Participation of Poly(ADP-ribose)-polymerase of nuclear matrix in DNA repair

The poly(ADP-ribose)-polymerase activity of brain and liver cell nuclei is changed during X-irradiation of rats. In the nuclear matrix, poly(ADP-ribose)-polymerase activity increases at a low dose of irradiation (1.7 Gy) and decreases at a high dose (6.7 Gy). A significant part of the activity of nuclear NMN-adenylyltransferase, a key enzyme for biosynthesis of NAD (the substrate of poly(ADP-ribose)-polymerase), has been found in the nuclear matrix. An interrelation between ADP-ribosylation taking place on the matrix level and eukaryotic cell DNA repair is suggested.     

Poly(ADP-ribose) in the cellular response to DNA damage

Poly(ADP-ribose) polymerase is a chromatin-bound enzyme which, on activation by DNA strand breaks, catalyzes the successive transfer of ADP-ribose units from NAD to nuclear proteins. Poly(ADP-ribose) synthesis is stimulated by DNA strand breaks, and the polymer may alter the structure and/or function of chromosomal proteins to facilitate the DNA repair process. Electronmicroscopic studies show that poly(ADP-ribose) unwinds the tightly packed nucleosomal structure of isolated chromatin. Recent studies also show that the presence of poly(ADP-ribose) enhances the activity of DNA ligase. This may increase the capacity of the cell to complete DNA repair. Inhibitors of poly(ADP-ribose) polymerase or deficiencies of the substrate, NAD, lead to retardation of the DNA repair process. When DNA strand breaks are extensive or when breaks fail to be repaired, the stimulus for activation of poly(ADP-ribose) persists and the activated enzyme is capable of totally consuming cellular pools of NAD. Depletion of NAD and consequent lowering of cellular ATP pools, due to activation of poly(ADP-ribose) polymerase, may account for rapid cell death before DNA repair takes place and before the genetic effects of DNA damage become manifest.     

Characterization of human poly(ADP-ribose) polymerase with autoantibodies

The addition of poly(ADP-ribose) chains to nuclear proteins has been reported to affect DNA repair and DNA synthesis in mammalian cells. The enzyme that mediates this reaction, poly(ADP-ribose) polymerase, requires DNA for catalytic activity and is activated by DNA with strand breaks. Because the catalytic activity of poly(ADP-ribose) polymerase does not necessarily reflect enzyme quantity, little is known about the total cellular poly(ADP-ribose) polymerase content and the rate of its synthesis and degradation. In the present experiments, specific human autoantibodies to poly(ADP-ribose) polymerase and a sensitive immunoblotting technique were used to determine the cellular content of poly(ADP-ribose) polymerase in human lymphocytes. Resting peripheral blood lymphocytes contained 0.5 X 10(6) enzyme copies per cell. After stimulation of the cells by phytohemagglutinin, the poly(ADP-ribose) polymerase content increased before DNA synthesis. During balanced growth, the T lymphoblastoid cell line CEM contained approximately 2 X 10(6) poly(ADP-ribose) polymerase molecules per cell. This value did not vary by more than 2-fold during the cell growth cycle. Similarly, mRNA encoding poly(ADP-ribose) polymerase was detectable throughout S phase. Poly(ADP-ribose) polymerase turned over at a rate equivalent to the average of total cellular proteins. Neither the cellular content nor the turnover rate of poly(ADP-ribose) polymerase changed after the introduction of DNA strand breaks by gamma irradiation. These results show that in lymphoblasts poly(ADP-ribose) polymerase is an abundant nuclear protein that turns over relatively slowly and suggest that most of the enzyme may exist in a catalytically inactive state.     

Changes in poly(ADP-ribose) level modulate the kinetics of DNA strand break rejoining

ADP-ribose polymers are rapidly synthesized in cell nuclei by the poly(ADP-ribose) polymerases PARP-1 and PARP-2 in response to DNA strand interruptions, using NAD(+) as precursor. The level of induced poly(ADP-ribose) formation is proportional to the level of DNA damage and can be decreased by NAD(+) or PARP deficiency, followed by poor DNA repair and genomic instability. Here we studied the correlation between poly(ADP-ribose) level and DNA strand break repair in lymphoblastoid Raji cells. Poly(ADP-ribose) synthesis was induced by 100 microM H(2)O(2) and intensified by the 1,4-dihydropyridine derivative AV-153. The level of poly(ADP-ribose) in individual cells was analyzed by quantitative in situ immunofluorescence and confirmed in whole-cell extracts by Western blotting, and DNA damage was assessed by alkaline comet assays. Cells showed a approximately 100-fold increase in poly(ADP-ribose) formation during the first 5 min of recovery from H(2)O(2) treatment, followed by a gradual decrease up to 15 min. This synthesis was completely inhibited by the PARP inhibitor NU1025 (100 microM) while the cells treated with AV-153, at non-genotoxic concentrations of 1 nM-10 microM, showed a concentration-dependent increase of poly(ADP-ribose) level up to 130% after the first minute of recovery. The transient increase in poly(ADP-ribose) level was strongly correlated with the speed and efficiency of DNA strand break rejoining (correlation coefficient r > or = 0.92, p<0.05). These results are consistent with the idea that poly(ADP-ribose) formation immediately after genome damage reflects rapid assembly and efficient functioning of repair machinery.     

PARP-2, A novel mammalian DNA damage-dependent poly(ADP-ribose) polymerase.

Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins in response to DNA damage that activates the base excision repair machinery. Poly(ADP-ribose) polymerase which we will now call PARP-1, has been the only known enzyme of this type for over 30 years. Here, we describe a cDNA encoding a 62-kDa protein that shares considerable homology with the catalytic domain of PARP-1 and also contains a basic DNA-binding domain. We propose to call this enzyme poly(ADP-ribose) polymerase 2 (PARP-2). The PARP-2 gene maps to chromosome 14C1 and 14q11.2 in mouse and human, respectively. Purified recombinant mouse PARP-2 is a damaged DNA-binding protein in vitro and catalyzes the formation of poly(ADP-ribose) polymers in a DNA-dependent manner. PARP-2 displays automodification properties similar to PARP-1. The protein is localized in the nucleus in vivo and may account for the residual poly(ADP-ribose) synthesis observed in PARP-1-deficient cells, treated with alkylating agents or hydrogen peroxide.     

Template specific inhibitor of mammalian DNA polymerases.

A study of the inhibition of mouse cellular DNA polymerases by poly-nucleotides and their vinyl analogs is presented. Poly(dT)-directed poly(dA) synthesis by representatives of all three classes of cellular DNA polymerase could be completely inhibited by poly(9-vinyladenine), although higher concentrations were required in the case of the gamma class enzyme. Studies on the mechanism of the inhibition using the alpha class DNA polymerase and different templates showed that the enzyme activity was inhibited in all cases where base-pairing between the vinyl polymer and the template occurred; poly(9-vinyladenine) did not interfere with the replication of templates to which it does not bind. The inhibition occurred shortly after addition of poly(9-vinyladenine) to ongoing reactions, yet the enzyme was not displaced from the template - primer complex.     

Effects of sodium azide on replicative and repair DNA synthesis in barley embryos

Sodium azide inhibited protein synthesis and replicative DNA synthesis at doses used for mutation induction (0.1–1 mM) in barley embryos. During subsequent treatment for 24 h using lower doses of azide, partial recovery from this inhibition was observed, especially in the case of replicative DNA synthesis. Contrary to expectation, cysteine acted as a weak enhancer of the inhibitory effect of azide on DNA replication. Under conditions of minimal replicative DNA synthesis, DNA-repair synthesis was detected after the azide action as measured by the BND-cellulose method. Benazzmide — an inhibitor of poly(ADP-ribose)-polymerase — stimulated the azide-dependent repair synthesis approximately 2-fold.     

Inhibition of poly(ADP-ribosyl)ation by overexpressing the poly(ADP-ribose) polymerase DNA-binding domain in mammalian cells

Poly(ADP-ribose) polymerase specifically recognizes DNA strand breaks by its DNA-binding domain. DNA binding activates the enzyme to catalyze the formation of poly(ADP-ribose) utilizing NAD as substrate. By a molecular genetic approach we set out to inhibit this enzyme activity in a highly specific manner, thus avoiding the inherent side effects of NAD analogs which have been used extensively as enzyme inhibitors. cDNA sequences coding for the human poly(ADP-ribose) polymerase DNA-binding domain were subcloned into eucaryotic expression plasmids and transiently transfected into monkey cells. Cells were fixed with ethanol followed by incubation with NAD. Indirect double immunofluorescence to detect both overexpressed protein and poly(ADP-ribose) in situ revealed that overexpression of the DNA-binding domain greatly inhibited poly(ADP-ribosyl)ation catalyzed by the resident enzyme during NAD postincubation. The same inhibition was observed when transfected cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine to induce DNA strand breaks in vivo and subjected to trichloroacetic acid/ethanol fixation and subsequent immunofluorescence analysis, a novel method we developed for the in situ detection of polymer synthesis in intact cells. This molecular genetic approach may prove to be a selective and efficient tool to investigate possible functions of poly(ADP-ribosyl)ation in living cells.     

TcPARP: A DNA damage-dependent poly(ADP-ribose) polymerase from Trypanosoma cruzi

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme present in most eukaryotes and has been involved in processes such as DNA repair and gene expression. The poly(ADP-ribose) polymer (PAR) is mainly catabolised by poly(ADP-ribose) glycohydrolase. Here, we describe the cloning and characterisation of a PARP from Trypanosoma cruzi (TcPARP). The recombinant enzyme (Mr=65) required DNA for catalytic activity and it was strongly enhanced by nicked DNA. Histones purified from T. cruzi increased TcPARP activity and the covalent attachment of [32P]ADP-ribose moieties to histones was demonstrated. TcPARP required no magnesium or any other metal ion cofactor for its activity. The enzyme was inhibited by 3-aminobenzamide, nicotinamide, theophylline and thymidine but not by menadione. We demonstrated an automodification reaction of TcPARP, and that the removal of attached PAR from this protein resulted in an increase of its activity. The enzyme was expressed in all parasite stages (amastigotes, epimastigotes and trypomastigotes). When T. cruzi epimastigotes were exposed to DNA-damaging agents such as hydrogen peroxide or beta-lapachone, PAR drastically increased in the nucleus, thus confirming PAR synthesis in vivo and suggesting a physiological role for PARP in trypanosomatid DNA repair signalling.     

Poly(ADP-ribose) synthesis and inhibition of DNA synthesis in Chinese hamster cells treated with methylating agents and thymidine

A possible role of poly(ADP-ribose) synthesis in modulating the response of V79 cells to DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) was investigated. Inhibition of [3H]thymidine (dThd) incorporation into DNA and lowering of NAD+ levels in intact cells were employed as parameters of DNA-synthesis inhibition and poly(ADP-ribose) synthesis, respectively. Dose responses of these parameters were studied in cells 2 and 24 h after treatment with the methylating agents in medium with or without dThd. The initial inhibition of DNA synthesis was uniformly associated with stimulation of poly(ADP-ribose) synthesis whether the cells were treated with MNNG or MMS, incubated with or without 20 microM dThd which did not inhibit poly(ADP-ribose) synthesis, or incubated with 3 mM dThd which did inhibit the latter synthesis. By contrast, the DNA-synthesis inhibition detected 24 h after treatment with MNNG was not associated with poly(ADP-ribose) synthesis. These data suggest that (i) the mechanism of this later inhibition of DNA synthesis is different from that of the initial inhibition, (ii) DNA-synthesis inhibition does not stimulate poly(ADP-ribose) synthesis, and (iii) single-strand breaks, resulting from N-methylation of the DNA, stimulate poly(ADP-ribose) synthesis, which may produce the initial inhibition of DNA synthesis. The initial inhibition of DNA synthesis was not uniformly associated with mutagenesis and dThd facilitation of MNNG-induced cytotoxicity and mutagenesis. This indicates that O-methylation of DNA does not stimulate poly(ADP-ribose) synthesis. Our data suggest that, in V79 cells treated with methylating agents, poly(ADP-ribose) synthesis is stimulated by single-strand breaks, inhibits DNA synthesis, and thereby serves to allow time for repair of the DNA prior to replication.     

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.     

Inhibition of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro

Incubation of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furthermore, the suppressed activities of DNA polymerase alpha, DNA polymerase beta, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes.     

Unscheduled synthesis of DNA and poly(ADP-ribose) in human fibroblasts following DNA damage

Unscheduled DNA synthesis has been measured in human fibroblasts under conditons of reduced rates of conversion of NAD to poly(ADP-ribose). Cells heterozygous for the xeroderma pigmentosum genotype showed normal rates of UV induced unscheduled DNA synthesis under conditions in which the rate of poly(ADP-ribose) synthesis was one-half the rate of normal cells. The addition of theophylline, a potent inhibitor of poly(ADP-ribose) polymerase, to the culture medium of normal cells blocked over 90% of the conversion of NAD to poly(ADP-ribose) following treatment with UV or N-methyl-N′-nitro-N-nitro-soguanidine but did not affect the rate of unscheduled DNA synthesis.     

Altered DNA damage response in Caenorhabditis elegans with impaired poly(ADP-ribose) glycohydrolases genes expression

Poly(ADP-ribosyl)ation is one of the first cellular responses induced by DNA damage. Poly(ADP-ribose) is rapidly synthesized by nick-sensor poly(ADP-ribose) polymerases, which facilitate DNA repair enzymes to process DNA damage. ADP-ribose polymers are rapidly catabolized into free ADP-ribose units by poly(ADP-ribose) glycohydrolase (PARG). The metabolism of poly(ADP-ribose) is a well-defined biochemical process for which a physiological role in animals is just beginning to emerge. Two Caenorhabditis elegans PARGs, PME-3 and PME-4, have been cloned by our group. The pme-3 gene encodes an enzyme of 89kDa having less than 18% overall identity with human PARG but 42% identity with the PARG signature motif. The pme-4 gene codes for a PARG of 55kDa with approximately 22% overall identity with human PARG and 40% identity with the PARG signature motif. Two alternatively spliced forms of PME-3 were identified with an SL1 splice leader on both forms of the mRNA and were found to be expressed throughout the worm's life cycle. Similarly, pme-4 was shown to be expressed in all developmental stages of the worm. Recombinant enzymes that were expressed in bacteria displayed a PARG activity that may partly account for the PARG activity measured in the total worm extract. Reporter gene analysis of pme-3 and pme-4 using a GFP fusion construct showed that pme-3 and pme-4 are mainly expressed in nerve cells. PME-3 was shown to be nuclear while PME-4 localized to the cytoplasm. Worms with pme-3 and pme-4 expression knocked-down by RNAi showed a significant sensitivity toward ionizing radiations. Taken together, these data provide evidence for a physiological role for PARGs in DNA damage response and survival. It also shows that PARGs are evolutionarily conserved enzymes and that they are part of an ancient cellular response to DNA damage.     

Depletion of the 110-kilodalton isoform of poly(ADP-ribose) glycohydrolase increases sensitivity to genotoxic and endotoxic stress in mice

Poly(ADP-ribosylation) is rapidly stimulated in cells following DNA damage. This posttranslational modification is regulated by the synthesizing enzyme poly(ADP-ribose) polymerase 1 (PARP-1) and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG). Although the role of PARP-1 in response to DNA damage has been studied extensively, the function of PARG and the impact of poly(ADP-ribose) homeostasis in various cellular processes are largely unknown. Here we show that by gene targeting in embryonic stem cells and mice, we specifically deleted the 110-kDa PARG protein (PARG(110)) normally found in the nucleus and that depletion of PARG(110) severely compromised the automodification of PARP-1 in vivo. PARG(110)-deficient mice were viable and fertile, but these mice were hypersensitive to alkylating agents and ionizing radiation. In addition, these mice were susceptible to streptozotocin-induced diabetes and endotoxic shock. These data indicate that PARG(110) plays an important role in DNA damage responses and in pathological processes.     

Relationship between poly(ADP-ribose) polymerase activity and DNA synthesis in cultured hepatocytes

Previous studies have demonstrated that an increase in poly(ADP-ribose) polymerase activity could be closely related to DNA replication during liver regeneration and to DNA repair synthesis in different experimental systems. This relationship was further investigated by studying the time course of endogenous and total poly(ADP-ribose) polymerase activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. A 6-fold increase in endogenous activity was observed early after epidermal growth factor addition, just before DNA synthesis. A subsequent 4-fold increment in total enzyme activity, concomitant with DNA synthesis, was detected. Orotic acid, which has recently shown mitoinhibitory effect, abolished the epidermal-growth-factor-induced increase in endogenous and total poly(ADP-ribose) polymerase activity, as well as DNA synthesis. On the contrary, 3-aminobenzamide inhibitor of poly(ADP-ribose) polymerase completely suppressed the endogenous activity but only partially modified the increase in total catalytic level and the overall pattern of thymidine incorporation. Taken together, these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by an increased poly(ADP-ribose) polymerase activity.     

Activation of poly(adenosine diphosphate ribose) polymerase by SV 40 minichromosomes: effects of deoxyribonucleic acid damage and histone H1

Poly(ADP-ribose) polymerase is a chromosomal enzyme that is completely dependent on added DNA for activity. The ability of DNA molecules to activate the polymerase appears to be enhanced by the presence of DNA damage. In the present study, we used SV 40 DNA and SV 40 minichromosomes to determine whether different types of DNA damage and different chromosomal components affect stimulation of polymerase activity. Treatment of SV 40 minichromosomes with agents or conditions that induced single-strand breaks increased their ability to stimulate poly(ADP-ribose) synthesis. This stimulation was enhanced by addition of histone H1 at a ratio of 1 microgram of histone H1 to 1 microgram of DNA. Higher ratios of histone H1 to DNA suppressed the ability of SV 40 minichromosomes containing single-strand breaks to stimulate enzyme activity. Treatment of SV 40 minichromosomes or SV 40 DNA with HaeIII restriction endonuclease to produce double-strand breaks markedly stimulated poly(ADP-ribose) polymerase activity. The stimulation of poly(ADP-ribose) polymerase by double-strand breaks occurred in the absence of histone H1 and was further enhanced by adding histone H1 up to ratios of 2 to 1 relative to DNA. At higher ratios of histone H1 to DNA, the presence of the histone continued to enhance the poly(ADP-ribose) synthesis stimulated by double-strand breaks.