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1.
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  相似文献   

2.
This paper describes the effect of an in-vitro poly(ADP-ribose) turnover system on the poly(ADP-ribosyl)ation of chromatin. Both poly(ADP-ribose)polymerase and poly(ADP-ribose)glycohydrolase were highly purified and used in 4 different turnover systems: non-turnover, slow, medium and fast turnover. These turnover systems were designed to reflect possible turnover conditions in intact cells. The major protein acceptors for poly(ADP-ribose) are histones and the polymerase itself, a process referred to as automodification. The level of poly(ADP-ribose) modification of polymerase, histone H1 and core histones has been measured. The size of the polymer for each of the 3 groups of acceptor proteins has been determined by gel electrophoresis. After many turnover cycles at medium and fast turnover, the histones (H1 and core) become the main poly(ADP-ribose) acceptor proteins. The rate at which steady-state polymer levels are reached and the total accumulation of polymer in a given turnover system are both inversely proportional to the amount of glycohydrolase present. Furthermore, increasing amounts of glycohydrolase in the turnover systems reduces average polymer size. The polymer synthesized in the medium and fast turnover systems is degraded by glycohydrolase in a biphasic fashion and in these systems the half-life of polymer agreed with results found in intact cells. Our results show that the relative levels of polymerase and glycohydrolase activities can regulate the proportional poly(ADP-ribose) distribution on chromatin-associated acceptor proteins during steady-state turnover conditions. The patterns of modification of polymerase and histones under turnover conditions agree with in vivo observations.  相似文献   

3.
A cDNA for human poly(ADP-ribose) polymerase was inserted into a plasmid, transfected and expressed in E. coli. A lysate of the E. coli cells containing the expression plasmid reacted with antibody against human poly(ADP-ribose) polymerase and synthesized poly(ADP-ribose). The partially purified poly(ADP-ribose) polymerase expressed in E. coli had the same molecular weight and enzymological properties as human placental poly(ADP-ribose) polymerase, including affinity for NAD, turnover number and DNA-dependency for activity. This expression system should be useful for structure-function analysis of poly(ADP-ribose) polymerase.  相似文献   

4.
Hydrolysis of protein-bound 32P-labelled poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase shows that there is differential accessibility of poly(ADP-ribosyl)ated proteins in chromatin to poly(ADP-ribose) glycohydrolase. The rapid hydrolysis of hyper(ADP-ribosyl)ated forms of histone H1 indicates the absence of an H1 dimer complex of histone molecules. When the pattern of hydrolysis of poly(ADP-ribosyl)ated histones was analyzed it was found that poly(ADP-ribose) attached to histone H2B is more resistant than the polymer attached to histone H1 or H2A or protein A24. Polymer hydrolysis of the acceptors, which had been labelled at high substrate concentrations (greater than or equal to 10 microM), indicate that the only high molecular weight acceptor protein is poly(ADP-ribose) polymerase and that little processing of the enzyme occurs. Finally, electron microscopic evidence shows that hyper(ADP-ribosyl)ated poly(ADP-ribose) polymerase, which is dissociated from its DNA-enzyme complex, binds again to DNA after poly(ADP-ribose) glycohydrolase action.  相似文献   

5.
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.  相似文献   

6.
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   

7.
Characterization of human poly(ADP-ribose) polymerase with autoantibodies   总被引:7,自引:0,他引:7  
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.  相似文献   

8.
9.
Previously it had been shown that poly(ADP-ribose) polymerase requires DNA for its activity and that this enzyme is auto-poly(ADP-ribosyl)ated. The studies reported here indicate that this self-modification inhibits the enzyme and decreases its affinity for DNA, as shown by sucrose gradient density centrifugation. The coupling of poly(ADP-ribose) polymerase with poly(ADP-ribose) glycohydrolase reactivates the polymerase by degrading poly(ADP-ribose) and restoring the polymerase-DNA complex. The assay of polymerase in the presence of glyco-hydrolase was made possible by use of a double-label assay involving release of 14C-labelled nicotinamide and the incorporation of 3H-labelled ADP-ribose from NAD+. These results provide the basis for a shuttle mechanism in which the polymerase can be moved on and off DNA by the action of these two enzymes. Mg2+ and histone H1 appear to activate the polymerase by increasing the affinity of the polymerase for DNA.  相似文献   

10.
A selection strategy to obtain cells deficient in poly(ADP-ribose) polymerase was developed based on the fact that treatment with high levels of N-methyl-N'-nitro-N-nitrosoguanidine results in sufficient activation of poly(ADP-ribose) polymerase to cause NAD and ATP depletion leading to cessation of all energy-dependent processes and rapid cell death. In contrast, cells with low levels of poly(ADP-ribose) polymerase should not consume their NAD and might therefore be more likely to survive the DNA damage. Using this approach, we have cloned a number of cell lines containing 37-82% enzyme activity. The apparent decrease in poly(ADP-ribose) polymerase activity is not due to increases in NAD glycohydrolase, poly(ADP-ribose) glycohydrolase, or phosphodiesterase activities. Further characterization of the poly(ADP-ribose) polymerase-deficient cells indicates that they have prolonged generation times and increased rates of spontaneous sister chromatid exchanges.  相似文献   

11.
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.  相似文献   

12.
13.
The post-translational poly ADP-ribosylation of proteins by the nuclear enzyme poly(ADP-ribose) polymerase (EC 2.4.2.30) involves a complex pattern of ADP-ribose polymers. We have determined how this enzyme produces the various polymer size patterns responsible for altered protein function. The results show that histone H1 and core histones are potent regulators of both the numbers and sizes of ADP-ribose polymers. Each histone induced the polymerase to synthesize a specific polymer size pattern. Various other basic and/or DNA binding proteins as well as other known stimulators of poly(ADP-ribose) polymerase (spermine, MgCl2, nicked DNA) were ineffective as polymer size modulators. Testing specific proteolytic fragments of histone H1, the polymer number and polymer size modulating activity could be mapped to specific polypeptide domains. The results suggest that histones specifically regulate the polymer termination reaction of poly(ADP-ribose) polymerase.  相似文献   

14.
Polymers of ADP-ribose bind chromatosomal histones in solution and may play a role in chromatin accessibility in vivo. We have enzymatically synthesized a poly(ADP-ribose) affinity resin to further characterize binding of nuclear proteins to ADP-ribose polymers. NAD+- and (ADP-ribose)-derivatized agarose beads were recognized as polymer acceptors by the nuclear enzyme poly(ADP-ribose) polymerase. This polymerase elongated the existing ligands by successive addition of exogenously available ADP-ribose residues to form polymers covalently linked to the agarose beads. Poly(ADP-ribose) formation on the beads was dependent on incubation time and the mode of ligand attachment to the agarose. The resulting poly(ADP-ribose)-derivatized agarose beads possessed polymers which closely resembled those modifying the ADP-ribose polymerase by the automodification reaction. Fractionation of rat liver nuclear lysate over the poly(ADP-ribose) resin revealed a strong affinity of H1 for ADP-ribose polymers, thereby supporting a role for poly(ADP-ribose) in chromatin functions. Poly(ADP-ribose)-agarose beads are extremely stable and will be useful not only for affinity studies, but also for mechanistic studies involving polymer elongation and catabolism.  相似文献   

15.
Isolated nuclei from HeLa cells can incorporate labeled ADP-ribose from NAD into an acid-precipitable product, poly(ADP-ribose). This reaction is stimulated by 4-6-fold by the addition of deoxyribonuclease I to the complete reaction mixture. If the nuclei are treated first with deoxyribonuclease I, no effect is seen; the stimulation is only apparent when the two enzymes deoxyribonuclease I and poly(ADP-ribose) polymerase, are operating at the same time. After making several minor modifications in the assay mixture, it was found that another endonuclease, micrococcal nuclease, can also stimulate the poly(ADP-ribose) polymerase activity of HeLa nuclei. A comparison of the two stimulatory effects indicated that the two endonucleases activated to the poly(ADP-ribose) polymerase activity of HeLa nuclei in the same way. Overall this evidence suggests that poly(ADP-ribose) polymerase may have a functional role in the process of DNA repair.  相似文献   

16.
The level of mRNA encoding the nuclear enzyme poly(ADP-ribose) polymerase (ADP-ribosyltransferase, EC 2.4.2.30) was found to be very low in quiescent human lymphocytes and to increase at least 10-fold between 1 and 2 dyas after stimulation with the mitogen phytohaemagglutinin, staying high for several days thereafter. This increase was inhibited by 3-methoxybenzamide (a competitive inhibitor of poly(ADP-ribose) polymerase) but was not affected significantly by aphidicolin. Incubation of activated cells with cycloheximide for 2 h increased the expression slightly. These data demonstrate that, during lymphocyte activation, the level of mRNA of the poly(ADP-ribose) polymerase gene correlates with, and hence is presumably responsible for, the increase in poly(ADP-ribose) polymerase protein detectable by enzyme assay or immunochemistry.  相似文献   

17.
Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins typical of most eukaryotic cells. This process participates in DNA replication and repair and is mainly regulated by two enzymes, poly(ADP-ribose) polymerase, which is responsible for the synthesis of polymers of ADP-ribose, and poly(ADP-ribose) glycohydrolase, which performs polymer degradation. The aim of this work was to investigate in the cockroach Periplaneta americana L. (Blattaria: Blattidae) the behaviour of poly(ADP-ribosylation). In particular, we addressed: (i) the possible modulation of poly(ADP-ribosylation) during the embryonic development; (ii) the expression of poly(ADP-ribose) polymerase and glycohydrolase in different tissues; and (iii) the role of poly(ADP-ribosylation) during spermatogenesis. In this work we demonstrated that: (i) as revealed by specific biochemical assays, active poly(ADP-ribose) polymerase and glycohydrolase are present exclusively in P. americana embryos at early stages of development; (ii) an activity carrying out poly(ADP-ribose) synthesis was found in extracts from testes; and (iii) the synthesis of poly(ADP-ribose) occurs preferentially in differentiating spermatids/spermatozoa. Collectively, our results indicate that the poly(ADP-ribosylation) process in P. americana, which is a hemimetabolous insect, displays catalytical and structural features similar to those described in the holometabolous insects and in mammalian cells. Furthermore, this process appears to be modulated during embryonic development and spermatogenesis.  相似文献   

18.
The biological function of poly(ADP-ribose) polymerase in DNA repair, cell-cycle regulation and cellular differentiation has yet to be defined. Isolation of cells which are deficient in poly(ADP-ribose) synthesis would greatly facilitate the determination of the biological role of this enzyme. A method is described for isolating Chinese hamster ovary (CHO) cells deficient in the poly(ADP-ribose) polymerase activity by direct screening of colonies for enzyme activity. Colonies with decreased production of poly(ADP-ribose) are recovered from nylon replicas for further analysis. Using this method we have isolated a series of CHO cells which have 50% or less poly(ADP-ribose) polymerase activity. These mutants have normal generation times and are 20% more sensitive to the effects of DNA (m)ethylating agents than the parental cell. However, these mutants display normal sensitivity to gamma-rays.  相似文献   

19.
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   

20.
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