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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., Competing Interests: Declaration of interests B.E.B., J.M.P., and T.T.T. are co-founders of Hysplex, Inc. with interests in PARPi development. B.E.B., J.M.P., and T.T.T. are co-inventors on a provisional patent application filed by UPenn that is related to this work. B.E.B. is on the scientific advisory board of Denovicon Therapeutics., (Copyright © 2025 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests J.M.P. is a co-founder of Hysplex with interests in PARP inhibitor development., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interests: The authors declare the following competing interests: N.C.K.V., B.D.G.P., and M.A. are inventors on a patent application describing CeTEAM and its uses (PCT/EP2019/073769). The remaining authors declare no competing interests., (© 2024. The Author(s).)

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 α-helix absent in PARP1. Only one clinical PARPi, Olaparib, stabilizes the PARP2 active site α-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 PARP1 and PARP2., Competing Interests: Declarations of interest B.E.B., J.M.P., and T.T.T. are co-founders of Hysplex, Inc. with interests in PARP inhibitor development. B.E.B., J.M.P., and T.T.T. are co-inventors on a provisional patent application filed by UPenn that is related to this work. B.E.B is on the scientific advisory board of Denovicon Therapeutics.

PARP1 and PARP2 detect DNA breaks, which activates their catalytic production of poly(ADP-ribose) that recruits repair factors and contributes to PARP1/2 release from DNA. PARP inhibitors (PARPi) are used in cancer treatment and target PARP1/2 catalytic activity, interfering with repair and increasing PARP1/2 persistence on DNA damage. In addition, certain PARPi exert allosteric effects that increase PARP1 retention on DNA. However, no clinical PARPi exhibit this allosteric behavior toward PARP1. In contrast, we show that certain clinical PARPi exhibit an allosteric effect that retains PARP2 on DNA breaks in a manner that depends on communication between the catalytic and DNA binding regions. Using a PARP2 mutant that mimics an allosteric inhibitor effect, we observed increased PARP2 retention at cellular damage sites. The PARPi AZD5305 also exhibited a clear reverse allosteric effect on PARP2. Our results can help explain the toxicity of clinical PARPi and suggest ways to improve PARPi moving forward.

Allosteric coupling between the DNA binding site to the NAD + -binding pocket drives PARP-1 activation. This allosteric communication occurs in the reverse direction such that NAD + mimetics can enhance PARP-1's affinity for DNA, referred to as type I inhibition. The cellular effects of type I inhibition are unknown, largely because of the lack of potent, membrane-permeable type I inhibitors. Here we identify the phthalazinone inhibitor AZ0108 as a type I inhibitor. Unlike the structurally related inhibitor olaparib, AZ0108 induces replication stress in tumorigenic cells. Synthesis of analogs of AZ0108 revealed features of AZ0108 that are required for type I inhibition. One analog, Pip6, showed similar type I inhibition of PARP-1 but was ∼90-fold more cytotoxic than AZ0108. Washout experiments suggest that the enhanced cytotoxicity of Pip6 compared with AZ0108 is due to prolonged target residence time on PARP-1. Pip6 represents a new class of PARP-1 inhibitors that may have unique anticancer properties., Competing Interests: Declaration of interests M.R.A. and M.S.C. are inventors on a patent related to the compounds generated from this manuscript. M.S.C. is a founder of Tilikum Therapeutics., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Human poly(ADP)-ribose polymerase-1 (PARP1) is a global regulator of various cellular processes, from DNA repair to gene expression. The underlying mechanism of PARP1 action during transcription remains unclear. Herein, we have studied the role of human PARP1 during transcription through nucleosomes by RNA polymerase II (Pol II) in vitro. PARP1 strongly facilitates transcription through mononucleosomes by Pol II and displacement of core histones in the presence of NAD+ during transcription, and its NAD+-dependent catalytic activity is essential for this process. Kinetic analysis suggests that PARP1 facilitates formation of "open" complexes containing nucleosomal DNA partially uncoiled from the octamer and allowing Pol II progression along nucleosomal DNA. Anti-cancer drug and PARP1 catalytic inhibitor olaparib strongly represses PARP1-dependent transcription. The data suggest that the negative charge on protein(s) poly(ADP)-ribosylated by PARP1 interact with positively charged DNA-binding surfaces of histones transiently exposed during transcription, facilitating transcription through chromatin and transcription-dependent histone displacement/exchange.

PARP1 and PARP2 produce poly(ADP-ribose) in response to DNA breaks. HPF1 regulates PARP1/2 catalytic output, most notably permitting serine modification with ADP-ribose. However, PARP1 is substantially more abundant in cells than HPF1, challenging whether HPF1 can pervasively modulate PARP1. Here, we show biochemically that HPF1 efficiently regulates PARP1/2 catalytic output at sub-stoichiometric ratios matching their relative cellular abundances. HPF1 rapidly associates/dissociates from multiple PARP1 molecules, initiating serine modification before modification initiates on glutamate/aspartate, and accelerating initiation to be more comparable to elongation reactions forming poly(ADP-ribose). This "hit and run" mechanism ensures HPF1 contributions to PARP1/2 during initiation do not persist and interfere with PAR chain elongation. We provide structural insights into HPF1/PARP1 assembled on a DNA break, and assess HPF1 impact on PARP1 retention on DNA. Our data support the prevalence of serine-ADP-ribose modification in cells and the efficiency of serine-ADP-ribose modification required for an acute DNA damage response., (© 2021. The Author(s).)

Poly(ADP-ribose) polymerase 1 (PARP1) is an enzyme involved in DNA repair, chromatin organization and transcription. During transcription initiation, PARP1 interacts with gene promoters where it binds to nucleosomes, replaces linker histone H1 and participates in gene regulation. However, the mechanisms of PARP1-nucleosome interaction remain unknown. Here, using spFRET microscopy, molecular dynamics and biochemical approaches we identified several different PARP1-nucleosome complexes and two types of PARP1 binding to mononucleosomes: at DNA ends and end-independent. Two or three molecules of PARP1 can bind to a nucleosome depending on the presence of linker DNA and can induce reorganization of the entire nucleosome that is independent of catalytic activity of PARP1. Nucleosome reorganization depends upon binding of PARP1 to nucleosomal DNA, likely near the binding site of linker histone H1. The data suggest that PARP1 can induce the formation of an alternative nucleosome state that is likely involved in gene regulation and DNA repair.

PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1's F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA replication forks use multiple mechanisms to deal with replication stress, but how the choice of mechanisms is made is still poorly understood. Here, we show that CARM1 associates with replication forks and reduces fork speed independently of its methyltransferase activity. The speeding of replication forks in CARM1-deficient cells requires RECQ1, which resolves reversed forks, and RAD18, which promotes translesion synthesis. Loss of CARM1 reduces fork reversal and increases single-stranded DNA (ssDNA) gaps but allows cells to tolerate higher replication stress. Mechanistically, CARM1 interacts with PARP1 and promotes PARylation at replication forks. In vitro, CARM1 stimulates PARP1 activity by enhancing its DNA binding and acts jointly with HPF1 to activate PARP1. Thus, by stimulating PARP1, CARM1 slows replication forks and promotes the use of fork reversal in the stress response, revealing that CARM1 and PARP1 function as a regulatory module at forks to control fork speed and the choice of stress response mechanisms., Competing Interests: Declaration of interests L.Z. is a member of the advisory board of Molecular Cell. The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as 'trapping'. To understand the molecular nature of 'trapping' in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The success of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors (PARPi) to treat cancer relates to their ability to trap PARP-1 at the site of a DNA break. Although different forms of PARPi all target the catalytic center of the enzyme, they have variable abilities to trap PARP-1. We found that several structurally distinct PARPi drive PARP-1 allostery to promote release from a DNA break. Other inhibitors drive allostery to retain PARP-1 on a DNA break. Further, we generated a new PARPi compound, converting an allosteric pro-release compound to a pro-retention compound and increasing its ability to kill cancer cells. These developments are pertinent to clinical applications where PARP-1 trapping is either desirable or undesirable., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Human tankyrase-1 (TNKS) is a member of the poly(ADP-ribose) polymerase (PARP) superfamily of proteins that posttranslationally modify themselves and target proteins with ADP-ribose (termed PARylation). The TNKS ankyrin repeat domain mediates interactions with a growing number of structurally and functionally diverse binding partners, linking TNKS activity to multiple critical cell processes, including Wnt signaling, Golgi trafficking, and telomere maintenance. However, some binding partners can engage TNKS without being modified, suggesting that separate parameters influence TNKS interaction and PARylation. Here, we present an analysis of the sequence and structural features governing TNKS interactions with two model binding partners: the PARylated partner telomeric repeat-binding factor 1 (TRF1) and the non-PARylated partner GDP-mannose 4,6-dehydratase (GMD). Using a combination of TNKS-binding assays, PARP activity assays, and analytical ultracentrifugation sedimentation analysis, we found that both the specific sequence of a given TNKS-binding peptide motif and the quaternary structure of individual binding partners play important roles in TNKS interactions. We demonstrate that GMD forms stable 1:1 complexes with the TNKS ankyrin repeat domain; yet, consistent with results from previous studies, we were unable to detect GMD modification. We also report in vitro evidence that TNKS primarily directs PAR modification to glutamate/aspartate residues. Our results suggest that TNKS-binding partners possess unique sequence and structural features that control binding and PARylation. Ultimately, our findings highlight the binding partner:ankyrin repeat domain interface as a viable target for inhibition of TNKS activity., (© 2019 Eisemann et al.)

PARP-1 is rapidly recruited and activated by DNA double-strand breaks (DSBs). Upon activation, PARP-1 synthesizes a structurally complex polymer composed of ADP-ribose units that facilitates local chromatin relaxation and the recruitment of DNA repair factors. Here, we identify a function for PARP-1 in DNA DSB resection. Remarkably, inhibition of PARP-1 leads to hyperresected DNA DSBs. We show that loss of PARP-1 and hyperresection are associated with loss of Ku, 53BP1 and RIF1 resection inhibitors from the break site. DNA curtains analysis show that EXO1-mediated resection is blocked by PARP-1. Furthermore, PARP-1 abrogation leads to increased DNA resection tracks and an increase of homologous recombination in cellulo. Our results, therefore, place PARP-1 activation as a critical early event for DNA DSB repair activation and regulation of resection. Hence, our work has direct implications for the clinical use and effectiveness of PARP inhibition, which is prescribed for the treatment of various malignancies.

Poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors are a class of anticancer drugs that block the catalytic activity of PARP proteins. Optimization of our lead compound 1 (( Z)-2-benzylidene-3-oxo-2,3-dihydrobenzofuran-7-carboxamide; PARP-1 IC 50 = 434 nM) led to a tetrazolyl analogue (51, IC 50 = 35 nM) with improved inhibition. Isosteric replacement of the tetrazole ring with a carboxyl group (60, IC 50 = 68 nM) gave a promising new lead, which was subsequently optimized to obtain analogues with potent PARP-1 IC 50 values (4-197 nM). PARP enzyme profiling revealed that the majority of compounds are selective toward PARP-2 with IC 50 values comparable to clinical inhibitors. X-ray crystal structures of the key inhibitors bound to PARP-1 illustrated the mode of interaction with analogue appendages extending toward the PARP-1 adenosine-binding pocket. Compound 81, an isoform-selective PARP-1/-2 (IC 50 = 30 nM/2 nM) inhibitor, demonstrated selective cytotoxic effect toward breast cancer gene 1 ( BRCA1)-deficient cells compared to isogenic BRCA1-proficient cells.

Poly(ADP-ribose) is a posttranslational modification and signaling molecule that regulates many aspects of human cell biology, and it is synthesized by enzymes known as poly(ADP-ribose) polymerases, or PARPs. A diverse collection of domain structures dictates the different cellular roles of PARP enzymes and regulates the production of poly(ADP-ribose). Here we primarily review recent structural insights into the regulation and catalysis of two family members: PARP-1 and Tankyrase. PARP-1 has multiple roles in the cellular response to DNA damage and the regulation of gene transcription, and Tankyrase regulates a diverse set of target proteins involved in cellular processes such as mitosis, genome integrity, and cell signaling. Both enzymes offer interesting modes of regulating the production and the target site selectivity of the poly(ADP-ribose) modification., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Despite significant advances in the development of mass spectrometry-based methods for the identification of protein ADP-ribosylation, current protocols suffer from several drawbacks that preclude their widespread applicability. Given the intrinsic heterogeneous nature of poly(ADP-ribose), a number of strategies have been developed to generate simple derivatives for effective interrogation of protein databases and site-specific localization of the modified residues. Currently, the generation of spectral signatures indicative of ADP-ribosylation rely on chemical or enzymatic conversion of the modification to a single mass increment. Still, limitations arise from the lability of the poly(ADP-ribose) remnant during tandem mass spectrometry, the varying susceptibilities of different ADP-ribose-protein bonds to chemical hydrolysis, or the context dependence of enzyme-catalyzed reactions. Here, we present a chemical-based derivatization method applicable to the confident identification of site-specific ADP-ribosylation by conventional mass spectrometry on any targeted amino acid residue. Using PARP-1 as a model protein, we report that treatment of ADP-ribosylated peptides with hydrofluoric acid generates a specific +132 Da mass signature that corresponds to the decomposition of mono- and poly(ADP-ribosylated) peptides into ribose adducts as a consequence of the cleavage of the phosphorus-oxygen bonds.

PARP-1 cleaves NAD + and transfers the resulting ADP-ribose moiety onto target proteins and onto subsequent polymers of ADP-ribose. An allosteric network connects PARP-1 multi-domain detection of DNA damage to catalytic domain structural changes that relieve catalytic autoinhibition; however, the mechanism of autoinhibition is undefined. Here, we show using the non-hydrolyzable NAD + analog benzamide adenine dinucleotide (BAD) that PARP-1 autoinhibition results from a selective block on NAD + binding. Following DNA damage detection, BAD binding to the catalytic domain leads to changes in PARP-1 dynamics at distant DNA-binding surfaces, resulting in increased affinity for DNA damage, and providing direct evidence of reverse allostery. Our findings reveal a two-step mechanism to activate and to then stabilize PARP-1 on a DNA break, indicate that PARP-1 allostery influences persistence on DNA damage, and have important implications for PARP inhibitors that engage the NAD + binding site.

ADP-ribosylation is a protein post-translational modification catalyzed by ADP-ribose transferases (ARTs). ART activity is critical in mediating many cellular processes, and is required for DNA damage repair. All five histone proteins are extensively ADP-ribosylated by ARTs upon induction of DNA damage. However, how these modifications aid in repair processes is largely unknown, primarily due to lack of knowledge about where they site-specifically occur on histones. Here, we conduct a comprehensive analysis of histone Asp/Glu ADP-ribosylation sites upon DNA damage induced by dimethyl sulfate (DMS). We also demonstrate that incubation of cell nuclei with NAD + , as has been done previously in the literature, leads to spurious ADP-ribosylation levels of histone proteins. Altogether, we were able to identify 30 modification sites, 20 of which are novel. We also quantify the abundance of these modification sites during the course of DNA damage insult to identify which sites are critical for mediating repair. We found that every quantifiable site increases in abundance over time and that each identified ADP-ribosylation site is located on the surface of the nucleosome. Together, the data suggest specific Asp/Glu residues are unlikely to be critical for DNA damage repair and rather that this process is likely dependent on ADP-ribosylation of the nucleosomal surface in general.

Human PARP-1, PARP-2, and PARP-3 are key players in the cellular response to DNA damage, during which their catalytic activities are acutely stimulated through interaction with DNA strand breaks. There are also roles for these PARPs outside of the DNA damage response, most notably for PARP-1 and PARP-2 in the regulation of gene expression. Here, we describe a general method to express and purify these DNA damage-dependent PARPs from E. coli cells for use in biochemical assays and for structural and functional analysis. The procedure allows for robust production of PARP enzymes that are free of contaminant DNA that can interfere with downstream analysis. The described protocols have been updated from our earlier reported methods, most importantly to introduce PARP inhibitors in the production scheme to cope with enzyme toxicity that can compromise the yield of purified protein.

DNA accessibility to various protein complexes is essential for various processes in the cell and is affected by nucleosome structure and dynamics. Protein factor PARP-1 (poly(ADP-ribose)polymerase 1) increases the accessibility of DNA in chromatin to repair proteins and transcriptional machinery, but the mechanism and extent of this chromatin reorganization are unknown. Here we report on the effects of PARP-1 on single nucleosomes revealed by spFRET (single-particle Förster Resonance Energy Transfer) microscopy. PARP-1 binding to a double-strand break in the vicinity of a nucleosome results in a significant increase of the distance between the adjacent gyres of nucleosomal DNA. This partial uncoiling of the entire nucleosomal DNA occurs without apparent loss of histones and is reversed after poly(ADP)-ribosylation of PARP-1. Thus PARP-1-nucleosome interactions result in reversible, partial uncoiling of the entire nucleosomal DNA., Competing Interests: Conflict of interest The authors declare no competing financial interests.

Mediator is a highly conserved transcriptional coactivator organized into four modules, namely Tail, Middle, Head, and Kinase (CKM). Previous work suggests regulatory roles for Tail and CKM, but an integrated model for these activities is lacking. Here, we analyzed the genome-wide distribution of Mediator subunits in wild-type and mutant yeast cells in which RNA polymerase II promoter escape is blocked, allowing detection of transient Mediator forms. We found that although all modules are recruited to upstream activated regions (UAS), assembly of Mediator within the pre-initiation complex is accompanied by the release of CKM. Interestingly, our data show that CKM regulates Mediator-UAS interaction rather than Mediator-promoter association. In addition, although Tail is required for Mediator recruitment to UAS, Tailless Mediator nevertheless interacts with core promoters. Collectively, our data suggest that the essential function of Mediator is mediated by Head and Middle at core promoters, while Tail and CKM play regulatory roles., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The poly(ADP-ribose) polymerase enzyme Tankyrase-1 (TNKS) regulates multiple cellular processes and interacts with diverse proteins using five ankyrin repeat clusters (ARCs). There are limited structural insights into functional roles of the multiple ARCs of TNKS. Here we present the ARC1-3 crystal structure and employ small-angle X-ray scattering (SAXS) to investigate solution conformations of the complete ankyrin repeat domain. Mutagenesis and binding studies using the bivalent TNKS binding domain of Axin1 demonstrate that only certain ARC combinations function together. The physical basis for these restrictions is explained by both rigid and flexible ankyrin repeat elements determined in our structural analysis. SAXS analysis is consistent with a dynamic ensemble of TNKS ankyrin repeat conformations modulated by Axin1 interaction. TNKS ankyrin repeat domain is thus an adaptable binding platform with structural features that can explain selectivity toward diverse proteins, and has implications for TNKS positioning of bound targets for poly(ADP-ribose) modification., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Tankyrase-1 (TNKS1/PARP-5a) is a poly(ADP-ribose) polymerase (PARP) enzyme that regulates multiple cellular processes creating a poly(ADP-ribose) posttranslational modification that can lead to target protein turnover. TNKS1 thereby controls protein levels of key components of signaling pathways, including Axin1, the limiting component of the destruction complex in canonical Wnt signaling that degrades β-catenin to prevent its coactivator function in gene expression. There are limited molecular level insights into TNKS1 regulation in cell signaling pathways. TNKS1 has a sterile α motif (SAM) domain that is known to mediate polymerization, but the functional requirement for SAM polymerization has not been assessed. We have determined the crystal structure of wild-type human TNKS1 SAM domain and used structure-based mutagenesis to disrupt polymer formation and assess the consequences on TNKS1 regulation of β-catenin-dependent transcription. Our data indicate the SAM polymer is critical for TNKS1 catalytic activity and allows TNKS1 to efficiently access cytoplasmic signaling complexes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Unexpectedly, a post-translational modification of DNA-binding proteins, initiating the cell response to single-strand DNA damage, was also required for long-term memory acquisition in a variety of learning paradigms. Our findings disclose a molecular mechanism based on PARP1-Erk synergism, which may underlie this phenomenon. A stimulation induced PARP1 binding to phosphorylated Erk2 in the chromatin of cerebral neurons caused Erk-induced PARP1 activation, rendering transcription factors and promoters of immediate early genes (IEG) accessible to PARP1-bound phosphorylated Erk2. Thus, Erk-induced PARP1 activation mediated IEG expression implicated in long-term memory. PARP1 inhibition, silencing, or genetic deletion abrogated stimulation-induced Erk-recruitment to IEG promoters, gene expression and LTP generation in hippocampal CA3-CA1-connections. Moreover, a predominant binding of PARP1 to single-strand DNA breaks, occluding its Erk binding sites, suppressed IEG expression and prevented the generation of LTP. These findings outline a PARP1-dependent mechanism required for LTP generation, which may be implicated in long-term memory acquisition and in its deterioration in senescence.

Poly(ADP-ribose) polymerase-1 (PARP-1) creates the posttranslational modification PAR from substrate NAD(+) to regulate multiple cellular processes. DNA breaks sharply elevate PARP-1 catalytic activity to mount a cell survival repair response, whereas persistent PARP-1 hyperactivation during severe genotoxic stress is associated with cell death. The mechanism for tight control of the robust catalytic potential of PARP-1 remains unclear. By monitoring PARP-1 dynamics using hydrogen/deuterium exchange-mass spectrometry (HXMS), we unexpectedly find that a specific portion of the helical subdomain (HD) of the catalytic domain rapidly unfolds when PARP-1 encounters a DNA break. Together with biochemical and crystallographic analysis of HD deletion mutants, we show that the HD is an autoinhibitory domain that blocks productive NAD(+) binding. Our molecular model explains how PARP-1 DNA damage detection leads to local unfolding of the HD that relieves autoinhibition, and has important implications for the design of PARP inhibitors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1's function remained obscure; inherent dynamics of SSBs and PARP-1's multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1's signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodification in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

An important feature of poly(ADP-ribose) polymerases (PARPs) is their ability to readily undergo automodification upon activation. Although a growing number of substrates were found to be poly(ADP-ribosyl)ated, including histones and several DNA damage response factors, PARPs themselves are still considered as the main acceptors of poly(ADP-ribose). By monitoring spectral counts of specific hydroxamic acid signatures generated after the conversion of the ADP-ribose modification onto peptides by hydroxylamine hydrolysis, we undertook a thorough mass spectrometry mapping of the glutamate and aspartate ADP-ribosylation sites onto automodified PARP-1, PARP-2 and PARP-3. Thousands of hydroxamic acid-conjugated peptides were identified with high confidence and ranked based on their spectral count. This semi-quantitative approach allowed us to locate the preferentially targeted residues in DNA-dependent PARPs. In contrast to what has been reported in the literature, automodification of PARP-1 is not predominantly targeted towards its BRCT domain. Our results show that interdomain linker regions that connect the BRCT to the WGR module and the WGR to the PRD domain undergo prominent ADP-ribosylation during PARP-1 automodification. We also found that PARP-1 efficiently automodifies the D-loop structure within its own catalytic fold. Interestingly, additional major ADP-ribosylation sites were identified in functional domains of PARP-1, including all three zinc fingers. Similar to PARP-1, specific residues located within the catalytic sites of PARP-2 and PARP-3 are major targets of automodification following their DNA-dependent activation. Together our results suggest that poly(ADP-ribosyl)ation hot spots make a dominant contribution to the overall automodification process., (Copyright © 2015 Elsevier B.V. All rights reserved.)

PARP-1, PARP-2 and PARP-3 are DNA-dependent PARPs that localize to DNA damage, synthesize poly(ADP-ribose) (PAR) covalently attached to target proteins including themselves, and thereby recruit repair factors to DNA breaks to increase repair efficiency. PARP-1, PARP-2 and PARP-3 have in common two C-terminal domains-Trp-Gly-Arg (WGR) and catalytic (CAT). In contrast, the N-terminal region (NTR) of PARP-1 is over 500 residues and includes four regulatory domains, whereas PARP-2 and PARP-3 have smaller NTRs (70 and 40 residues, respectively) of unknown structural composition and function. Here, we show that PARP-2 and PARP-3 are preferentially activated by DNA breaks harboring a 5' phosphate (5'P), suggesting selective activation in response to specific DNA repair intermediates, in particular structures that are competent for DNA ligation. In contrast to PARP-1, the NTRs of PARP-2 and PARP-3 are not strictly required for DNA binding or for DNA-dependent activation. Rather, the WGR domain is the central regulatory domain of PARP-2 and PARP-3. Finally, PARP-1, PARP-2 and PARP-3 share an allosteric regulatory mechanism of DNA-dependent catalytic activation through a local destabilization of the CAT. Collectively, our study provides new insights into the specialization of the DNA-dependent PARPs and their specific roles in DNA repair pathways., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

PARP-1 is a nuclear protein that has important roles in maintenance of genomic integrity. During genotoxic stress, PARP-1 recruits to sites of DNA damage where PARP-1 domain architecture initiates catalytic activation and subsequent poly(ADP-ribose)-dependent DNA repair. PARP-1 inhibition is a promising new way to selectively target cancers harboring DNA repair deficiencies. However, current inhibitors target other PARPs, raising important questions about long-term off-target effects. Here, we propose a new strategy that targets PARP-1 allosteric regulation as a selective way of inhibiting PARP-1. We found that disruption of PARP-1 domain-domain contacts through mutagenesis held no cellular consequences on recruitment to DNA damage or a model system of transcriptional regulation, but prevented DNA-damage-dependent catalytic activation. Furthermore, PARP-1 mutant overexpression in a pancreatic cancer cell line (MIA PaCa-2) increased sensitivity to platinum-based anticancer agents. These results not only highlight the potential of a synergistic drug combination of allosteric PARP inhibitors with DNA-damaging agents in genomically unstable cancer cells (regardless of homologous recombination status), but also signify important applications of selective PARP-1 inhibition. Finally, the development of a high-throughput PARP-1 assay is described as a tool to promote discovery of novel PARP-1 selective inhibitors.

ADP-ribosylation of proteins regulates protein activities in various processes including transcription control, chromatin organization, organelle assembly, protein degradation, and DNA repair. Modulating the proteins involved in the metabolism of ADP-ribosylation can have therapeutic benefits in various disease states. Protein crystal structures can help understand the biological functions, facilitate detailed analysis of single residues, as well as provide a basis for development of small molecule effectors. Here we review recent advances in our understanding of the structural biology of the writers, readers, and erasers of ADP-ribosylation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Poly(ADP-ribose) polymerase 1 (PARP-1) regulates gene transcription, cell death signaling, and DNA repair through production of the posttranslational modification poly(ADP-ribose). During the cellular response to genotoxic stress PARP-1 rapidly associates with DNA damage, which robustly stimulates poly(ADP-ribose) production over a low basal level of PARP-1 activity. DNA damage-dependent PARP-1 activity is central to understanding PARP-1 biological function, but structural insights into the mechanisms underlying this mode of regulation have remained elusive, in part due to the highly modular six-domain architecture of PARP-1. Recent structural studies have illustrated how PARP-1 uses specialized zinc fingers to detect DNA breaks through sequence-independent interaction with exposed nucleotide bases, a common feature of damaged and abnormal DNA structures. The mechanism of coupling DNA damage detection to elevated poly(ADP-ribose) production has been elucidated based on a crystal structure of the essential domains of PARP-1 in complex with a DNA strand break. The multiple domains of PARP-1 collapse onto damaged DNA, forming a network of interdomain contacts that introduce destabilizing alterations in the catalytic domain leading to an enhanced rate of poly(ADP-ribose) production., (Copyright © 2013. Published by Elsevier Ltd.)

Poly(ADP-ribose) polymerase-1 (PARP-1) (ADP, adenosine diphosphate) has a modular domain architecture that couples DNA damage detection to poly(ADP-ribosyl)ation activity through a poorly understood mechanism. Here, we report the crystal structure of a DNA double-strand break in complex with human PARP-1 domains essential for activation (Zn1, Zn3, WGR-CAT). PARP-1 engages DNA as a monomer, and the interaction with DNA damage organizes PARP-1 domains into a collapsed conformation that can explain the strong preference for automodification. The Zn1, Zn3, and WGR domains collectively bind to DNA, forming a network of interdomain contacts that links the DNA damage interface to the catalytic domain (CAT). The DNA damage-induced conformation of PARP-1 results in structural distortions that destabilize the CAT. Our results suggest that an increase in CAT protein dynamics underlies the DNA-dependent activation mechanism of PARP-1.

The flap domain of multisubunit RNA polymerases (RNAPs), also called the wall, forms one side of the RNA exit channel. In bacterial RNAP, the mobile part of the flap is called the flap tip and makes essential contacts with initiation and elongation factors. Cocrystal structures suggest that the orthologous part of eukaryotic RNAPII, called the flap loop, contacts transcription factor IIB (TFIIB), but the function of the flap loop has not been assessed. We constructed and tested a deletion of the flap loop in human RNAPII (subunit RPB2 Δ873-884) that removes the flap loop interaction interface with TFIIB. Genome-wide analysis of the distribution of the RNAPII with the flap loop deletion expressed in a human embryonic kidney cell line (HEK 293) revealed no effect of the flap loop on global transcription initiation, RNAPII occupancy within genes, or the efficiency of promoter escape and productive elongation. In vitro, the flap loop deletion had no effect on promoter binding, abortive initiation or promoter escape, TFIIS-stimulated transcript cleavage, or inhibition of transcript elongation by the complex of negative elongation factor (NELF) and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF). A modest effect on transcript elongation and pausing was suppressed by TFIIF. Although similar to the flap tip of bacterial RNAP, the RNAPII flap loop is not equivalently essential.

Poly(ADP-ribose) polymerase-1 (PARP-1) has two homologous zinc finger domains, Zn1 and Zn2, that bind to a variety of DNA structures to stimulate poly(ADP-ribose) synthesis activity and to mediate PARP-1 interaction with chromatin. The structural basis for interaction with DNA is unknown, which limits our understanding of PARP-1 regulation and involvement in DNA repair and transcription. Here, we have determined crystal structures for the individual Zn1 and Zn2 domains in complex with a DNA double strand break, providing the first views of PARP-1 zinc fingers bound to DNA. The Zn1-DNA and Zn2-DNA structures establish a novel, bipartite mode of sequence-independent DNA interaction that engages a continuous region of the phosphodiester backbone and the hydrophobic faces of exposed nucleotide bases. Biochemical and cell biological analysis indicate that the Zn1 and Zn2 domains perform distinct functions. The Zn2 domain exhibits high binding affinity to DNA compared with the Zn1 domain. However, the Zn1 domain is essential for DNA-dependent PARP-1 activity in vitro and in vivo, whereas the Zn2 domain is not strictly required. Structural differences between the Zn1-DNA and Zn2-DNA complexes, combined with mutational and structural analysis, indicate that a specialized region of the Zn1 domain is re-configured through the hydrophobic interaction with exposed nucleotide bases to initiate PARP-1 activation.

A general method to express and purify full-length human poly(ADP-ribose) polymerase-1 (PARP-1), individual PARP-1 domains, and groups of PARP-1 domains from Escherichia coli cells is described. The procedure allows for robust production of highly pure PARP-1 that is free of DNA contamination and well-suited for biochemical experiments and for structural and biophysical analysis. Two biochemical assays for monitoring PARP-1 automodification activity are presented that can be used to evaluate purified PARP-1, combinations of PARP-1 domains, or PARP-1 mutants.

PARP-1 is involved in multiple cellular processes, including transcription, DNA repair, and apoptosis. PARP-1 attaches ADP-ribose units to target proteins, including itself as a post-translational modification that can change the biochemical properties of target proteins and mediate recruitment of proteins to sites of poly(ADP-ribose) synthesis. Independent of its catalytic activity, PARP-1 binds to chromatin and promotes compaction affecting RNA polymerase II transcription. PARP-1 has a modular structure composed of six independent domains. Two homologous zinc fingers, Zn1 and Zn2, form the DNA-binding module. Zn1-Zn2 binding to DNA breaks triggers catalytic activity. Recently, we have identified a third zinc binding domain in PARP-1, the Zn3 domain, which is essential for DNA-dependent PARP-1 activity. The crystal structure of the Zn3 domain revealed a novel zinc-ribbon fold and a homodimeric Zn3 structure that formed in the crystal lattice. Structure-guided mutagenesis was used here to investigate the roles of these two features of the Zn3 domain. Our results indicate that the zinc-ribbon fold of the Zn3 domain mediates an interdomain contact crucial to assembly of the DNA-activated conformation of PARP-1. In contrast, residues located at the Zn3 dimer interface are not required for DNA-dependent activation but rather make important contributions to the chromatin compaction activity of PARP-1. Thus, the Zn3 domain has dual roles in regulating the functions of PARP-1.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a chromatin-associated enzyme with multiple cellular functions, including DNA repair, transcriptional regulation, and cell signaling. PARP-1 has a modular architecture with six independent domains comprising the 113-kDa polypeptide. Two zinc finger domains at the N terminus of PARP-1 bind to DNA and thereby activate the catalytic domain situated at the C terminus of the enzyme. The tight coupling of DNA binding and catalytic activities is critical to the cellular regulation of PARP-1 function; however, the mechanism for coordinating these activities remains an unsolved problem. Here, we demonstrate using spectroscopic and crystallographic analysis that human PARP-1 has a third zinc-binding domain. Biochemical mutagenesis and deletion analysis indicate that this region mediates interdomain contacts important for DNA-dependent enzyme activation. The crystal structure of the third zinc-binding domain reveals a zinc ribbon fold and suggests conserved residues that could form interdomain contacts. The new zinc-binding domain self-associates in the crystal lattice to form a homodimer with a head-totail arrangement. The structure of the homodimer provides a scaffold for assembling the activated state of PARP-1 and suggests a mechanism for coupling the DNA binding and catalytic functions of PARP-1.

High-resolution crystallographic structures of multisubunit RNA polymerases (RNAPs) have increased our understanding of transcriptional mechanisms. Based on a thorough review of the literature, we have compiled the mutations affecting the function of multisubunit RNA polymerases, many of which having been generated and studied prior to the publication of the first high-resolution structure, and highlighted the positions of the altered amino acids in the structures of both the prokaryotic and eukaryotic enzymes. The observations support many previous hypotheses on the transcriptional process, including the implication of the bridge helix and the trigger loop in the processivity of RNAP, the importance of contacts between the RNAP jaw-lobe module and the downstream DNA in the establishment of a transcription bubble and selection of the transcription start site, the destabilizing effects of ppGpp on the open promoter complex, and the link between RNAP processivity and termination. This study also revealed novel, remarkable features of the RNA polymerase catalytic mechanisms that will require additional investigation, including the putative roles of fork loop 2 in the establishment of a transcription bubble, the trigger loop in start site selection, and the uncharacterized funnel domain in RNAP processivity.

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.

During transcription by RNA polymerase (RNAP) II, the incoming ribonucleoside triphosphate (NTP) enters the catalytic center in association with an Mg2+ ion, termed metal B [Mg(B)]. When bound to RNAP II, Mg(B) is coordinated by the beta and gamma phosphates of the NTP, Rpb1 residues D481 and D483 and Rpb2 residue D837. Rpb2 residue D837 is highly conserved across species. Notably, its neighboring residue, E836 (E791 in human RNAP II), is also highly conserved. To probe the role of E791 in transcription, we have affinity purified and characterized a human RNAP II mutant in which this residue was substituted for alanine. Our results indicate that the transcription activity of the Rpb2 E791A mutant is impaired at low NTP concentrations both in vitro and in vivo. They also revealed that both its NTP polymerization and transcript cleavage activities are decreased at low Mg concentrations. Because Rpb2 residue E791 appears to be located too far from the NTP-Mg(B) complex to make direct contact at either the entry (E) or addition (A) site, we propose alternative mechanisms by which this highly conserved residue participates in loading NTP-Mg(B) in the active site during transcription.

The interaction of many proteins with genomic DNA is required for the expression, replication, and maintenance of the integrity of mammalian genomes. These proteins participate in processes as diverse as gene transcription and mRNA processing, as well as in DNA replication, recombination, and repair. This intricate system, where the various nuclear machineries interact with one another and bind to either common or distinct DNA regions to create an impressive network of protein-protein and protein-DNA interactions, is made even more complex by the need for a very stringent control in order to ensure normal cell growth and differentiation. A general methodology based on the in vivo pull-down of tagged components of nuclear machines and regulatory proteins was used to study genome-wide protein-protein and protein-DNA interactions in mammalian cells. In particular, this approach has been used in defining the interaction networks (or "interactome") formed by RNA polymerase II, a molecular machine that decodes the human genome. In addition, because this methodology allows for the purification of variant forms of tagged complexes having site-directed mutations in key elements, it can also be used for deciphering the relationship between the structure and the function of the molecular machines, such as RNA polymerase II, that by binding DNA play a central role in the pathway from the genome to the organism.

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

The topological organization of a TATA binding protein-TFIIB-TFIIF-RNA polymerase II (RNAP II)-TFIIE-promoter complex was analyzed using site-specific protein-DNA photo-cross-linking of gel-purified complexes. The cross-linking results for the subunits of RNAP II were used to determine the path of promoter DNA against the structure of the enzyme. The results indicate that promoter DNA wraps around the mobile clamp of RNAP II. Cross-linking of TFIIF and TFIIE both upstream of the TATA element and downstream of the transcription start site suggests that both factors associate with the RNAP II mobile clamp. TFIIE alpha closely approaches promoter DNA at nucleotide -10, a position immediately upstream of the transcription bubble in the open complex. Increased stimulation of transcription initiation by TFIIE alpha is obtained when the DNA template is artificially premelted in the -11/-1 region, suggesting that TFIIE alpha facilitates open complex formation, possibly through its interaction with the upstream end of the partially opened transcription bubble. These results support the central roles of the mobile clamp of RNAP II and TFIIE in transcription initiation.

A topological model for transcription initiation by RNA polymerase II (RNAPII) has recently been proposed. This model stipulates that wrapping of the promoter DNA around RNAPII and the general initiation factors TBP, TFIIB, TFIIE, TFIIF and TFIIH induces a torsional strain in the DNA double helix that facilitates strand separation and open complex formation. In this report, we show that TFIIA, a factor previously shown to both stimulate basal transcription and have co-activator functions, is located near the cross-point of the DNA loop where it can interact with TBP, TFIIE56, TFIIE34, and the RNAPII-associated protein (RAP) 74. In addition, we demonstrate that TFIIA can stimulate basal transcription by stimulating the functions of both TFIIE34 and RAP74 during the initiation step of the transcription reaction. These results provide novel insights into mechanisms of TFIIA function.