Expression of human poly(ADP-ribose) polymerase in Saccharomyces cerevisiae

The coding sequence for human poly(ADP-ribose) polymerase was expressed inducibly in Saccharomyces cerevisiae from a low-copy-number plasmid vector. Cell free extracts of induced cells had poly(ADPribose) polymerase activity when assayed under standard conditions; activity could not be detected in non-induced cell extracts. Induced cells formed poly(ADP-ribose) in vivo, and levels of these polymers increased when cells were treated with the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The cytotoxicity of this agent was increased in induced cells, and in vivo labelling with [³H]adenine further decreased their viability. Increased levels of poly(ADP-ribose) found in cells treated with the alkylating agent were not accompanied by lowering of the NAD concentration.     

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     

Nuclear incorporation of [adenine 14C]NAD is altered by compounds which affect poly(ADP-ribose) formation.

The use of a DNA alkylating agent, which induces poly(ADP-ribose) formation, has been employed to study the incorporation of [adenine 14C]NAD into pea root meristem nuclei, which is a prerequisite for poly(ADP-ribose) synthesis. The incorporation of [adenine 14C]NAD is significantly reduced when the poly(ADP-ribose)polymerase inhibitors, 7-methylxanthine and 3-methoxybenzamide are present and this incorporation is augmented when the DNA alkylating agent methyl methanesulfonate is added. Such information supports the hypothesis that poly(ADP-ribose) may be involved in the cell cycle regulation of pea root meristem nuclei.     

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.     

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.     

The time-dependent effect of 2,2′-dichlorodiethyl sulfide (sulfur mustard, HD, 1,1′-thiobis [2-chloroethane]) on the lymphocyte viability and the kinetics of protection by poly(ADP-ribose) polymerase inhibitors

2,2-Dichlorodiethyl sulfide (sulfur mustard, HD, 1,1-thiobis [2-chloroethane]) is a potent vesicant which can cause severe lesions to skin, lung, and eyes. Due to the high number of debilitating exposures during the Iran — Iraq war to the alkylating agent, HD, there is an increased interest in its mechanism of action and in the development of therapeutic interventions to prevent HD-induced lesions. Recently we reported anin vitro assay using human mononuclear leukocytes for studying HD-induced pathology. To study the time dependence of HD-induced mononuclear leukocyte cell death and to determine the parameters of any potential therapeutic intervention, an assay was developed and automated using a flow cytometer to measure propidium iodide exclusion by mononuclear cells. This assay demonstrated that HD-initiated cell death did not begin before 4 h post-exposlure, but after 4 h proceeded in a concentration-dependent manner. In this assay both niacinamide and 3-aminobenzamide, poly(ADP-ribose) polymerase inhibitors, were shown to be effective in blocking HD-induced cell death when added to the cultures during the first 4 h post-exposure. They offered partial protection when added between 6 and 12 h and were of no benefit when added after 12 h post-exposure.Abbreviations ADP adenosine diphosphate - DNA deoxyribonucleic acid - HD 2,2-dichlorodiethyl sulfide - NAD nicotinamide-adenine dinucleotide - PI propidium iodide - SD standard deviation     

Activation of Cysteine Proteases in Cowpea Plants during the Hypersensitive Response-A Form of Programmed Cell Death

There is increasing evidence that the hypersensitive response during plant–pathogen interactions is a form of programmed cell death. In an attempt to understand the biochemical nature of this form of programmed cell death in the cowpea–cowpea rust fungus system, proteolytic activity in extracts of fungus-infected and uninfected cowpea plants was investigated, using exogenously added poly(ADP-ribose) polymerase as a marker. Unlike the proteolytic cleavage pattern of endogenous poly(ADP-ribose) polymerase in apoptotic animal cells, exogenously added poly(ADP-ribose) polymerase in extracts of fungus-infected plants was proteolytically cleaved into fragments of molecular masses 77, 52, 47, and 45 kDa.In vitroandin vivoprotease inhibitor experiments revealed the activation of cysteine proteases, and possibly a regulatory role, during the hypersensitive response.     

Cellular regulation of poly ADP-ribosylation of proteins: II. Augmentation of poly(ADP-ribose) polymerase in SV40 3T3 cells following methotrexate-induced G1/S inhibition of cell cycle progression

SV40-3T3 cells were exposed in monolayer cultures to 5 × 10⁻⁷ M methotrexate (MTX), that inhibited thymidylate synthetase, arrested cell growth without cell killing in 24 h and did not induce single- (ss) or double-strand (ds) breaks in DNA. Following 24, up to 72 h, the poly(ADP-ribose) polymerase content of attached cells was induced by 5 × 10⁻⁷ M MTX and the augmentation of the enzyme increased with the time of exposure to the drug. Inhibition of protein or RNA synthesis abolished augmentation of enzymatic activity; so too did the initiation of maximal cell growth by thymidine + hypoxanthine, by-passing the inhibitory site of MTX. Isolation of the ADP-ribosylated enzyme protein by gel electrophoresis identified poly(ADP-ribose) polymerase protein as the molecule that was induced by 5 × 10⁻⁷ M MTX. Under identical conditions, the poly(ADP-ribose) polymerase induction in 3T3 cells could not be demonstrated. A possible cell-cycle-dependent biosynthesis of the enzyme protein is proposed in SV40 3T3 cells.     

Poly(ADP-ribosylation) at successive stages of rooster spermatogenesis. Levels of polymeric ADP-ribose in vivo and poly(ADP-ribose) polymerase activity and turnover of ADP-ribosyl residues in vitro

Rooster testis cells were separated by sedimentation at unit gravity and the in vivo levels of polymeric ADP-ribose were determined both in intact cells and isolated nuclei by fluorescence methods. Poly(ADP-ribose) polymerase activity was assayed after cell permeabilization or after isolation of nuclei. The turnover of ADP-ribosyl residues was determined in isolated nuclei using benzamide. The content of poly(ADP-ribose), the poly(ADP-ribose) polymerase activity, and the turnover of ADP-ribosyl residues, decreased during the differentiation of the germinal cell line, especially at the end of spermiogenesis. Treatment of cells with 1 mM dimethyl sulfate for 1 h resulted in a marked stimulation of poly(ADP-ribose) polymerase activity in meiotic and premeiotic cells and also in round and late spermatids. The enzymatic activity was not detected and could not be induced in mature spermatozoa. These cells, however, still contained polymeric ADP-ribose with a 2% of branched form.     

MNNG-induced cell death is controlled by interactions between PARP-1, poly(ADP-ribose) glycohydrolase, and XRCC1

PARP-1 (poly(ADP-ribose) polymerases) modifies proteins with poly(ADP-ribose), which is an important signal for genomic stability. ADP-ribose polymers also mediate cell death and are degraded by poly(ADP-ribose) glycohydrolase (PARG). Here we show that the catalytic domain of PARG interacts with the automodification domain of PARP-1. Furthermore, PARG can directly down-regulate PARP-1 activity. PARG also interacts with XRCC1, a DNA repair factor that is recruited by DNA damage-activated PARP-1. We investigated the role of XRCC1 in cell death after treatment with supralethal doses of the alkylating agent MNNG. Only in XRCC1-proficient cells MNNG induced a considerable accumulation of poly(ADP-ribose). Similarly, extracts of XRCC1-deficient cells produced large ADP-ribose polymers if supplemented with XRCC1. Consequently, MNNG triggered in XRCC1-proficient cells the translocation of the apoptosis inducing factor from mitochondria to the nucleus followed by caspase-independent cell death. In XRCC1-deficient cells, the same MNNG treatment caused non-apoptotic cell death without accumulation of poly(ADP-ribose). Thus, XRCC1 seems to be involved in regulating a poly(ADP-ribose)-mediated apoptotic cell death.     

Inhibition of cell proliferation in Saccharomyces cerevisiae by expression of human NAD+ ADP-ribosyltransferase requires the DNA binding domain ("zinc fingers").

Summary Constitutive expression of human nuclear NAD⁺: protein ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose)polymerase; EC 2.4.2.30] as an active enzyme in Saccharomyces cerevisiae, under the control of the alcohol dehydrogenase promoter, was only possible with simultaneous inhibition of ADP-ribosylation by 3-methoxybenzamide. Induction of fully active pADPRT from the inducible galactose epimerase promoter resulted in inhibition of cell division and morphological changes reminiscent of cell cycle mutants. Expression of a pADPRT cDNA truncated at its 5end had no influence on cell proliferation at all. Obviously the amino-terminal part of the DNA binding domain containing the first zinc finger, which is essential for inducibility of pADPRT activity by DNA breaks, is also required for inhibition of cell growth on expression in yeast. Full-length as well as truncated pADPRT molecules were directed to the cell nucleus where the fully active enzyme produced large amounts of poly(ADP-ribose) by automodification. Since pADPRT turned out to be the only target for ADP-ribosylation in these cells, elevated levels of poly(ADP-ribose) were the most likely cause of inhibition of cell division, presumably resulting from interaction with chromosomal proteins.     

Catalytic activities of human poly(ADP-ribose) polymerase from normal and mutagenized cells detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

An activity gel procedure is described to identify functional polypeptides of human poly(ADP-ribose) polymerase. Purified or crude enzyme preparations from HeLa cells were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels containing gapped DNA. After renaturation of the peptides in situ, the intact gel was incubated in a poly(ADP-ribose) polymerase reaction mixture containing [32P]NAD. Autoradiograms of the gels consistently exhibited a major activity band at Mr = 116,000-120,000; in many runs, three minor distinct bands at Mr = 125,000, 135,000, and 145,000 were also seen. [32P]NAD appeared to be incorporated into poly(ADP-ribose) since: (i) the activity bands were not detectable when the enzyme-inhibitor 3-aminobenzamide was added to the gel incubation mixture; and (ii) the radioactive polymer, electroeluted from the bands, was completely digested by phosphodiesterase I. Preliminary activity gel analysis of extracts of HeLa cells treated with different DNA-damaging agents revealed that the apparent activity of the Mr = 116,000 form increased by about 10-fold in cells treated with 1 mM dimethyl sulfate and 10-20-fold in cells treated with 10 microM mitomycin C. Only a small increase was obtained in cells treated with 1 mM methyl methanesulfonate, and no change in the activity band pattern was observed after 50 and 100 J/m-2 of UV irradiation.     

Effect of poly(ADP-ribose)polymerase inhibitors on the frequency of sister-chromatid exchanges in Bloom syndrome cells

The effect of inhibitors of poly(ADP-ribose)polymerase, benzamide (Bam) and m-aminobenzamide (m-AB), on sister-chromatid exchanges (SCEs) and cell growth, was examined in lymphoblastoid cell lines from a normal adult (KS-64) and from a Bloom syndrome patient (BS1-2). The presence of Bam and m-AB increased the levels of SCEs in KS-64 and BS1-2 lymphoblastoid cells. Though the net increase was similar in the two types of cell, the relative increase was much lower in the BS1-2 cells. Bam and m-AB increased the number of SCEs in BS1-2 cells to levels of 95.4 +/- 3.24 and 98.1 +/- 3.23 per cell, respectively, as compared with the baseline level of 75.5 +/- 2.16. On the other hand, when KS-64 cells were treated with Bam and m-AB, the number of SCEs increased to 27.1 +/- 1.98 and 28.6 +/- 2.71 per cell, respectively, compared with the baseline number of 6.7 +/- 0.41 per cell. These inhibitors of poly(ADP-ribose)polymerase also inhibited cell growth at concentrations which induced SCEs in KS-64 as well as in BS1-2 cells. No significant decrease in the poly(ADP-ribose)polymerase activity or in the amount of poly-(ADP-ribose) was detected in BS1-2 cells as compared with KS-64 cells. The mechanism by which SCEs are increased in BS1-2 cells is discussed.     

Poly(ADP-ribose) polymerase: Structural conservation among different classes of animals and its implications

Poly(ADP-ribose) polymerase cDNAs have been isolated from different classes of animals. Cloning of genes from lower eukaryotes has allowed us to investigate directly the biological functions of poly(ADP-ribosyl)ationin vivo. The conservation of specific regions among mammals, chicken,Xenopus laevis, andDrosophila melanogaster reveals the essential structural elements required for recognition of breaks in DNA and for catalytic activity. Cys, His and basic residues in the zinc-finger consensus region are conserved. The carboxyl terminal region corresponding to an NAD-binding domain is strongly conserved. The dinucleotide-binding consensus sequence and 1-A-2, Rossmann fold structure, and -sheet structures are completely conserved from mammals to insect. InDrosophila, a putative leucine-zipper motif has been identified, and other poly(ADP-ribose) polymerases also contain an -helical, amphipathic structure in the auto-modification domain. In this article, we review the recent structural analyses of the functional domains of poly(ADP-ribose) polymerase in phylogenetically divergent species, and discuss the implications of structural conservation for its biological functions.Abbreviations aa amino acid(s) - D. melanogaster Drosophila melanogaster - PARP poly(ADP-ribose) polymerase [EC 2.4.2.30] - PCR polymerase chain reaction - X. laevis Xenopus laevis     

Evidence for an involvement of glutamine synthetase in regulation of nitrogenase activity in Rhodopseudomonas capsulata

In the presnet studies with whole cells and extracts of the photosynthetic bacterium Rhodopseudomonas capsulata the rapid inhibition of nitrogenase dependent activities (i.e. N₂-fixation acetylene reduction, or photoproduction of H₂) by ammonia was investigated. The results suggest, that the regulation of the nitrogenase activity by NH ₄ ⁺ in R. capsulata is mediated by glutamine synthetase (GS). (i) The glutamate analogue methionine sulfoximine (MSX) inhibited GS in situ and in vitro, and simultaneously prevented nitrogenase activity in vivo. (ii) When added to growing cultures ammonia caused rapid adenylylation of GS whereas MSX abolished the activity of both the adenylylated and unadenylylated form of the enzyme. (iii) Recommencement of H₂ production due to an exhaustion of ammonia coincided with the deadenylylation of GS. (iv) In extracts, the nitrogenase was found to be inactive only when NH ₄ ⁺ or MSX were added to intact cells. Subsequently the cells had to be treated with cetyltrimethylammonium bromide (CTAB). (v) In extracts the nitrogenase activity declined linearily with an increase of the ration of adenylylated vs. deadenylylated GS. A mechanism for inhibition of nitrogenase activity by ammonia and MSX is discussed.Abbreviations BSA bovin serum albumine - CTAB cetyltrimethylammonium bromide - GOGAT l-glutamine: 2-oxoglutarate amino transferase - GS glutamine synthetase - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MSX l-methionine-d,l-sulfoximine     

Trehalase activity in mycorrhizal and nonmycorrhizal roots of leek and soybean

Root extracts of leek (Allium porrum L.) and soybean (Glycine max L. Merr.) showed trehalase activity which was inhibited by phloridzin and was several times higher than the activity of general -glucosidase. The activity had an acidic optimum. Trehalase activity in extracts of sporocarps and extraradical mycelium of the arbuscular mycorrhizal fungus Glomus mosseae Nicol. & Gerd. (Trappe & Gerd.) was higher than in root extracts and had an optimum at pH 7. Following inoculation with G. mosseae, trehalase activity increased in mycorrhizal roots above the levels observed in nonmycorrhizal roots. Irrespective of fungal colonization, root trehalase activity increased in the presence of Mg²⁺, decreased in the presence of Mn²⁺ and Zn²⁺, and was unaffected by Na₂EDTA.     

Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.

The zinc-finger DNA-binding domain (DBD) of poly (ADP-ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA-damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP-ribose linked to nuclear proteins during DNA repair. The 46 kDa DBD of human PARP, and several derivatives thereof mutated in its first or second zinc-finger, were overproduced in Escherichia coli, in CV-1 monkey cells or in human fibroblasts to study their DNA-binding properties, the trans-dominant inhibition of resident PARP activity, and the consequences on DNA repair, respectively. A positive correlation was found between the in vitro DNA-binding capacity of the recombinant DBD polypeptides and their inhibitory effect on PARP activity stimulated by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Furthermore, overproduced wild-type DBD blocked unscheduled DNA synthesis induced in living cells by MNNG treatment, but not that induced by UV irradiation. These results define a critical role for the second zinc-finger of PARP for DNA single-stranded break binding and furthermore underscore the importance for PARP to act as a critical regulatory component in the repair of DNA damage induced by alkylating agents.     

Poly(ADP-ribose) immunostaining to detect apoptosis induced by a neurotoxic fragment of prion protein

PrP106–126 is a synthetic peptide representing codons 106–126 of the prion protein, which spontaneously forms amyloid fibrils and exerts neurotoxic effects on primary mouse brain cell cultures. Neurotoxicity by this peptide is commonly used as a model for the neurotoxicity observed in prion diseases and involves the formation of reactive oxygen species which, in turn, can cause DNA damage, including DNA strand breaks. Strand breaks in nuclear DNA can activate poly(ADP-ribose) polymerase to covalently modify nuclear proteins with poly(ADP-ribose). We, therefore, examined by immunofluorescence whether or not PrP106–126 triggers poly(ADP-ribose) formation. We observed strong poly(ADP-ribose) immunofluorescence signals in a fraction of cells, typically arranged in a clustered pattern, by 30–48h after peptide addition. A few positive cells were also present in untreated cultures. Cell morphology was suggestive of apoptosis, and this was confirmed by positivity in the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) assay. On the other hand, our immunofluorescence assay did not detect any early activation of poly(ADP-ribose) polymerase in morphologically normal cells that could have resulted from peptide-induced formation of reactive oxygen species. We conclude that poly(ADP-ribose) immunostaining is a convenient and reliable method for visualizing cells undergoing apoptosis induced by PrP106–126.     

PARG dysfunction enhances DNA double strand break formation in S-phase after alkylation DNA damage and augments different cell death pathways

Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for the degradation of poly(ADP-ribose). PARG dysfunction sensitizes cells to alkylating agents and induces cell death; however, the details of this effect have not been fully elucidated. Here, we investigated the mechanism by which PARG deficiency leads to cell death in different cell types using methylmethanesulfonate (MMS), an alkylating agent, and Parg^−/− mouse ES cells and human cancer cell lines. Parg^−/− mouse ES cells showed increased levels of γ-H2AX, a marker of DNA double strand breaks (DSBs), accumulation of poly(ADP-ribose), p53 network activation, and S-phase arrest. Early apoptosis was enhanced in Parg^−/− mouse ES cells. Parg^−/− ES cells predominantly underwent caspase-dependent apoptosis. PARG was then knocked down in a p53-defective cell line, MIAPaCa2 cells, a human pancreatic cancer cell line. MIAPaCa2 cells were sensitized to MMS by PARG knockdown. Enhanced necrotic cell death was induced in MIAPaCa2 cells after augmenting γ-H2AX levels and S-phase arrest. Taken together, these data suggest that DSB repair defect causing S-phase arrest, but p53 status was not important for sensitization to alkylation DNA damage by PARG dysfunction, whereas the cell death pathway is dependent on the cell type. This study demonstrates that functional inhibition of PARG may be useful for sensitizing at least particular cancer cells to alkylating agents.