Effect of N-2-acetylaminofluorene on poly(ADP-ribose) polymerase activity and DNA synthesis in cultured rat hepatocytes exposed to epidermal growth factor.

The nuclear enzyme poly(ADP-ribose) polymerase is involved in basic cellular processes such as DNA replication and repair, cell differentiation and transformation, gene expression. We have studied the effect of 2AAF, a genotoxic aromatic amine, on pADPRP activity during DNA synthesis stimulated by EGF, using the cultured rat hepatocytes model. DNA synthesis was measured as [3H]thymidine incorporated/microgram DNA while pADPRP activity was expressed in pmol[32P]NAD incorporated/min/microgram DNA. Our results show that 2AAF treatment of EGF-stimulated rat hepatocytes induces a full block of DNA replication which is preceded and accompanied by a net inhibition of endogenous and total pADPRP activity, respectively. A block in pADPRP activity in normal hepatocytes, exposed to 2AAF in vitro or in vivo, could play a key role in cell transformation. Our data add further information on the possible involvement of this nuclear catalytic activity during DNA replication.     

Simulated microgravity conditions affect poly(ADP-ribose) polymerase activity in cultured human lymphocytes

Human peripheral blood lymphocytes (PBL), activated with concanavalin A (ConA), were used to determine the effects of simulated microgravity on poly(ADP-ribose) polymerase (PARP) activity. Results indicate that the ConA stimulation of human cultured PBL induces a partial but signitficant inhibition of PARP-1 acitvity (-30%). In control PBL, not exposed to ConA, after 24 hours, there was a clear decrease in PARP-1 acitivty (-40%). In PBL exposed to ConA and simulated weightlessness, activity decreased by -37%.     

Molecular interactions between DNA, poly(ADP-ribose) polymerase, and histones

Molecular interactions between purified poly(ADP-ribose) polymerase, whole thymus histones, histone H1, rat fibroblast genomic DNA, and closed circular and linearized SV40 DNA were determined by the nitrocellulose filter binding technique. Binding of the polymerase protein or histones to DNA was augmented greatly when both the enzyme protein and histones were present simultaneously. The polymerase protein also associated with histones in the absence of DNA. The cooperative or promoted binding of histones and the enzyme to relaxed covalently closed circular SV40 DNA was greater than the binding to the linearized form. Binding of the polymerase to SV40 DNA fragments in the presence of increasing concentrations of NaCl indicated a preferential binding to two restriction fragments as compared to the others. Polymerase binding to covalently closed relaxed SV40 DNA resulted in the induction of superhelicity. The simultaneous influence of the polymerase and histones on DNA topology were more than additive. Topological constraints on DNA induced by poly(ADP-ribose) polymerase were abolished by auto ADP-ribosylation of the enzyme. Benzamide, by inhibiting poly(ADP-ribosylation), reestablished the effect of the polymerase protein on DNA topology. Polymerase binding to in vitro-assembled core particle-like nucleosomes was also demonstrated.     

Poly(ADP-ribose) polymerase activity in mouse cells which exhibit temperature-sensitive DNA synthesis

The poly(ADP-ribose) polymerase activity of wild-type mouse L cells and of Balb/C-3T3 mouse fibroblasts remained relatively unchanged (at approx. 400 nmol substrate utilized/mg DNA per h) in actively-growing cells incubated at 34 degrees C or at 38.5 degrees C for at least 72 h. A similar result was obtained with the following temperature-sensitive cells grown at the permissive temperature (34 degrees C): ts A1S9 mouse L cells, ts C1 mouse L cells and Balb/C-3T3 ts mouse fibroblasts. The poly(ADP-ribose) polymerase activity of the temperature-sensitive cells was little affected during incubation for 20-24 h at the non-permissive temperature of 38.5 degrees C under which conditions temperature-inactivation of DNA replication was complete. Thereafter, this enzyme activity was found to increase some 2-fold, at a time when normal semi-conservative DNA synthesis was totally suppressed and replaced by repair replication (Sheinin, R. and Guttman, S. (1977) Biochim. Biophys. Acta 479, 105-118; Sheinin, R., Dardick, I. and Doane, F.W. (1980) Exp. Cell. Res., in the press).     

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     

Acetaldehyde activation of poly(ADP-ribose)polymerase in hepatocytes of mice treated in vivo

The activities of poly(ADP-ribose) polymerase and of DNA polymerases alpha and beta and the level of cytochrome P450 were determined in mouse parenchymal liver cells 5 h after treatment with 0.1, 0.3, 1.0, and 3.0 mumole of acetaldehyde. Injection with 1.0 and 3.0 mumole of acetaldehyde induced an increase in poly(ADP-ribose) polymerase activity and in the P450 level, but had no effect on DNA polymerases. The stimulation of poly(ADP-ribose) polymerase activity can be used as an index of induced DNA damage. The possibility of using this experimental approach with other cells derived from mice treated in vivo with different xenobiotics is discussed.     

Influence of poly(ADP-ribose) polymerase on the enzymatic synthesis of SV40 DNA.

The influence of poly(ADP-ribose) polymerase (PARP) on the replication of DNA containing the SV40 origin of replication has been examined. Extensive replication of SV40 DNA can be carried out in the presence of T antigen, topoisomerase I, the multimeric human single strand DNA-binding protein (HSSB), and DNA polymerase alpha-DNA primase (pol alpha-primase) complex (the monopolymerase system). In the monopolymerase system, both small products (Okazaki fragments), arising from lagging strand synthesis, and long products, arising from leading strand synthesis, are formed. The synthesis of long products requires the presence of relatively high levels of pol alpha-primase complex. In the presence of PARP, the synthesis of long products was blocked and only small Okazaki fragments accumulated, arising from the replication of the lagging strand template. The inhibition of leading strand synthesis by PARP can be effectively reversed by supplementing the monopolymerase system with the multimeric activator 1 protein (A1), the proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta (the dipolymerase system). The inhibition of leading strand synthesis in the monopolymerase system was caused by the binding of PARP to the ends of DNA chains, which blocked their further extension by pol alpha. The selective accumulation of Okazaki fragments was shown to be due to the coupled synthesis of primers by DNA primase and their immediate extension by pol alpha complexed to primase. PARP had little effect on this coupled reaction, but did inhibit the subsequent elongation of products, presumably after pol alpha dissociated from the 3'-end of the DNA fragments. PARP inhibited several other enzymatic reactions which required free ends of DNA chains. PARP inhibited exonuclease III, DNA ligase, the 5' to 3' exonuclease, and the elongation of primed DNA templates by pol alpha. In contrast, PARP only partly competed with the elongation of primed DNA templates by the pol delta elongation system which required SSB, A1, and PCNA. These results suggest that the binding of PARP at the ends of nascent DNA chains can be displaced by the binding of A1 and PCNA to primer ends. HSSB can be poly(ADP-ribosylated) in vivo as well as in vitro. However, the selective effect of PARP in blocking leading strand synthesis in the monopolymerase system was shown to depend primarily on its DNA binding property rather than on its ability to synthesize poly(ADP-ribose).     

Effect of simulated microgravity conditions on poly(ADP-ribose) polymerase activity in primary cultures of adult rat hepatocytes

The possible involvement of poly(ADP-ribose) polymerase [PARP; E.C. 2.4.2.30] in the adaptive response to low-g conditions was studied in cultured adult rat hepatocytes exposed to simulated microgravity produced by the random positioning machine (RPM-3D-clinostat). Four different poly(ADP-ribose) polymerases (PARPs) have been identified recently. The best-studied member of this family is PARP-1, a highly conserved, multimodular 113 kDa protein. In multicellular organisms PARPs catalyze poly(ADP-ribose) synthesis from NAD+ to a number of structural and catalytic proteins. Moreover, PARP-1 can control its protein and DNA interactions by catalyzing its automodification with poly(ADP-ribose) molecules that can include up to 200 ADP-ribose residues and several branching points; by these polymers, PARP-1 may nocovalently interact with other proteins and alter their functions. PARP-1 binds to DNA and is activated by free ends interacting with several other DNA damage checkpoint proteins. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions. Poly(ADP-ribosyl)ation plays a central role in genome stability and is involved in DNA replication and repair, gene expression, cell differentiation and transformation. We have shown that a loss of PARP-1 activity is a critical event in the early molecular steps of the hepatocarcinogenesis process. Moreover, a prompt increase in this enzymatic activity is linked not only to the presence of DNA free ends but is linked also to the start of DNA synthesis. More recently, we have reported that PARP-1 is involved in hormone-mediated gene expression in vitro and in vivo during rat liver regeneration.     

Increased ionic strength: effects on DNA fragmentation and poly(ADP-ribose) polymerase activity in HeLa nuclei

Poly(ADP-ribose) polymerase activity was measured in a crude nuclear fraction isolated from HeLa cells. It was found that the addition of ammonium sulfate or other salts to the standard incubation medium inhibited the formation of poly(ADP-ribose). Through the use of alkaline sucrose density gradients it was also noted that this same increase in ionic strength inhibited the in vitro breakdown of the HeLa DNA. Additional experiments with alkaline sucrose density gradients and deoxyribonuclease I showed that the in vitro activity of poly(ADP-ribose) polymerase is largely dependent upon DNA fragmentation but that DNA fragmentation at least in vitro is not dependent upon the formation of poly(ADP-ribose). These observations imply that this nuclear enzyme is not extremely sensitive to changes in the ionic strength of the reaction media but is affected indirectly, supposedly through changes in the endonuclease activity of the HeLa nuclei. If this proves to be true, then the addition of salt to the incubation medium for poly(ADP-ribose) polymerase could prove to be a valuable tool for the study of ADP-ribosylation reactions.     

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.     

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.     

Human base excision repair enzymes apurinic/apyrimidinic endonuclease1 (APE1), DNA polymerase beta and poly(ADP-ribose) polymerase 1: interplay between strand-displacement DNA synthesis and proofreading exonuclease activity

We examined interactions between base excision repair (BER) DNA intermediates and purified human BER enzymes, DNA polymerase β (pol β), apurinic/apyrimidinic endonuclease (APE1) and poly(ADP-ribose) polymerase-1 (PARP-1). Studies under steady-state conditions with purified BER enzymes and BER substrates have already demonstrated interplay between these BER enzymes that is sensitive to the respective concentrations of each enzyme. Therefore, in this study, using conditions of enzyme excess over substrate DNA, we further examine the question of interplay between BER enzymes on BER intermediates. The results reveal several important differences compared with data obtained using steady-state assays. Excess PARP-1 antagonizes the action of pol β, producing a complete block of long patch BER strand-displacement DNA synthesis. Surprisingly, an excess of APE1 stimulates strand-displacement DNA synthesis by pol β, but this effect is blocked by PARP-1. The APE1 exonuclease function appears to be modulated by the other BER proteins. Excess APE1 over pol β may allow APE1 to perform both exonuclease function and stimulation of strand-displacement DNA synthesis by pol β. This enables pol β to mediate long patch sub-pathway. These results indicate that differences in the stoichiometry of BER enzymes may regulate BER.     

Regulatory mechanisms of poly(ADP-ribose) polymerase

Here, we describe the latest developments on the mechanistic characterization of poly(ADP-ribose) polymerase (PARP) [EC 2.4.2.30], a DNA-dependent enzyme that catalyzes the synthesis of protein-bound ADP-ribose polymers in eucaryotic chromatin. A detailed kinetic analysis of the automodification reaction of PARP in the presence of nicked dsDNA indicates that protein-poly(ADP-ribosyl)ation probably occurs via a sequential mechanism since enzyme-bound ADP-ribose chains are not reaction intermediates. The multiple enzymatic activities catalyzed by PARP (initiation, elongation, branching and self-modification) are the subject of a very complex regulatory mechanism that may involve allosterism. For instance, while the NAD+ concentration determines the average ADP-ribose polymer size (polymerization reaction), the frequency of DNA strand breaks determines the total number of ADP-ribose chains synthesized (initiation reaction). A general discussion of some of the mechanisms that regulate these multiple catalytic activities of PARP is presented below.     

Cytological detection of poly (ADP-ribose) polymerase

An attempt was made to demonstrate poly (ADP-ribose) polymerase cytologically. In vitro incorporation from the nucleotide, [³H]NAD was detected in frozen sections of onion embryo and meristematic tissue by autoradiography. In meristematic tissue, there was a correlation between the number of cells displaying intensein vitro incorporation from [³H]NAD and cytological DNA polymerase activity. Performed enzymes effecting a distinct incorporation from [³H]NAd were localized in the nuclei of all tissues of the ungerminated seed except the endosperm. Evidence for poly (ADP-ribose) polymerase has been obtained for the first time from higher plant cells and localized cytologically.