Domain Of Poly ADP-ribose Polymerase Research Articles

Abstract 2754: Antibody-based tools for in vitro and live cell analysis of endogenous PARP1, an essential human DNA repair enzyme

Abstract DNA damage caused by normal cell activity or exogenous genotoxic agents is a constant threat to the genome. The nuclear enzyme Poly(ADP-ribose) polymerase 1 (PARP1) is rapidly activated by DNA lesions such as single-strand breaks and signals their presence by attaching ADP-ribose units to chromatin-associated proteins. To improve our understanding of PARP1 within a disease context, there is an ongoing need to develop novel PARP1 detection systems. Here we describe the development of a VHH directed against human PARP1. The variable domain of single chain antibodies (VHH or nanobody) is a versatile research tool for a variety of applications. The high binding affinity of VHHs, their small size (15 kDa) and their robust expression in various cellular systems make them preferable to conventional antibodies. Moreover, VHH domains can be selected to recognize and bind their target structures within living cells. This highly specific PARP1 VHH was selected by phage display using a VHH library from an immunized alpaca. We characterized its affinity, selectivity and activity both in vitro and in live cells. To validate the in vitro interactions of this PARP1-specific VHH, we developed a PARP1 immunoprecipitation reagent by conjugating the PARP1-specific VHH to an immobilizing matrix (termed PARP1-Trap). This PARP1-Trap was shown to bind with high specificity to human PARP1 and not to other members of the PARP family. Importantly, the binding of the PARP1-Trap to PARP1 leaves the enzymatic activity of PARP1 unaffected. The epitope of the PARP1-Trap was localized to the N-terminal domain of PARP1 and consists of the three-dimensional motif of zinc fingers 2 and 3 together. To determine whether the PARP1 VHH recognizes its target structure also in a cellular environment, the VHH was genetically fused to a fluorescent protein and expressed in living cells (termed Chromobody). The interaction of the PARP1 Chromobody with PARP1 was visualized using a protein-protein interaction assay called fluorescent two-hybrid (F2H). The F2H principle is based on a tethering strategy: a GFP-tagged protein (here GFP-PARP1) is enriched at a protein interaction platform engineered into F2H-BHK cells and serves as bait, whereas the RFP-tagged PARP1 VHH serves as a prey. Using the F2H assay, we could show that the PARP1 Chromobody is functional within living cells and specifically recognizes its antigen in a cellular environment. Moreover, by monitoring the PARP1 Chromobody signal after microirradiation, we were for the first time able to follow the recruitment of endogenous PARP1 to sites of DNA damage in living cells. In summary, we developed a novel PARP1 VHH for both biochemical and live cell analysis of human PARP1. We anticipate that PARP1-VHH based reagents will provide new insights into the PARP1 enzyme. For example, the use of the PARP1-Trap coupled with mass spectrometry analysis may lead to the identification of hitherto unknown PARP1 interaction partners. Citation Format: Andrea Buchfellner, Larisa Yurlova, Stephanie Dennison, Benjamin Ruf, Ulrich Rothbauer, Tina Romer. Antibody-based tools for in vitro and live cell analysis of endogenous PARP1, an essential human DNA repair enzyme. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2754.

Recent advances on the research of PARP-1 inhibitors

Poly(ADP-ribose)polymerase-1 (PARP-1) plays significant roles in the DNA repair process by catalyzing the transfer of ADP-ribose from NAD+ to its receptors. It is a promising anticancer drug target and many PARP-1 inhibitors have been developed and used in the clinical trials. PARP-1 inhibitors could be used not only as chemo/ radiotherapy sensitizers, but also as single agents to selectively kill BRCA deficient cancer cells. In this review, 6 classes of PARP-1 inhibitors with distinct structure scaffold were described in terms of the structure-activity relationships and their binding modes within the catalytic domain of PARP-1.

Insights into the binding of PARP inhibitors to the catalytic domain of human tankyrase-2.

The poly(ADP-ribose) polymerase (PARP) family represents a new class of therapeutic targets with diverse potential disease indications. PARP1 and PARP2 inhibitors have been developed for breast and ovarian tumors manifesting double-stranded DNA-repair defects, whereas tankyrase 1 and 2 (TNKS1 and TNKS2, also known as PARP5a and PARP5b, respectively) inhibitors have been developed for tumors with elevated β-catenin activity. As the clinical relevance of PARP inhibitors continues to be actively explored, there is heightened interest in the design of selective inhibitors based on the detailed structural features of how small-molecule inhibitors bind to each of the PARP family members. Here, the high-resolution crystal structures of the human TNKS2 PARP domain in complex with 16 various PARP inhibitors are reported, including the compounds BSI-201, AZD-2281 and ABT-888, which are currently in Phase 2 or 3 clinical trials. These structures provide insight into the inhibitor-binding modes for the tankyrase PARP domain and valuable information to guide the rational design of future tankyrase-specific inhibitors.

Binding to WGR Domain by Salidroside Activates PARP1 and Protects Hematopoietic Stem Cells from Oxidative Stress

A component of the base excision repair pathway, poly(ADP-ribose) polymerase-1 (PARP1) functions in multiple cellular processes, including DNA repair and programmed cell death. We previously showed that Salidroside, a phenylpropanoid glycoside isolated from medicinal plants, prevented the loss of hematopoietic stem cells (HSCs) in native mice and rescued HSCs repopulating in transplanted recipients under oxidative stress. The aim of this study was to investigate the mechanism by which PARP1 activation by Salidroside maintains HSCs under oxidative stress. We found that although there were no spontaneous defects in hematopoiesis in Parp1(-/-) mice, oxidative stress compromised the repopulating capacity of Parp1(-/-) HSCs in transplanted recipient mice. A biochemical study using truncated proteins lacking the defined functional domains of PARP1 showed that the tryptophan-glycine-arginine-rich (WGR) domain of PARP1 was critical for Salidroside binding and subsequent PARP1 activation under oxidative stress. Functionally, complementation of Parp1(-/-) HSCs with full-length PARP1WT, but not the PARP1R591K mutant in WGR domain restored Salidroside-stimulated PARP1 activation in vitro. Mechanistically, activated PARP1 by Salidroside enhanced the repopulating capacity of the stressed HSCs by accelerating oxidative DNA damage repair. INNOVATIONS AND CONCLUSION: Our findings reveal the action of mechanism for Salidroside in PARP1 stimulation and a novel role of PARP1 activation in maintaining HSC function under oxidative stress.

Interferon-Stimulated Poly(ADP-Ribose) Polymerases Are Potent Inhibitors of Cellular Translation and Virus Replication

The innate immune response is the first line of defense against most viral infections. Its activation promotes cell signaling, which reduces virus replication in infected cells and leads to induction of the antiviral state in yet-uninfected cells. This inhibition of virus replication is a result of the activation of a very broad spectrum of specific cellular genes, with each of their products usually making a small but detectable contribution to the overall antiviral state. The lack of a strong, dominant function for each gene product and the ability of many viruses to interfere with the development of the antiviral response strongly complicate identification of the antiviral activity of the activated individual cellular genes. However, we have previously developed and applied a new experimental system which allows us to define a critical function of some members of the poly(ADP-ribose) polymerase (PARP) family in clearance of Venezuelan equine encephalitis virus mutants from infected cells. In this new study, we demonstrate that PARP7, PARP10, and the long isoform of PARP12 (PARP12L) function as important and very potent regulators of cellular translation and virus replication. The translation inhibition and antiviral effect of PARP12L appear to be mediated by more than one protein function and are a result of its direct binding to polysomes, complex formation with cellular RNAs (which is determined by both putative RNA-binding and PARP domains), and catalytic activity. IMPORTANCE The results of this study demonstrate that interferon-stimulated gene products PARP7, PARP10, and PARP12L are potent inhibitors of the replication of Venezuelan equine encephalitis virus and other alphaviruses. The inhibitory functions are determined by more than a single mechanism, and one of them is based on the ability of these proteins to regulate cellular translation. Interference with the cellular translational machinery depends on the integrity of both the amino-terminal domain, containing a number of putative RNA-binding motifs, and the catalytic function of the carboxy-terminal PARP domain. The PARP-induced changes in translation efficiency appear to have a more potent effect on the synthesis of virus-specific proteins than on that of cellular proteins, thus making PARP-specific translational downregulation an important contributor to the overall development of the antiviral response.

Exploring the effect of PARP-1 flexibility in docking studies

Poly(ADP-ribose)polymerase-1 (PARP-1) is an enzyme belonging to the ADP-ribosyltransferase family. A large body of works has validated PARP-1 as an attractive drug target for different therapeutic areas, including cancers and ischemia. Accordingly, sampling the conformational space of the enzyme is pivotal to understand its functions and improve structure-based drug discovery approaches.In the first part of this study we apply replica exchange molecular dynamic (REMD) simulations to sample the conformational space of the catalytic domain of PARP-1 in the ligand-bound and unbound forms. In the second part, we assess how and to what extend the emerging enzyme flexibility affects the performance of docking experiments of a library of PARP-1 inhibitors. This study pinpoints a putative key role of conformational shifts of Leu324, Tyr325 and Lys242 in opening an additional binding site pocket that affects the binding of ligands to the catalytic cleft of PARP-1. Furthermore, it highlights the improvement of the enrichment factor of active ligands obtained in docking experiments when using conformations generated with REMD simulations of ligand-bound PARP-1.

PARP-1 mechanism for coupling DNA damage detection to poly(ADP-ribose) synthesis

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.

(PARP)-1 N-Terminal Fragment Down Regulates Endogenous PARP-1 Expression and Activity and Sensitises Cells to Oxidative Stress

Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays a vital role in DNA repair. This makes it an attractive anticancer therapeutic target. It is activated by DNA breaks and catalyses the synthesis of homopolymers of ADP-ribose from NAD+. Competitive inhibitors as well as the non-catalytic, DNA-binding domain of PARP-1 abolish its polymer synthesising function. Such inhibitors of PARP-1 have been used in clinical trials and are known to cause nausea, fatigue and haematological events and with chronic use the risk of drug-induced DNA damage and tumorigenesis. In this study, I have investigated the effect of PARP-1-N-terminal fragment on endogenous PARP-1 activity and expression in mammalian cells. To elicit DNA damage response, different concentrations of H2O2 were used. For visualisation of the effects by live imaging, 750 bp PARP-1-N-terminal fragment was tagged to EGFPN1 vector. My data shows an inverse correlation between the expression of this fragment and poly (ADP-ribose) synthesis at lower concentrations of H2O2 and induction of apoptosis at higher concentrations. My experimental evidence also supports regulation of endogenous PARP-1 expression by the fragment in the absence of DNA damage. This construct has allowed for the visualisation of its functional ability to sensitise cells to oxidative damage and induce apoptosis as observed by the formation of apoptotic precursors and caspase cleavage products in live cells. The data also provides direct evidence for its therapeutic potential in chemotherapy or radiotherapy to avert necrosis induced inflammatory response.

The 3'-5' Exonuclease, TREX1, Interacts with Poly(ADP-ribose) Polymerase-1 (PARP1) in Response to DNA Damage

AbstractAbstract 1046 Background:Recognition of nucleic acids plays a key role in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). The major 3’-5’ DNA exonuclease, TREX1, degrades single- and double-stranded DNA to minimize potential immune activation by persistent self or retroelement DNA. Mutations in the human TREX1gene have been linked to four diseases: SLE; Aicardi-Goutières syndrome (AGS); retinal vasculopathy and cerebral leukodystrophy (RVCL); and familial chilblain lupus (CBL). SLE, AGS and CBL have overlapping features characterized by expression of interferon-alpha and autoantibody production, but are distinct clinical conditions. RVCL is a non-inflammatory endotheliopathy. Methods:As part of our efforts to determine the molecular mechanisms underlying TREX1 involvement in the pathogenesis of these diseases, we identified a novel TREX1-binding partner protein by Mass Spectrometry and Co-Immunoprecipitation (Co-IP) assays. We examined the interaction domains and the effects of mutations in TREX1 associated with these diseases. Results:We found that TREX1 interacted with poly(ADP-ribose) polymerase-1 (PARP1), a nuclear DNA repair enzyme involved in the DNA damage response. The interaction domain of PARP1 was mapped to the amino-terminal zinc finger domains. Subcellular localization analysis showed that TREX1 normally localized to the cytoplasm and cytomembranes, but translocated to the nucleus (with some bound to chromatin) in response to sublethal DNA damage. Co-IP assays revealed that the interaction was increased following DNA damage in concert with the post-translational PARlation of PARP1. Functional studies showed that TREX1 induced the PARlation of PARP1 in a TREX1 dose-dependent manner. Furthermore, siRNA knockdown of TREX1 promoted PARP1 cleavage and loss of PARP1 enzymatic activity, resulting in the induction of apoptosis. We further analyzed the effects of TREX1-PARP1 interaction in relation to the pathogenesis of AGS or RVCL. Co-IP assays revealed that the RVCL-related TREX1-mutant protein, V235fs, bound to PARP1 as well as wild-type TREX1, whereas the AGS-related R114H mutant protein did not bind PARP1. Conclusion:These findings demonstrate that TREX1 contributes to the regulation of PARP1 in the DNA damage response and that mutation-induced alterations in the function of TREX1 may be a factor in the development or progression of these autoimmune diseases by affecting PARP1 activity. Disclosures:No relevant conflicts of interest to declare.

Coordination of DNA repair by NEIL1 and PARP-1: a possible link to aging

Oxidative DNA damage accumulates with age and is repaired primarily via the base excision repair (BER) pathway. This process is initiated by DNA glycosylases, which remove damaged bases in a substrate-specific manner. The DNA glycosylases human 8-oxoguanine-DNA glycosylase (OGG1) and NEIL1, a mammalian homolog ofEscherichia coli endonuclease VIII, have overlapping yet distinct substrate specificity. Recently, we reported that OGG1 binds to the Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA damage sensor protein that poly(ADP-ribosyl)ates nuclear proteins in response to DNA damage and other cellular signals. Here, we show that NEIL1 and PARP-1 bind both in vitro and in vivo. PARP-1 binds to the C-terminal-100 amino acids of NEIL1 and NEIL1 binds to the BRCT domain of PARP-1. NEIL1 stimulates the poly(ADP-ribosyl)ation activity of PARP-1. Furthermore, NEIL-deficient fibroblasts have impaired poly(ADP-ribosyl)ation of cellular proteins after DNA damage, which can be rescued by NEIL1 expression. Additionally, PARP-1 inhibits NEIL1 incision activity in a concentration-dependent manner. Consistent with the idea of impaired DNA repair during aging, we observed differential binding of PARP-1 to recombinant NEIL1 in older mice compared to younger mice. These data further support the idea that dynamic interplay between different base excision repair proteins is important for efficient BER.

ADP Ribosylation by PARP-1 Suppresses HOXB7 Transcriptional Activity

Interactions with cofactors regulate transcriptional activity and also help HOX proteins to achieve the specificity required for transcriptional regulation of target genes. In this study, we describe a novel protein/protein interaction of HOXB7 with poly (ADP-ribose) polymerase-1 (PARP-1) that involves the homeodomain of HOXB7 and the first zinc finger domain of PARP-1. Upon binding to PARP-1, HOXB7 undergoes poly(ADP-ribosyl)altion resulting in a reduction of its transcriptional activity. Since aspartic acid and glutamic acid residues are acceptors of the ADP ribose moiety transferred by PARP-1, deletion of the evolutionarily conserved C-terminal Glu-rich tail of HOXB7 dramatically attenuates ADP-ribosylation of HOXB7 by PARP-1. Further, a mutant of HOXB7 without the Glu-rich tail loses the ability to be negatively regulated by PARP-1 and becomes transcriptionally more active in luciferase reporter assays. Since the homeodomain is highly conserved among HOX proteins, five other HOX proteins were tested. All six showed interaction with, and were poly(ADP-ribosyl)ated by PARP-1. However, among them, this modification altered the DNA binding activity of only HOXA7 and HOXB7. In summary, this study identifies a new interacting partner of HOX proteins. More importantly, this study reveals a novel mechanism whereby polyADP-ribosylation regulates transcriptional activities of HOX proteins such as HOXB7 and HOXA7.

Tankyrase 1 regulates centrosome function by controlling CPAP stability

CPAP--a gene mutated in primary microcephaly--is required for procentriole formation. Here we show that CPAP degradation and function is controlled by the poly(ADP-ribose) polymerase tankyrase 1. CPAP is PARsylated by tankyrase 1 in vitro and in vivo. Overexpression of tankyrase 1 leads to CPAP proteasomal degradation, preventing centriole duplication, whereas depletion of tankyrase 1 stabilizes CPAP in G1, generating elongated procentrioles and multipolarity. Tankyrase 1 localizes to centrosomes exclusively in G1, coinciding with CPAP degradation. Hence, tankyrase 1-mediated PARsylation regulates CPAP levels during the cell cycle to limit centriole elongation and ensure normal centrosome function.

The zinc-finger domains of PARP1 cooperate to recognize DNA strand breaks.

Poly(ADP-ribose) polymerase 1 (PARP1) is a primary DNA damage sensor whose (ADP-ribose) polymerase activity is acutely regulated by interaction with DNA breaks. Upon activation at sites of DNA damage, PARP1 modifies itself and other proteins by covalent addition of long, branched polymers of ADP-ribose, which in turn recruit downstream DNA repair and chromatin remodeling factors. PARP1 recognizes DNA damage through its N-terminal DNA-binding domain (DBD), which consists of a tandem repeat of an unusual zinc-finger (ZnF) domain. We have determined the crystal structure of the human PARP1-DBD bound to a DNA break. Along with functional analysis of PARP1 recruitment to sites of DNA damage in vivo, the structure reveals a dimeric assembly whereby ZnF1 and ZnF2 domains from separate PARP1 molecules form a strand-break recognition module that helps activate PARP1 by facilitating its dimerization and consequent trans-automodification.

Interdependent genotoxic mechanisms of monomethylarsonous acid: Role of ROS-induced DNA damage and poly(ADP-ribose) polymerase-1 inhibition in the malignant transformation of urothelial cells

Exposure of human bladder urothelial cells (UROtsa) to 50 nM of the arsenic metabolite, monomethylarsonous acid (MMA III), for 12 weeks results in irreversible malignant transformation. The ability of continuous, low-level MMA III exposure to cause an increase in genotoxic potential by inhibiting repair processes necessary to maintain genomic stability is unknown. Following genomic insult within cellular systems poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger protein, is rapidly activated and recruited to sites of DNA strand breaks. When UROtsa cells are continuously exposed to 50 nM MMA III, PARP-1 activity does not increase despite the increase in MMA III-induced DNA single-strand breaks through 12 weeks of exposure. When UROtsa cells are removed from continuous MMA III exposure (2 weeks), PARP-1 activity increases coinciding with a subsequent decrease in DNA damage levels. Paradoxically, PARP-1 mRNA expression and protein levels are elevated in the presence of continuous MMA III indicating a possible mechanism to compensate for the inhibition of PARP-1 activity in the presence of MMA III. The zinc finger domains of PARP-1 contain vicinal sulfhydryl groups which may act as a potential site for MMA III to bind, displace zinc ion, and render PARP-1 inactive. Mass spectrometry analysis demonstrates the ability of MMA III to bind a synthetic peptide representing the zinc-finger domain of PARP-1, and displace zinc from the peptide in a dose-dependent manner. In the presence of continuous MMA III exposure, continuous 4-week zinc supplementation restored PARP-1 activity levels and reduced the genotoxicity associated with MMA III. Zinc supplementation did not produce an overall increase in PARP-1 protein levels, decrease the levels of MMA III-induced reactive oxygen species, or alter Cu–Zn superoxide dismutase levels. Overall, these results present two potential interdependent mechanisms in which MMA III may increase the susceptibility of UROtsa cells to genotoxic insult and/or malignant transformation: elevated levels of MMA III-induced DNA damage through the production of reactive oxygen species, and the direct MMA III-induced inhibition of PARP-1.

The Key Residues of Active Sites on the Catalytic Fragment for Paclitaxel Interacting with Poly (ADP-Ribose) Polymerase

Poly(ADP-ribose) polymerase (PARP) is regarded as a target protein for paclitaxel (PTX) to bind. An important issue is to identify the key residues as active sites for PTX interacting with PARP, which will help to understand the potential drug activity of PTX against cancer cells. Using docking method and MD simulation, we have constructed a refined structure of PTX docked on the catalytic function domain of PARP (PDB code: 1A26). The residues Glu327(988), Tyr246(907), Lys242(903), His165(826), Asp105(766), Gln102(763) and Gln98(759) in PARP are identified as potential sites involved in interaction with PTX according to binding energy (Eb) between PTX and single residue calculated with B3LYP/6- 31G(d,p). These residues form an active binding pocket located on the surface of the catalytic fragment, possibly interacting with the required groups of PTX leading to its activity against cancer cells. It is noted that most of the active sites make conatct with the “southern hemisphere” of PTX except for one residue, Tyr246(907), which interacts with the “nourthern hemisphere” of PTX. The conformation of PTX in complex with the catalytic fragment is observed as being T-shaped, similar to that complexed with β-tubulin. The total Eb of −269.9 kJ/mol represents the potent interaction between PTX and the catalytic fragment, implying that PTX can readily bind to the active pocket. The tight association of PTX with the catalytic fragment would inhibit PARP activation, suggesting a potential application of PTX as an effective antineoplastic agent.

Double-stranded DNA Binding Domain of Poly(ADP-ribose) Polymerase-1 and Molecular Insight into the Regulation of Its Activity

Poly(ADP-ribose) polymerase-1 (PARP-1) modifies various proteins, including itself, with ADP-ribose polymers (automodification). Polymer synthesis is triggered by binding of its zinc finger 1 (Zn1) and 2 (Zn2) to DNA breaks and is followed by inactivation through automodification. The multiple functional domains of PARP-1 appear to regulate activation and automodification-mediated inactivation of PARP-1. However, the roles of these domains in activation-inactivation processes are not well understood. Our results suggest that Zn1, Zn2, and a domain identified in this study, the double-stranded DNA binding (DsDB) domain, are involved in DNA break-dependent activation of PARP-1. We found that binding of the DsDB domain to double-stranded DNA and DNA break recognition by Zn1 and Zn2, whose actual binding targets are likely to be single-stranded DNA, lead to the activation of PARP-1. In turn, the displacement of single- and double-stranded DNA from Zn2 and the DsDB domain caused by ADP-ribose polymer synthesis results in the dissociation of PARP-1 from DNA breaks and thus its inactivation. We also found that the WGR domain is one of the domains involved in the RNA-dependent activation of PARP-1. Furthermore, because zinc finger 3 (Zn3) has the ability to bind to single-stranded RNA, it may have an indirect role in RNA-dependent activation. PARP-1 functional domains, which are involved in oligonucleic acid binding, therefore coordinately regulate PARP-1 activity depending on the status of the neighboring oligonucleic acids. Based on these results, we proposed a model for the regulation of PARP-1 activity.

The DNA-Binding Domain of Human PARP-1 Interacts with DNA Single-Strand Breaks as a Monomer through Its Second Zinc Finger

Poly(ADP-ribose)polymerase-1 (PARP-1) is a highly abundant chromatin-associated enzyme present in all higher eukaryotic cell nuclei, where it plays key roles in the maintenance of genomic integrity, chromatin remodeling and transcriptional control. It binds to DNA single- and double-strand breaks through an N-terminal region containing two zinc fingers, F1 and F2, following which its C-terminal catalytic domain becomes activated via an unknown mechanism, causing formation and addition of polyadenosine-ribose (PAR) to acceptor proteins including PARP-1 itself. Here, we report a biophysical and structural characterization of the F1 and F2 fingers of human PARP-1, both as independent fragments and in the context of the 24-kDa DNA-binding domain (F1+F2). We show that the fingers are structurally independent in the absence of DNA and share a highly similar structural fold and dynamics. The F1+F2 fragment recognizes DNA single-strand breaks as a monomer and in a single orientation. Using a combination of NMR spectroscopy and other biophysical techniques, we show that recognition is primarily achieved by F2, which binds the DNA in an essentially identical manner whether present in isolation or in the two-finger fragment. F2 interacts much more strongly with nicked or gapped DNA ligands than does F1, and we present a mutational study that suggests origins of this difference. Our data suggest that different DNA lesions are recognized by the DNA-binding domain of PARP-1 in a highly similar conformation, helping to rationalize how the full-length protein participates in multiple steps of DNA single-strand breakage and base excision repair.

The BRCT Domain of PARP-1 Is Required for Immunoglobulin Gene Conversion

Author SummaryTo produce a limitless diversity of antibodies within the constraints of a finite genome, activated B cells introduce random mutations into antibody genes through a process of targeted DNA damage and subsequent mutagenic repair. At the same time, the rest of the genome must be protected from mutagenesis to prevent off-target mutations which can lead to the development of lymphoma or leukemia. How antibody genes are specifically targeted is still largely unknown. A potential player in this process is the DNA-damage-sensing enzyme PARP-1, which recruits DNA repair enzymes to sites of damage. Using a chicken B cell lymphoma cell line because it has only a single PARP isoform and constitutively mutates its antibody genes, we compared the types of mutations accumulated in PARP-1−/− cells to wild type. We found that in cells lacking PARP-1, the major pathway of mutagenic repair was disrupted and fewer mutations than normal were introduced into their antibody genes. To identify what might be important for mutagenesis, we tested different factors for their ability to rescue this mutagenic deficiency and found a role for the BRCT (BRCA1 C-terminal) domain of PARP-1, a consensus protein domain known to be involved in directing protein-protein interactions. Our evidence suggests that PARP-1 may be interacting with another hypothetical protein via its BRCT domain that is required for the mutagenic rather than faithful repair of DNA lesions in the antibody genes.

PIASy Mediates SUMO-2/3 Conjugation of Poly(ADP-ribose) Polymerase 1 (PARP1) on Mitotic Chromosomes

PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins.

P21 CDKN1A participates in base excision repair by regulating the activity of poly(ADP-ribose) polymerase-1

The cell cycle inhibitor p21 CDKN1A has been shown to participate in nucleotide excision repair by interacting with PCNA. Here we have investigated whether p21 plays a role in base excision repair (BER), by analyzing p21 interactions with BER factors, and by assessing the response of p21 −/− human fibroblasts to DNA damage induced by alkylating agents. Absence of p21 protein resulted in a higher sensitivity to alkylation-induced DNA damage, as indicated by reduced clonogenic efficiency, defective DNA repair (assessed by the comet test), and by persistence of histone H2AX phosphorylation. To elucidate the mechanisms at the basis of the function of p21 in BER, we focused on its interaction with poly(ADP-ribose) polymerase-1 (PARP-1), an important player in this repair process. p21 was found to bind the automodification/DNA binding domain of PARP-1, although some interaction occurred also with the catalytic domain after DNA damage. This association was necessary to regulate PARP-1 activity since poly(ADP-ribosylation) induced by DNA damage was higher in p21 −/− human fibroblasts than in parental p21 +/+ cells, and in primary fibroblasts after p21 knock-down by RNA interference. Concomitantly, recruitment of PARP-1 and PCNA to damaged DNA was greater in p21 −/− than in p21 +/+ fibroblasts. This accumulation resulted in persistent interaction of PARP-1 with BER factors, such as XRCC1 and DNA polymerase β, suggesting that prolonged association reduced the DNA repair efficiency. These results indicate that p21 regulates the interaction between PARP-1 and BER factors, to promote efficient DNA repair.