Detection of poly(ADP-ribose) polymerase and its reaction product poly(ADP-ribose) by immunocytochemistry

Summary Poly(ADP-ribose) polymerase catalyses the formation of ADP-ribose polymers covalently attached to various nuclear proteins, using NAD⁺ as substrate. The activity of this enzyme is strongly stimulated upon binding to DNA single or double strand breaks. Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage and is thought to be involved in DNA repair, genetic recombination, apoptosis and other processes during which DNA strand breaks are formed. In recent years we and others have established cell culture systems with altered poly(ADP-ribose) polymerase activity. Here we describe immunocytochemistry protocols based on the use of antibodies against the DNA-binding domain of human poly(ADP-ribose) polymerase and against its reaction product poly(ADP-ribose). These protocols allow for the convenient mass screening of cell transfectants with overexpression of poly(ADP-ribose) polymerase or of a dominant-negative mutant for this enzyme, i.e. the DNA-binding domain. In addition, the immunocytochemical detection of poly(ADP-ribose) allows screening for cells with altered enzyme activity.     

Cytoplasmic poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase in AEV-transformed chicken erythroblasts

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase activities were both investigated in chicken erythroblasts transformed by Avian Erythroblastosis Virus. Respectively 21% and 58% of these activities were found to be present in the post-mitochondrial supernatant (PMS). Fractionation of the PMS on sucrose gradients and poly(A⁺) mRNA detection by hybridization to [³H] poly(U) show that cytoplasmic poly(ADP-ribose) polymerase is exclusively localized in free mRNP. The glycohydrolase activity sedimented mostly in the 6 S region but 1/3 of the activity was in the free mRNP zone. Seven poly(ADP-ribose) protein acceptors were identified in the PMS in the Mr 21000–120000 range. The Mr 120000 protein corresponds to automodified poly(ADP-ribose) polymerase. A Mr 21000 protein acceptor is abundant in PMS and a Mr 34000 is exclusively associated with ribosomes and ribosomal subunits. The existence of both poly(ADP-ribose) polymerase and glycohydrolase activities in free mRNP argues in favour of a role of poly(ADP-ribosylation) in mRNP metabolism. A possible involvement of this post translational modification in the mechanisms of repression-derepression of mRNA is discussed.Abbreviations ADP-ribose adenosine (5) diphospho(5)--D ribose - poly(ADP-ribose) polymer of ADP-ribose - mRNP messenger ribonucleoprotein particles - PMSF phenylmethylsulfonyl fluoride - LDS lithium dodecyl sulfate - TCA trichloroacetic acid     

Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes

Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli.     

Initiation of poly(ADP-ribosyl) histone synthesis by poly(ADP-ribose) synthetase

Initiation of poly(ADP-ribosyl) histone synthesis was achieved in vitro using an apparently homogeneous preparation of poly(ADP-ribose) synthetase. When poly(ADP-ribose) was synthesized in the presence of DNA and increase amounts of histone H1, increasing portions (up to about 55%) of the product were found associated with the histone, judging from solubility in 5% HClO4 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Most of the polymers were directly attached to the histone protein and not produced by elongation from pre-existing ADP-ribose; the cohesive end of poly(ADP-ribose), isolated as ribose 5-phosphate with snake venom phosphodiesterase digestion, was labeled almost quantitatively with [ribose (NMN)-14C]NAD. The poly(ADP-ribose) . histone linkage was labile in mild alkali and neutral NH2OH, suggesting that the same bond, probably ester, was formed in this system as in crude chromatin or isolated nuclei. Elongation of a histone-bound monomer into a polymer by this enzyme was previously demonstrated (Ueda, K., Kawaichi, M., Okayama, H., and Hayaishi, O. (1979) J. Biol. Chem. 254, 679-687), but initiation of ADP-ribose chains on histone has never been shown with a purified enzyme. This appeared to be due to the low concentrations of histone so far used. These findings indicated that a single enzyme catalyzes two different types of reaction, i.e. an attachment of ADP-ribose to histone and its elongation into a polymer.     

Alteration of poly(ADP-ribose) metabolism by hyperthermia

The intracellular levels of poly(ADP-ribose) in cultured mouse cells were increased in response to hyperthermic treatment (43 degrees C). When hyperthermia was combined with other stressful treatments such as with ethanol and/or an alkylating agent, a dramatic synergistic increase in polymer levels was observed. The effect of hyperthermia did not appear to be related to the presence of DNA strand breaks. A possible involvement of poly(ADP-ribose) metabolism in the general cellular response to environmental stress is suggested.     

Presence of poly (ADP-ribose) polymerase and poly (ADP-ribose) glycohydrolase in the dinoflagellate Crypthecodinium cohnii

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase have been detected in chromatin extracts from the dinoflagellate Crypthecodinium cohnii. Poly(ADP-ribose) glycohydrolase was detected by the liberation of ADP-ribose from poly(ADP-ribose). Poly(ADP-ribose) polymerase was proved by (a) demonstration of phosphoribosyl-AMP in the phosphodiesterase digest of the reaction product, (b) demonstration of ADP-ribose oligomers by fractionation of the reaction product on DEAE-Sephadex. The (ADP-ribose)-protein transfer is dependent on DNA; it is inhibited by nicotinamide, thymidine, theophylline and benzamide. The protein-(ADP-ribose bond is susceptible to 0.1 M NaOH (70%) and 0.4 M NH2OH (33%). Dinoflagellates, nucleated protists, are unique in that their chromatin lacks histones and shows a conformation like bacterial chromatin [Loeblich, A. R., III (1976) J. Protozool. 23, 13--28]; poly(ADP-ribose) polymerase, however, has been found only in eucaryotes. Thus our results suggest that histones were not relevant to the establishment of poly(ADP-ribose) during evolution.     

Importance of poly(ADP-ribose) glycohydrolase in the control of poly(ADP-ribose) metabolism.

Poly(ADP-ribosyl)ation is a posttranslational modification that alters the functions of the acceptor proteins and is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes. Following DNA damage, activated poly(ADP-ribose) polymerase-1 (PARP-1) catalyzes the elongation and branching of poly(ADP-ribose) (pADPr) covalently attached to nuclear target proteins. Although the biological role of poly(ADP-ribosyl)ation has not yet been defined, it has been implicated in many important cellular processes such as DNA repair and replication, modulation of chromatin structure, and apoptosis. The transient nature and modulation of poly(ADP-ribosyl)ation depend on the activity of a unique cytoplasmic enzyme called poly(ADP-ribose) glycohydrolase which hydrolyzes pADPr bound to acceptor proteins in free ADP-ribose residues. While the PARP homologues have been recently reviewed, there are relatively scarce data about PARG in the literature. Here we summarize the latest advances in the PARG field, addressing the question of its putative nucleo-cytoplasmic shuttling that could enable the tight regulation of pADPr metabolism. This would contribute to the elucidation of the biological significance of poly(ADP-ribosyl)ation.     

Poly(etheno ADP-ribose) blocks poly(ADP-ribose) glycohydrolase activity

Poly(ADP-ribose) is a biopolymer synthesized by poly(ADP-ribose) polymerases. Recent findings suggest the possibility for modulation of cellular functions including cell death and mitosis by poly(ADP-ribose). Derivatization of poly(ADP-ribose) may be useful for investigating the effects of poly(ADP-ribose) on various cellular processes. We prepared poly(etheno ADP-ribose) (poly(epsilonADP-ribose)) by converting the adenine moiety of poly(ADP-ribose) to 1-N(6)-etheno adenine residues. Poly(epsilonADP-ribose) is shown to be highly resistant to digestion by poly(ADP-ribose) glycohydrolase (Parg). On the other hand, poly(epsilonADP-ribose) could be readily digested by phosphodiesterase. Furthermore, poly(epsilonADP-ribose) inhibited Parg activity to hydrolyse ribose-ribose bonds of poly(ADP-ribose). This study suggests the possibility that poly(epsilonADP-ribose) might be a useful tool for studying the poly(ADP-ribose) dynamics and function of Parg. This study also implies that modification of the adenine moiety of poly(ADP-ribose) abrogates the susceptibility to digestion by Parg.     

3'-Deoxy-NAD+ as a substrate for poly(ADP-ribose)polymerase and the reaction mechanism of poly(ADP-ribose) elongation

Adenyl-32P-Labeled 3'-deoxy-NAD+ was utilized as a substrate by pure DNA-dependent poly(ADP-ribose)polymerase (EC 2.4.2.30) from calf thymus in the automodification reaction with an apparent Km of 20 microM and a Vmax of 80 nmol/min/mg of protein. Analysis by lithium lauryl sulfate-polyacrylamide gel electrophoresis revealed a single 32P-labeled protein of 116-kDa which comigrated with automodified enzyme. Addition of increasing amounts of histone H1 up to a concentration of 15 micrograms/ml stimulated the synthesis of protein-bound polymers of 3'-deoxy-ADP-ribose. However, the average polymer size was equal to 2 in the presence and 4 in the absence of histone H1, respectively. The synthesis of protein-bound oligomers of 3'-deoxy-ADP-ribose was inhibited by the polymerase inhibitors benzamide, nicotinamide, thymidine, and NaCl. A pulse labeling of polymer synthesis with 40 microM [32P]3'-deoxy-NAD+ either in the presence or absence of 15 micrograms/ml of histone H1, followed by a chase with 1 mM [3H]NAD+, was used to determine the mechanism of poly(ADP-ribose) elongation. Following enzyme digestion of these polymers with phosphodiesterase, it was found that 52 and 24% of the total 32P radiolabel was associated with the 3'-deoxy-AMP termini of the polymers synthesized in the pulse reactions, in the presence or absence of histone H1, respectively. In contrast, less than 10% of the total radioactivity was associated with 3'-deoxy-AMP in the product of the chase reactions. These results are consistent with the conclusion that the initially attached residue of 3'-deoxy-ADP-ribose to either the polymerase or histone H1, is elongated by the "protein-distal" addition of ADP-ribose residues to the AMP terminus of the growing polymer chain.     

Control of histone H1 dimer-poly(ADP-ribose) complex formation by poly(ADP-ribose) glycohydrolase

In nuclei incubated in vitro with [³H]NAD to promote poly(ADP-ribose) synthesis, about 6% of the polymer synthesized is differentially extracted into cold 5% PCA along with the H1 histone. Polyacrylamide gel electrophoresis of the extracts revealed large differences in the mobility of the incorporated radioactivity depending on the source of the nuclei used. With rat mammary tumors, the radioactivity co-migrated with the H1 histone on both acid-urea and SDS-urea gels. In contrast, the labeled polymer from HBL-100 mammary cell nuclei co-electrophoresed with a minor protein component which moved more slowly than H1. With lactating mammary glands, an intermediate profile was seen. The difference in mobility on the gels was found to be due to differences in the chain lengths of the poly(ADP-ribose) attached in the H1 protein. The difference in chain length produced was inversely related to the level of poly(ADP-ribose) degrading activity in the various nuclear preparations.     

Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V_max) and the michaelis constant (k_m) of PARG reaction were 4.46 μM and 128.33 μmol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 μM. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.     

Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase activity on poly(ADP-ribosyl)ated nucleosomal proteins

Hydrolysis of protein-bound 32P-labelled poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase shows that there is differential accessibility of poly(ADP-ribosyl)ated proteins in chromatin to poly(ADP-ribose) glycohydrolase. The rapid hydrolysis of hyper(ADP-ribosyl)ated forms of histone H1 indicates the absence of an H1 dimer complex of histone molecules. When the pattern of hydrolysis of poly(ADP-ribosyl)ated histones was analyzed it was found that poly(ADP-ribose) attached to histone H2B is more resistant than the polymer attached to histone H1 or H2A or protein A24. Polymer hydrolysis of the acceptors, which had been labelled at high substrate concentrations (greater than or equal to 10 microM), indicate that the only high molecular weight acceptor protein is poly(ADP-ribose) polymerase and that little processing of the enzyme occurs. Finally, electron microscopic evidence shows that hyper(ADP-ribosyl)ated poly(ADP-ribose) polymerase, which is dissociated from its DNA-enzyme complex, binds again to DNA after poly(ADP-ribose) glycohydrolase action.     

Altered poly(ADP-ribose) metabolism impairs cellular responses to genotoxic stress in a hypomorphic mutant of poly(ADP-ribose) glycohydrolase

Genotoxic stress activates nuclear poly(ADP-ribose) (PAR) metabolism leading to PAR synthesis catalyzed by DNA damage activated poly(ADP-ribose) polymerases (PARPs) and rapid PAR turnover by action of nuclear poly(ADP-ribose) glycohydrolase (PARG). The involvement of PARP-1 and PARP-2 in responses to DNA damage has been well studied but the involvement of nuclear PARG is less well understood. To gain insights into the function of nuclear PARG in DNA damage responses, we have quantitatively studied PAR metabolism in cells derived from a hypomorphic mutant mouse model in which exons 2 and 3 of the PARG gene have been deleted (PARG-Delta2,3 cells), resulting in a nuclear PARG containing a catalytic domain but lacking the N-terminal region (A domain) of the protein. Following DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we found that the activity of both PARG and PARPs in intact cells is increased in PARG-Delta2,3 cells. The increased PARG activity leads to decreased PARP-1 automodification with resulting increased PARP activity. The degree of PARG activation is greater than PARP, resulting in decreased PAR accumulation. Following MNNG treatment, PARG-Delta2,3 cells show reduced formation of XRCC1 foci, delayed H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. Our results show that a precise coordination of PARPs and PARG activities is important for normal cellular responses to DNA damage and that this coordination is defective in the absence of the PARG A domain.     

Structural and functional basis for ADP-ribose and poly(ADP-ribose) binding by viral macro domains

Macro domains constitute a protein module family found associated with specific histones and proteins involved in chromatin metabolism. In addition, a small number of animal RNA viruses, such as corona- and toroviruses, alphaviruses, and hepatitis E virus, encode macro domains for which, however, structural and functional information is extremely limited. Here, we characterized the macro domains from hepatitis E virus, Semliki Forest virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). The crystal structure of the SARS-CoV macro domain was determined at 1.8-Angstroms resolution in complex with ADP-ribose. Information derived from structural, mutational, and sequence analyses suggests a close phylogenetic and, most probably, functional relationship between viral and cellular macro domain homologs. The data revealed that viral macro domains have relatively poor ADP-ribose 1"-phosphohydrolase activities (which were previously proposed to be their biologically relevant function) but bind efficiently free and poly(ADP-ribose) polymerase 1-bound poly(ADP-ribose) in vitro. Collectively, these results suggest to further evaluate the role of viral macro domains in host response to viral infection.     

Determination of poly(ADP-ribose) chain length distribution on polyacrylamide gels by silver staining.

Silver staining and polyacrylamide gel electrophoresis were used to visualize chain length distribution of poly(ADP-ribose) enzymatically synthesized from NAD by rat liver nuclei. The method described has the advantage that synthesis does not require radioactive-labeled NAD, and microgram quantities (greater than 5 micrograms) of poly(ADP-ribose) can be resolved and visualized as discrete bands according to chain lengths which range from 8 to 60 residues. This method can be applied to estimate size distribution of poly(ADP-ribose) chains in cells or tissues.     

Reprogramming cellular events by poly(ADP-ribose)-binding proteins

Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions.     

Purification and characterization of poly(ADP-ribose) glycohydrolase. Different modes of action on large and small poly(ADP-ribose)

Poly(ADP-ribose) glycohydrolase was purified approximately 74,000-fold to apparent homogeneity from calf thymus with a yield of 3.2%. The enzyme was a monomeric protein of Mr = 59,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The action of glycohydrolase on poly(ADP-ribose) was exoglycosidic in the direction of adenosine terminus----ribose terminus; radioactive ADP-ribose monomers were immediately produced from evenly labeled poly(ADP-ribose), but not from the polymer labeled selectively at the ribose terminus. The enzymatic degradation of large poly(ADP-ribose) (greater than 20 ADP-ribose residues) proceeded in a biphasic as well as bimodal manner. In the early and rapid phase, the enzyme degraded part of large polymers successively, leaving the remainder completely intact, and accumulated ADP-ribose monomers and small polymers of the size less than half of original polymers, indicating that the enzyme action was processive up to a certain extent. In the late and 20-fold slower phase, by contrast, the enzyme degraded the accumulated small polymers gradually and evenly, i.e. in a nonprocessive manner. The Km for large polymers was approximately 100-fold lower than that for small polymers. Similar rates and processivities were observed with large and small polymers bound to various proteins. These results suggested that the glycohydrolase may regulate differentially the levels of large and small poly(ADP-ribose) in the cell.