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1.
Poly(ADP-ribose) synthetase has been purified 2,000-fold to apparent homogeneity from human placenta. The purification procedure involves affinity chromatography with 3-aminobenzamide as the ligand. The purified enzyme absolutely requires DNA for the catalytic activity and catalyzes poly(ADP-ribosyl)ation of the synthetase itself (automodification) and histone H1. Mg2+ enhances both the automodification and poly(ADP-ribosyl)ation of histone H1. The enzyme is a monomeric protein with a pI of 10.0 and an apparent molecular weight of 116,000. The sedimentation coefficient and Strokes radius are 4.6 S and 5.9 nm, respectively. The frictional ratio is 1.82. Amino acid analysis and limited proteolysis with papain and alpha-chymotrypsin indicate that the human placental enzyme is very similar to the enzyme from calf thymus, although some differences are noted. Mouse antibody raised against the placental enzyme completely inhibits the activity of enzymes from human placenta and HeLa cells and cross-reacts with the enzymes from calf thymus and mouse testis. Immunoperoxidase staining with this antibody demonstrates the intranuclear localization of the enzyme in human leukemia cells. All these results indicate that molecular properties as well as antigenic determinants of poly(ADP-ribose) synthetase are highly conserved in various animal cells.  相似文献   

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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.  相似文献   

4.
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.  相似文献   

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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.  相似文献   

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The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase.   总被引:21,自引:0,他引:21  
Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of approximately 350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.  相似文献   

10.
Because mammary epithelium from virgin mice must undergo DNA synthesis prior to differentiation and because poly(ADP-ribosyl)ation has been linked to the cell cycle, it was hypothesized that this requirement for DNA synthesis might be related to the poly(ADP-ribosyl)ation of nuclear proteins. However, 3-methoxybenzamide, an inhibitor of poly(ADP-ribosyl)ation, stimulates alpha-lactalbumin accumulation even when added after DNA replication is completed. Furthermore, in parous mice this compound is still effective when DNA synthesis is blocked by cytosine arabinoside-beta-D-arabinofuranoside. Therefore, poly(ADP-ribosyl)ation appears to be associated, not with DNA synthesis, but with some other event in mammary gland differentiation.  相似文献   

11.
APLF is a novel protein of unknown function that accumulates at sites of chromosomal DNA strand breakage via forkhead-associated (FHA) domain-mediated interactions with XRCC1 and XRCC4. APLF can also accumulate at sites of chromosomal DNA strand breaks independently of the FHA domain via an unidentified mechanism that requires a highly conserved C-terminal tandem zinc finger domain. Here, we show that the zinc finger domain binds tightly to poly(ADP-ribose), a polymeric posttranslational modification synthesized transiently at sites of chromosomal damage to accelerate DNA strand break repair reactions. Protein poly(ADP-ribosyl)ation is tightly regulated and defects in either its synthesis or degradation slow global rates of chromosomal single-strand break repair. Interestingly, APLF negatively affects poly(ADP-ribosyl)ation in vitro, and this activity is dependent on its capacity to bind the polymer. In addition, transient overexpression in human A549 cells of full-length APLF or a C-terminal fragment encoding the tandem zinc finger domain greatly suppresses the appearance of poly(ADP-ribose), in a zinc finger-dependent manner. We conclude that APLF can accumulate at sites of chromosomal damage via zinc finger-mediated binding to poly(ADP-ribose) and is a novel component of poly(ADP-ribose) signaling in mammalian cells.  相似文献   

12.
The emerging role of poly(ADP-ribose) polymerase-1 in longevity   总被引:3,自引:0,他引:3  
In the present paper, the involvement of the family of poly(ADP-ribose) polymerases (PARPs), and especially of PARP-1, in mammalian longevity is reviewed. PARPs catalyse poly(ADP-ribosyl)ation, a covalent post-translational protein modification in eukaryotic cells. PARP-1 and PARP-2 are activated by DNA strand breaks, play a role in DNA base-excision repair (BER) and are survival factors for cells exposed to low doses of ionising radiation or alkylating agents. PARP-1 is the main catalyst of poly(ADP-ribosyl)ation in living cells under conditions of DNA breakage, accounting for about 90% of cellular poly(ADP-ribose). DNA-damage-induced poly(ADP-ribosyl)ation also functions as a negative regulator of DNA damage-induced genomic instability. Cellular poly(ADP-ribosyl)ation capacity in permeabilised mononuclear blood cells (MNC) is positively correlated with life span of mammalian species. Furthermore PARP-1 physically interacts with WRN, the protein deficient in Werner syndrome, a human progeroid disorder, and PARP-1 and WRN functionally cooperate in preventing carcinogenesis in vivo. Some of the other members of the PARP family have also been revealed as important regulators of cellular functions relating to ageing/longevity. In particular, tankyrase-1, tankyrase-2, PARP-2 as well as PARP-1 have been found in association with telomeric DNA and are able to poly(ADP-ribosyl)ate the telomere-binding proteins TRF-1 and TRF-2, thus blocking their DNA-binding activity and controlling telomere extension by telomerase.  相似文献   

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The role of poly(ADP-ribosyl)ation in the adaptive response   总被引:2,自引:0,他引:2  
An involvement of the poly(ADP-ribosyl)ation system in the expression of the adaptive response has been demonstrated with inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase. This enzyme is a key component of a reaction cycle in chromatin, involving dynamic synthesis and degradation of variably sized ADP-ribose polymers in response to DNA strand breaks. The present report reviews recent work focussing on the response of the poly(ADP-ribosyl)ation system in low dose adaptation. The results suggest that adaptation of human cells to minute concentrations of an alkylating agent involves a different activation mechanism for poly(ADP-ribose) polymerase than DNA break-mediated stimulation after high dose treatment. Moreover, adaptation induces the formation of branched polymers with a very high binding affinity for histone tails and selected other proteins. High dose challenge treatment of adapted cells further enhances formation of branched polymers. We propose that apart from sensing DNA nicks, poly(ADP-ribose) polymerase may be part of pathway protecting cells from downstream events of DNA damage.  相似文献   

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Here, we report the biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase (PARP) (EC 2.4.2. 30). PARP was effectively mono(ADP-ribosyl)ated both in solution and via an activity gel assay following SDS-PAGE with 20 microM or lower concentrations of [32P]-3'-dNAD+ as the ADP-ribosylation substrate. We observed the exclusive formation of [32P]-3'-dAMP and no polymeric ADP-ribose molecules following chemical release of enzyme-bound ADP-ribose units and high-resolution polyacrylamide gel electrophoresis. The reaction in solution (i) was time-dependent, (ii) was activated by nicked dsDNA, and (iii) increased with the square of the enzyme concentration. Stoichiometric analysis of the reaction indicated that up to four amino acid residues per mole of enzyme were covalently modified with single units of 3'-dADP-ribose. Peptide mapping of mono(3'-dADP-ribosyl)ated-PARP following limited proteolysis with either papain or alpha-chymotrypsin indicated that the amino acid acceptor sites for chain initiation with 3'-dNAD+ as a substrate are localized within an internal 22 kDa automodification domain. Neither the amino-terminal DNA-binding domain nor the carboxy-terminal catalytic fragment became ADP-ribosylated with [32P]-3'-dNAD+ as a substrate. Finally, the apparent rate constant of mono(ADP-ribosyl)ation in solution indicates that the initiation reaction catalyzed by PARP proceeds 232-fold more slowly than ADP-ribose polymerization.  相似文献   

16.
A new method to determine oligo- and poly(ADP-ribosyl)ated enzymes and proteins in vitro has been developed. This method is based on the facts that in Mg2+-depleted condition automodification of poly(ADP-ribose)polymerase is minimized and exogenously added acceptor protein is oligo(ADP-ribosyl)ated predominantly, and in Mg2+-fortified conditions the exogenous acceptor can be poly(ADP-ribosyl)ated. When 13 proteins, including several enzymes, were subjected to this system, dimeric bovine seminal RNase and micrococcal nuclease were found to be oligo(ADP-ribosyl)ated under Mg2+-depleted conditions but their activity was unchanged. Under Mg2+-fortified conditions however, the RNase was deactivated concomitantly with its extensive poly(ADP-ribosyl)ation. When dimeric bovine seminal RNase was monomerized in advance by treatment with dithiothreitol and urea, the enzyme lost ADP-ribose-accepting ability in spite of a significant residual enzyme activity. As used here successfully, the Mg2+-depleted and Mg2+-fortified ADP-ribosylation and subsequent chromatographic analysis of various proteins and enzymes might be an useful method for proving their oligo- and poly(ADP-ribosyl)ation.  相似文献   

17.
Poly(ADP-ribosyl)ation of nuclear proteins is catalyzed by poly(ADP-ribose) polymerase. This enzyme is involved in the regulation of basic cellular functions of DNA metabolism. DNA breaks induced by DNA-damaging agents trigger the activation of poly(ADP-ribose) polymerase increasing its endogenous level. This increase modifies the pattern of poly(ADP-ribosyl)ated chromatin proteins. In this paper we describe a procedure for the isolation of intact nuclei from rat liver to be used for the endogenous activity assay. Artifactual activation of the enzyme was avoided since a very low level of DNA-strand breaks occurs during the isolation of nuclei. We present a series of experiments which prove the ability of this procedure to detect increases in endogenous liver activity without modification of the total level. The application of this technique can be useful for a better understanding of the role of early changes in poly(ADP-ribose) polymerase level in physiological conditions and during exposure to DNA-damaging agents.  相似文献   

18.
Modulation of chromatin structure by poly(ADP-ribosyl)ation   总被引:5,自引:0,他引:5  
Poly(ADP-ribose) polymerase is a nuclear enzyme that is highly conserved in eucaryotes. Its activity is totally dependent on the presence of DNA containing single or double stranded breaks. We have shown that this activation results in a decondensation of chromatin superstructure in vitro, which is caused mainly by hyper(ADP-ribosy)ation of histone H1. In core particles, the modification of histone H2B leads to a partial dissociation of DNA from core histones. The conformational change of native chromatin by poly(ADP-ribosyl)ation is reversible upon degradation of the histone H1-bound poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase. We propose that cuts produced in vivo on DNA during DNA repair activate poly(ADP-ribose) polymerase, which then synthesizes poly(ADP-ribose) on histone H1, in particular, and contributes to the opening of the 25-nm chromatin fiber, resulting in the increased accessibility of DNA to excision repair enzymes. This mechanism is fast and reversible.  相似文献   

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The addition of thymidine as well as nicotinamide to isolated nuclei resulted in a strong inhibition of poly(ADP-ribosyl)ation, whereas that of hydroxyurea and amethopterin has essentially no effect. The nuclei isolated from the cells immediately after release from thymidine synchronization exhibited a significantly increased activity of poly(ADP-ribosyl)ation. Thereafter, the fluctuation pattern of the activity of poly(ADP-ribosyl)ation in isolated nuclei during the cell cycle was essentially the same as in the case of hydroxyurea synchronization. The activity of poly(ADP-ribosyl)ation in isolated nuclei after treatment with thymidine in vivo increased with the treatment time. The time-dependent increase was also evident in the case of nicotinamide treatment. Little increase in the activity was observed in hydroxyurea and amethopterin treatment. When poly(ADP-ribose) polymerase was extracted from the nuclei isolated from the cells which were pretreated with each of the four compounds, there was no significant difference in the amount among these compounds. The reason for the increase in the poly(ADP-ribosyl)ation in vitro by the in vivo treatment with thymidine is discussed.  相似文献   

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