amino acid mutations in PARP13, which reduce its PAR-binding activity, lead to the formation of smaller yet more numerous stress granules than observed in the wild-type. This fragmented stress granule phenotype is also apparent in PARP13 variants with cancer-associated single-nucleotide polymorphisms (SNPs) that disrupt PAR binding. Notably, this fragmented phenotype is conserved across a variety of stresses that trigger stress granule formation via diverse pathways. Furthermore, this PAR-binding mutant diminishes condensate dynamics and impedes fusion. Overall, our study uncovers the important role of PAR-protein interactions in stress granule dynamics and maturation, mediated through PARP13. Stress granules, cellular structures essential for stress response, require poly(ADP-ribose) as a multivalent scaffold. Here, the authors show that disrupting poly(ADP-ribose) binding to PARP13 alters granule size, dynamics, and maturation, despite PARP13 lacking ADPribosyltransferase activity.

Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 recognize DNA breaks immediately upon their formation, generate a burst of local PARylation to signal their location, and are co-targeted by all current FDA-approved forms of PARP inhibitors (PARPi) used in the cancer clinic. Recent evidence indicates that the same PARPi molecules impact PARP2 differently from PARP1, raising the possibility that allosteric activation may also differ. We find that, unlike for PARP1, destabilization of the autoinhibitory domain of PARP2 is insufficient for DNA damage-induced catalytic activation. Rather, PARP2 activation requires further unfolding of an active site helix. In contrast, the corresponding helix in PARP1 only transiently forms, even prior to engaging DNA. Only one clinical PARPi, Olaparib, stabilizes the PARP2 active site helix, representing a structural feature with the potential to discriminate small molecule inhibitors. Collectively, our findings reveal unanticipated differences in local structure and changes in activation-coupled backbone dynamics between human PARP1 and PARP2.

The increase of antimicrobial resistance constitutes a significant threat to human health. One of the mechanisms responsible for the spread of resistance to antimicrobials is the transfer of plasmids between bacteria by conjugation. This process is mediated by type IV secretion systems (T4SS) and previous studies have provided in vivo evidence for interactions between DNA and components of the T4SS. Here, we purified TraD and TraE, two inner membrane proteins from the Escherichia coli pKM101 T4SS. Using electrophoretic mobility shift assays and fluorescence polarization we showed that the purified proteins both bind single-stranded and double-stranded DNA in the nanomolar affinity range. The previously identified conjugation inhibitor BAR-072 inhibits TraE DNA binding in vitro, providing evidence for its mechanism of action. Site-directed mutagenesis identified conserved amino acids that are required for conjugation that may be targets for the development of more potent conjugation inhibitors.

Poly(ADP-ribose) or PAR is a versatile signaling molecule with a broad impact on human biology. PAR is a prominent indicator of cellular DNA damage and genomic transactions such as replication and transcription. Canonically, human PARP enzymes create PAR as a modification on proteins. Recently, PARP enzymes were found to create free PAR molecules that are not attached to protein. Free PAR has been implicated in cell death signaling, but the production of free PAR was assumed to be generated by glycohydrolases breaking down protein-linked PAR into smaller fragments. The direct de novo production of free PAR by PARP1 occurs alongside the synthesis of protein-linked PAR in response to DNA damage, suggesting a more prevalent role for free PAR in DNA damage signaling. This review outlines the discovery of free PAR synthesis in biochemical reactions and in cellular models of the DNA damage response. The implications for this finding are summarized in the context of DNA damage signaling and associated processes of biomolecular condensate formation and Parthanatos cell death signaling.

Cellular target engagement technologies enable quantification of intracellular drug binding; however, simultaneous assessment of drug-associated phenotypes has proven challenging. Here, we present cellular target engagement by accumulation of mutant as a platform that can concomitantly evaluate drug-target interactions and phenotypic responses using conditionally stabilized drug biosensors. We observe that drug-responsive proteotypes are prevalent among reported mutants of known drug targets. Compatible mutants appear to follow structural and biophysical logic that permits intra-protein and paralogous expansion of the biosensor pool. We then apply our method to uncouple target engagement from divergent cellular activities of MutT homolog 1 (MTH1) inhibitors, dissect Nudix hydrolase 15 (NUDT15)-associated thiopurine metabolism with the R139C pharmacogenetic variant, and profile the dynamics of poly(ADP-ribose) polymerase 1/2 (PARP1/2) binding and DNA trapping by PARP inhibitors (PARPi). Further, PARP1-derived biosensors facilitated high-throughput screening for PARP1 binders, as well as multimodal ex vivo analysis and non-invasive tracking of PARPi binding in live animals. This approach can facilitate holistic assessment of drug-target engagement by bridging drug binding events and their biological consequences.

Catalytic poly(ADP-ribose) production by PARP1 is allosterically activated through interaction with DNA breaks, and PARP inhibitor compounds have the potential to influence PARP1 allostery in addition to preventing catalytic activity. Using the benzimidazole-4-carboxamide pharmacophore present in the first generation PARP1 inhibitor veliparib, a series of 11 derivatives was designed, synthesized, and evaluated as allosteric PARP1 inhibitors, with the premise that bulky substituents would engage the regulatory helical domain (HD) and thereby promote PARP1 retention on DNA breaks. We found that core scaffold modifications could indeed increase PARP1 affinity for DNA; however, the bulk of the modification alone was insufficient to trigger PARP1 allosteric retention on DNA breaks. Rather, compounds eliciting PARP1 retention on DNA breaks were found to be rigidly held in a position that interferes with a specific region of the HD domain, a region that is not targeted by current clinical PARP inhibitors. Collectively, these compounds highlight a unique way to trigger PARP1 retention on DNA breaks and open a path to unveil the pharmacological benefits of such inhibitors with novel properties.

PARP enzymes transfer ADP-ribose from NAD+ onto proteins as a covalent modification that regulates multiple aspects of cell biology. Here, we identify an undiscovered catalytic activity for human PARP1: de novo generation of free PAR molecules that are not attached to proteins. Free PAR production arises when a molecule of NAD+ or ADP-ribose docks in the PARP1 acceptor site and attaches to an NAD+ molecule bound to the donor site, releasing nicotinamide and initiating ADP-ribose chains that emanate from NAD+/ADP-ribose rather than protein. Free PAR is also produced by human PARP2 and the PARP enzyme Tankyrase. We demonstrate that free PAR in cells is generated mostly by PARP1 de novo synthesis activity rather than by PAR-degrading enzymes PAR glycohydrolase (PARG), ARH3, and TARG1 releasing PAR from protein. The coincident production of free PAR and protein-linked modifications alters models for PAR signaling and broadens the scope of PARP enzyme signaling capacity.

Abstract PARP4 is an ADP-ribosyltransferase that resides within the vault ribonucleoprotein organelle. Our knowledge of PARP4 structure and biochemistry is limited relative to other PARPs. PARP4 shares a region of homology with PARP1, an ADP-ribosyltransferase that produces poly(ADP-ribose) from NAD+ in response to binding DNA breaks. The PARP1-homology region of PARP4 includes a BRCT fold, a WGR domain, and the catalytic (CAT) domain. Here, we have determined X-ray structures of the PARP4 catalytic domain and performed biochemical analysis that together indicate an active site that is open to NAD+ interaction, in contrast to the closed conformation of the PARP1 catalytic domain that blocks access to substrate NAD+. We have also determined crystal structures of the minimal ADP-ribosyltransferase fold of PARP4 that illustrate active site alterations that restrict PARP4 to mono(ADP-ribose) rather than poly(ADP-ribose) modifications. We demonstrate that PARP4 interacts with vault RNA, and that the BRCT is primarily responsible for the interaction. However, the interaction does not lead to stimulation of mono(ADP-ribosylation) activity. The BRCT–WGR–CAT of PARP4 has lower activity than the CAT alone, suggesting that the BRCT and WGR domains regulate catalytic output. Our study provides first insights into PARP4 structure and regulation and expands understanding of PARP structural biochemistry.

PDF file - 410K, Dose responses, transcriptional, biological, biochemical, and in vivo supplemental data

HuR silencing results in downregulation of PARG expression, which prevents efficient removal of PAR polymers with or without PARPi stress. Expression and function of other PAR removing enzymes is not affected with HuR silencing.

ADP-ribose is a versatile modification that plays a critical role in diverse cellular processes. The addition of this modification is catalyzed by ADP-ribosyltransferases, among which notable poly(ADP-ribose) polymerase (PARP) enzymes are intimately involved in the maintenance of genome integrity. The role of ADP-ribose modifications during DNA damage repair is of significant interest for the proper development of PARP inhibitors targeted toward the treatment of diseases caused by genomic instability. More specifically, inhibitors promoting PARP persistence on DNA lesions, termed PARP "trapping," is considered a desirable characteristic. In this review, we discuss key classes of proteins involved in ADP-ribose signaling (writers, readers, and erasers) with a focus on those involved in the maintenance of genome integrity. An overview of factors that modulate PARP1 and PARP2 persistence at sites of DNA lesions is also discussed. Finally, we clarify aspects of the PARP trapping model in light of recent studies that characterize the kinetics of PARP1 and PARP2 recruitment at sites of lesions. These findings suggest that PARP trapping could be considered as the continuous recruitment of PARP molecules to sites of lesions, rather than the physical stalling of molecules. Recent studies and novel research tools have elevated the level of understanding of ADP-ribosylation, marking a coming-of-age for this interesting modification.

PARP enzymes create ADP-ribose modifications to regulate multiple facets of human biology, and some prominent PARP family members are best known for the nucleic acid interactions that regulate their activities and functions. Recent structural studies have highlighted PARP interactions with nucleic acids, in particular for PARP enzymes that detect and respond to DNA strand break damage. These studies build on our understanding of how DNA break detection is linked to the catalysis of ADP-ribose modifications, provide insights into distinct modes of DNA interaction, and shed light on the mechanisms of PARP inhibitor action. PARP enzymes have several connections to RNA biology, including the detection of the genomes of RNA viruses, and recent structural work has highlighted how PARP13/ZAP specifically targets viral genomes enriched in CG dinucleotides.

PARPi treatment mediated apoptosis and chromatin- associated trapping of PARP1 is significantly enhanced upon HuR and PARG inhibition.

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