Poly(ADP-ribose) polymerase-1: what have we learned from the deficient mouse model?

Poly (ADP-ribose) polymerase (113 kDa; PARP-1) is a constitutive factor of the DNA damage surveillance network developed by the eukaryotic cell to cope with the numerous environmental and endogenous genotoxic agents. This enzyme recognizes and is activated by DNA strand breaks. This original property plays an essential role in the protection and processing of the DNA ends as they arise in DNA damage that triggers the base excision repair (BER) pathway. The generation, by homologous recombination, of three independent deficient mouse models have confirmed the caretaker function of PARP-1 in mammalian cells under genotoxic stress. Unexpectedly, the knockout strategy has revealed the instrumental role of PARP-1 in cell death after ischemia-reperfusion injury and in various inflammation process. Moreover, the residual PARP activity found in PARP-1 deficient cells has been recently attributed to a novel DNA damage-dependent poly ADP-ribose polymerase (62 kDa; PARP-2), another member of the expanding PARP family that, on the whole, appears to be involved in the genome protection. The present review summarizes the recent data obtained with the three PARP knockout mice in comparison with the chemical inhibitor approach.

Werner's syndrome T lymphocytes display a normal in vitro life-span.

Werner's syndrome (WS) is an autosomal recessive disorder displaying many features consistent with accelerated ageing. Fibroblasts from WS patients show a distinct mutator phenotype (characterised by the production of large chromosomal deletions) and a profound reduction in proliferative capacity. The disorder results from a mutation in a novel ReqQ helicase. Recently, we demonstrated that the proliferative defect was corrected by the ectopic expression of telomerase. From these data, we propose that mutations in the wrn gene lead to deletions at or near the telomere which reduce the cells replicative life-span. This hypothesis predicts that cell types which retain the ability to upregulate telomerase as part of their response to a proliferative stimulus would fail to show any significant effect of wrn gene mutations upon life-span. Human T lymphocytes represent a well-characterised example of such a cell type. To test the hypothesis, WS T lymphocytes were cultured until they reached replicative senescence. These cultures displayed life-spans which did not differ significantly from those of normal controls. These findings are consistent with the hypothesis that the effects of wrn mutations on replicative life-span are telomere-mediated.

Regulation by phosphorylation of Xenopus laevis poly(ADP-ribose) polymerase enzyme activity during oocyte maturation.

Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme that is dependent on DNA breaks and nicks for its enzyme activity. These DNA nicks and breaks function as allosteric effectors of the enzyme activity. This reaction is important for efficient DNA base excision repair, although it is not a component of the elementary repair pathway itself. The physiological relevance of this reaction might be to ensure correct and efficient DNA repair. We have examined the enzyme activity of PARP in oocytes and eggs of Xenopus laevis. Although both oocytes and eggs contain approximately the same amounts of enzyme protein, there is no detectable enzyme activity in the oocytes, whereas in the eggs the enzyme is active. Enzyme activity appears during oocyte maturation, approx. 4 h after induction by progesterone. This enzyme activation coincides with the appearance of active maturation-promoting factor. Enzyme activation is accompanied by a shift in the electrophoretic mobility of the polypeptide, from an apparent molecular mass of 116 kDa to 125 kDa. Treatment with either bacterial or potato phosphatase reverses the mobility shift and abolishes enzyme activity. Incubation of maturing X. laevis eggs with radioactive inorganic phosphate and subsequent immunoprecipitation demonstrate that the PARP protein is phosphorylated in vivo. We show that maturation-promoting factor (Cyclin B/cdc2) cannot itself be responsible for the phosphorylation and activation of PARP in maturing X. laevis eggs. Together, these results demonstrate that the enzyme activity of PARP in X. laevis oocytes and eggs is regulated by post-translational, covalent phosphorylation.

Post-translational activation of non-homologous DNA end-joining in Xenopus oocyte extracts.

We have analysed the recircularisation of plasmid DNA, cut with two different endonucleases to generate non-homologous DNA ends, in extracts of unfertilised eggs and oocytes of Xenopus. We found that the capacity to join non-homologous DNA ends, generating diagnostic covalently closed monomer circles, appeared during oocyte maturation at the time of germinal vesicle breakdown. This enzyme function was post-translationally activated in oocyte extracts incubated with unfertilised egg extract containing active cdc2/cyclin B, or by incubation with purified cdc2/cyclin B. Dephosphorylation of egg proteins by alkaline phosphatase inhibited the ability to join non-homologous DNA ends. We show that most linear non-homologous DNA ends repaired to form closed-circular supercoiled monomers, are joined without loss of nucleotides. Following partial purification, the activity was inhibited by inhibitors of poly(ADP-Rib) polymerase, an enzyme that is inactive in oocytes, but phosphorylated and activated during maturation. Competitive inhibition of poly(ADP-Rib) polymerase by > 50 microM 3-aminobenzamide prevented the joining of both matched and non-homologous DNA ends. We conclude that post-translational phosphorylation provides one route by which end-joining of non-homologous DNA can be regulated.

Purification, characterisation, and molecular cloning of a chicken erythroblast mono(ADP-ribosyl)transferase.

We have purified an arginine-specific mono(ADP-ribosyl)transferase from chicken erythrocytes. The purified transferase was free from poly (ADP-ribose) polymerase activity. The molecular weight of the purified enzyme was estimated to be 27.5 kDa by gel filtration through Sephadex G-75 in a non-denaturing solvent. Activity gel experiments indicate that the active enzyme has an apparent molecular weight in SDS gels of about 28 kDa. The optimum pH of the reaction is about 8.0. The K(m) value for NAD+ of the purified enzyme is about 130 microM. Small molecular weight inhibitors of poly (ADP-ribose) polymerase have no significant effect on the mono ADP-ribosyl transferase enzyme activity. A number of inhibitors of the arginine-specific mono(ADP-ribosyl)transferase activity have been identified. Among the more effective inhibitors are 1,4 naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, 4-amino-1-naphthol and 1,2-naphthoquinone. We have also cloned a mono(ADP-ribosyl)transferase from chicken erythroblasts. This gene has been expressed in E. coli and ADP-ribosylation activity has been demonstrated using histones as substrate. The activity is shown to be arginine-specific by the use of poly-L-arginine as substrate. Use of a specific inhibitor has shown that this enzyme is indeed a mono(ADP-ribosyl)transferase and not a NAD glycohydrolase activity. The sequence of this gene is very similar to several other mono(ADP-ribosyl)transferase genes. There are thus at least three different chicken mono(ADP-ribosyl)transferase genes in the blood system alone; this suggests that there is a quite large family of mono(ADP-ribosyl)transferase genes in animals. We have also isolated the promoter region of this chicken gene and are able to identify several standard motifs in this promoter.

The limited reproductive life span of normal human cells in culture.

It has been suggested that the limited reproductive life span of normal (diploid) cells in culture may be explained by an inevitable shortening of one or more telomeres. The hypothesis is that one of the shortened telomeres will either generate a specific signal or will invoke a DNA damage checkpoint, in either case causing that cell to leave the cell cycle irreversibly. To assess this hypothesis, I review what constitutes the limited life span of cells in culture. Careful inspection of the kinetics of the life span of diploid cells in culture has shown that the limited life span arises because a fraction of newborn cells irreversibly leave the cell cycle at each division; and this fraction of reproductively sterile cells increases steadily throughout the life span of the culture. Cell fusion experiments suggest that only a small number of genes are involved in preventing continued cell growth, but that at least two independent mutation events are required to immortalize human cells, although only one event is sufficient in some rodent species. Human genetic diseases such as Werner's syndrome indicate that the duration of the life span is also genetically regulated, and is independent of the cessation of cell proliferation.

XRCC1 polypeptide interacts with DNA polymerase beta and possibly poly (ADP-ribose) polymerase, and DNA ligase III is a novel molecular 'nick-sensor' in vitro.

The DNA repair proteins XRCC1 and DNA ligase III are physically associated in human cells and directly interact in vitro and in vivo. Here, we demonstrate that XRCC1 is additionally associated with DNA polymerase-beta in human cells and that these polypeptides also directly interact. We also present data suggesting that poly (ADP-ribose) polymerase can interact with XRCC1. Finally, we demonstrate that DNA ligase III shares with poly (ADP-ribose) polymerase the novel function of a molecular DNA nick-sensor, and that the DNA ligase can inhibit activity of the latter polypeptide in vitro. Taken together, these data suggest that the activity of the four polypeptides described above may be co-ordinated in human cells within a single multiprotein complex.

Efficient retroviral infection of mammalian cells is blocked by inhibition of poly(ADP-ribose) polymerase activity.

Integration of proviral DNA into the host cell genome is a characteristic feature of the retroviral life cycle. This process involves coordinate DNA strand break formation and rejoining reactions. The full details of the integration process are not yet fully understood. However, the endonuclease and DNA strand-joining activities of the virus-encoded integrase protein (IN) are thought to act in concert with other, as-yet-unidentified, endogenous nuclear components which are involved in the DNA repair process. The nuclear enzyme poly(ADP-ribose) polymerase (PARP), which is dependent on DNA strand breaks for its activity, is involved in the efficient repair of DNA strand breaks, and maintenance of genomic integrity, in nucleated eukaryotic cells. In the present work, we examine the possible involvement of PARP in the retroviral life cycle and demonstrate that inhibition of PARP activity, by any one of three independent mechanisms, blocks the infection of mammalian cells by recombinant retroviral vectors. This requirement for PARP activity appears to be restricted to processes involved in the integration of provirus into the host cell DNA. PARP inhibition does not affect viral entry into the host cell, reverse transcription of the viral RNA genome, postintegration synthesis of viral gene products, synthesis of the viral RNA genome, or the generation of infective virions. Therefore, efficient retroviral infection of mammalian cells is blocked by inhibition or PARP activity.

Sequence of a chicken erythroblast mono(ADP-ribosyl)transferase-encoding gene and its upstream region.

We have cloned the MADPRT gene encoding the 300-amino-acid mono(ADP-ribosyl)transferase (MADPRT) from chicken erythroblasts. The protein has homology to the rabbit and human skeletal muscle (50% identity) and two chicken heterophil (52% identity) NAD+:arginine MADPRT. The active site region is particularly conserved. The upstream region of the MADPRT gene from erythroblasts has several features characteristic of promoter sequences.

ADP-ribosylation reactions.

ADP-ribosylation reactions have been studied now for over 30 years. They came to light originally in studies of some bacterial toxins, which turned out to be mono-ADP-ribosyl transferases. Subsequently, endogenous mono-ADP-ribosyltransferases were discovered. Although the substrates of the toxins are always so-called G-proteins, the substrate for a muscle mono-ADP-ribosyl transferase has been shown to be an extra-cellular cell adhesion molecule. A new pathway of NAD catabolism is the recently described cyclic ADP-ribose; this seems to be involved in calcium metabolism. Just under 30 years ago, poly(ADP-ribose) polymerase was discovered. From protein studies as well as from recent molecular biology, some amino acids essential for the enzyme activity or for binding to DNA have been identified. I suggest that poly(ADP-ribose) polymerase has several related functions in maintaining the integrity of the genome in eukaryotic cells. The highly-charged polymer, poly(ADP-ribose), is always made when free, ie naked DNA ends appear, in order to ensure the correct processing of these DNA breaks. In particular, the polymer may act to prevent DNA recombination reactions that would interfere with DNA repair. In addition, the polymer may protect the free DNA end from exonuclease action, and thirdly, it may unravel the chromatin structure. This suggests that this enzyme is not a necessary component of the process of DNA excision repair, but that this enzyme is required for correct and efficient DNA excision repair.(ABSTRACT TRUNCATED AT 250 WORDS)

The function of poly (ADP-ribosylation) in DNA breakage and rejoining.

Poly (ADP-ribose) polymerase has an obligatory requirement for DNA strand-breaks in order to show full enzyme activity. Exposure of cells to DNA damaging agents activates this enzyme presumably through the production of DNA strand-breaks, either directly or via cellular enzymes. Recent evidence from manipulations of the cloned cDNA of this enzyme confirm the earlier evidence, obtained using enzyme inhibitors, that this enzyme is involved in DNA excision repair, probably at or near the ligation step. A very unusual human genetic disease has provided direct evidence for a link between the enzyme activities of poly (ADP-ribose) polymerase and of DNA ligase I. There is also some evidence that this enzyme may be involved in other cases of DNA breakage and rejoining, such as homologous and non-homologous DNA recombination, for example, in sister chromatid exchanges, in DNA transfection, in the integration of retroviral proviral DNA and in variable antigen switching in African trypanosomes.

Poly(ADP-ribose) polymerase in plant nuclei.

We show that poly(ADP-ribose) polymerase is present in maize, pea and wheat nuclei. We have identified the enzyme product as poly(ADP-ribose) by purification and electrophoresis on a DNA sequencing gel. This reveals a polymer ladder consisting of up to 45 residues. The polymer product from maize, after digestion with snake venom phosphodiesterase, gave only 5'-AMP and (phosphoribosyl)-AMP; the mean chain length of the polymer was 5 and 11 residues in two separate experiments. The optimum pH of the plant enzyme is greater than pH 7.0 in pea, wheat and maize; the optimum temperature for enzyme activity is approximately 15 degrees C. The Km for NAD+ for the enzyme from maize is estimated to be approximately 50 microM under optimal conditions. Several compounds (nicotinamide, deoxythymidine, 3-aminobenzamide, 3-methoxybenzamide and 5-bromodeoxyuridine) that specifically inhibit the animal enzyme also inhibit the enzyme from plants. The ratio of the IC50 for 5-bromodeoxyuridine to the IC50 for 3-aminobenzamide in maize is similar to that of the animal enzyme indicating that the enzyme involved is poly(ADP-ribose) polymerase and not mono(ADP-ribosyl) transferase. SDS gel electrophoresis and gel filtration analysis of a crude extract of maize nuclei indicate a molecular mass for poly(ADP-ribose) polymerase of approximately 114 kDa.

The expression of proliferation-dependent antigens during the lifespan of normal and progeroid human fibroblasts in culture.

Normal human fibroblasts display a limited lifespan in culture, which is due to a steadily decreasing fraction of cells that are able to proliferate. Using antibodies that react with antigens present in proliferating cells only, in an indirect immunofluorescence assay, we have estimated the fraction of proliferating cells in cultures of normal human fibroblasts. Furthermore, we have estimated the rate of decline in the fraction of proliferating cells during the process of cellular ageing by application of the assay to normal human fibroblasts throughout their lifespan in culture. Werner's Syndrome is an autosomal recessive disease in which individuals display symptoms of ageing prematurely. Werner's Syndrome fibroblasts display a reduced lifespan in culture compared with normal human fibroblasts. Like normal human fibroblasts, the growth of Werner's Syndrome fibroblasts is characterised by a decreasing fraction of cells reacting with the proliferation-associated antibodies throughout their lifespan in culture. However, the rate of loss of proliferating cells in Werner's Syndrome fibroblasts during the process of cellular ageing is accelerated 5- to 6-fold compared with the rate determined for normal human fibroblasts.