Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.4.2.30 (PARP)
 13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The influence of poly(ADP-ribose) polymerase (PARP) on the replication of DNA containing the SV40 origin of replication has been examined. Extensive replication of SV40 DNA can be carried out in the presence of T antigen, topoisomerase I, the multimeric human single strand DNA-binding protein (HSSB), and DNA polymerase alpha-DNA primase (pol alpha-primase) complex (the monopolymerase system). In the monopolymerase system, both small products (Okazaki fragments), arising from lagging strand synthesis, and long products, arising from leading strand synthesis, are formed. The synthesis of long products requires the presence of relatively high levels of pol alpha-primase complex. In the presence of PARP, the synthesis of long products was blocked and only small Okazaki fragments accumulated, arising from the replication of the lagging strand template. The inhibition of leading strand synthesis by PARP can be effectively reversed by supplementing the monopolymerase system with the multimeric activator 1 protein (A1), the proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta (the dipolymerase system). The inhibition of leading strand synthesis in the monopolymerase system was caused by the binding of PARP to the ends of DNA chains, which blocked their further extension by pol alpha. The selective accumulation of Okazaki fragments was shown to be due to the coupled synthesis of primers by DNA primase and their immediate extension by pol alpha complexed to primase. PARP had little effect on this coupled reaction, but did inhibit the subsequent elongation of products, presumably after pol alpha dissociated from the 3'-end of the DNA fragments. PARP inhibited several other enzymatic reactions which required free ends of DNA chains. PARP inhibited exonuclease III, DNA ligase, the 5' to 3' exonuclease, and the elongation of primed DNA templates by pol alpha. In contrast, PARP only partly competed with the elongation of primed DNA templates by the pol delta elongation system which required SSB, A1, and PCNA. These results suggest that the binding of PARP at the ends of nascent DNA chains can be displaced by the binding of A1 and PCNA to primer ends. HSSB can be poly(ADP-ribosylated) in vivo as well as in vitro. However, the selective effect of PARP in blocking leading strand synthesis in the monopolymerase system was shown to depend primarily on its DNA binding property rather than on its ability to synthesize poly(ADP-ribose).
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PMID:Influence of poly(ADP-ribose) polymerase on the enzymatic synthesis of SV40 DNA. 167 70

The endogenous poly(ADP-ribosyl)--nonhistone protein conjugates were isolated from dimethyl-sulfate-treated rat hepatoma AH 7974 cells using aminophenylboronic-acid--agarose chromatography. Seven major components could be discerned on dodecyl sulfate gels (molecular mass 43, 60, 66, 86, 100, 110 and 170 kDa) while control cells indicated only slight staining at above 200 kDa. The most abundant conjugate formed in response to alkylation damage was further purified using preparative gel electrophoresis and identified on the basis of its intrinsic enzymic activity as automodified poly(APD-ribose) synthase. In addition, topoisomerase I activity was found associated with a 60-kDa peptide. ADP-ribosylated endonuclease and actin were not detect-able. The purified conjugate fraction contained maximally 8.8 nmol/mg ADP-ribose and 7.9 nmol/mg oligo(ADP-ribose) with a mean chain length of 2.3 residues. The modifying (ADP-ribosyl)n groups were attached to its acceptors by a hydroxylamine-insensitive bond and had practically no effect on the DNA affinity of either poly(ADP-ribose) synthase or topoisomerase I.
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PMID:Poly(ADP-ribose) synthase is the major endogenous nonhistone acceptor for poly(ADP-ribose) in alkylated rat hepatoma cells. 312 14

Mammalian cells contain activities that amplify the effects of activators on class II gene transcription in vitro. The molecular identity of several of these cofactor activities is still unknown. Here we identify poly(ADP-ribose) polymerase (PARP) as one functional component of the positive cofactor 1 activity. PARP enhances transcription by acting during preinitiation complex formation, but at a step after binding of transcription factor IID. This transcriptional activation requires the amino-terminal DNA-binding domain, but not the carboxyl-terminal catalytic region. In purified systems, coactivator function requires a large molar excess of PARP over the number of templates, as reported for other DNA-binding cofactors such as topoisomerase I. PARP effects on supercoiled templates are DNA concentration-dependent and do not depend on damaged DNA. The PARP coactivator function is suppressed by NAD+, probably as a result of auto-ADP-ribosylation. These observations provide another example of the potentiation of trancription by certain DNA-binding cofactors and may point to interactions of PARP with RNA polymerase II-associated factors in special situations.
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PMID:Poly(ADP-ribose) polymerase enhances activator-dependent transcription in vitro. 912 82

We have evaluated the influence of anchorage status together with endogenous levels of bcl-2 family members on the ability of the topoisomerase I inhibitor, topotecan (TPT), to induce programmed cell death (PCD) in human colon, breast, lymphoid, and cervical cancer cell lines. As part of this study, we assessed the use of measuring poly(ADP-ribose) polymerase (PARP) cleavage by Western blot, as an index of apoptosis, relative to measuring chromatin condensation by acridine orange analysis. Our results show a strong correlation between both assays, indicating that PARP cleavage is an accurate method to examine PCD. We have encountered a strong association between cell attachment and sensitivity to TPT-induced PCD. Cells growing attached to flasks appear to be relatively more resistant than suspension-growing cells in spite of endogenous bcl-2, bax, or bcl-x levels. Furthermore, we demonstrate that interference with attachment status alters the sensitivity of cells to TPT-induced PCD. Although cell attachment to ProNectin F confers protection against TPT-induced chromatin condensation and cleavage of PARP, cell detachment by poly(2-hydroxyethyl methacrylate) stimulates TPT-induced PCD and PARP cleavage.
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PMID:Factors affecting topotecan-induced programmed cell death: adhesion protects cells from apoptosis and impairs cleavage of poly(ADP-ribose)polymerase. 918 15

Poly(ADP-ribose) polymerase (PARP) has been suggested to play a regulatory role in vivo, in DNA replication and/or DNA repair based mainly on its capacity to bind to DNA strand breaks. This interaction is modulated through auto poly(ADP-ribosylation). However, the biological function of PARP may also involve interactions with proteins such as topoisomerase I or DNA polymerase alpha, which may or may not be themselves ADP-ribosylated. Using the yeast two-hybrid method search for other proteins interacting with PARP, we have isolated a full-length cDNA clone coding for a protein of 158 amino acid residues. This amino acid sequence is 66 and 56% identical to yeast ubiquitin-conjugating enzymes Hus5 and Ubc9 of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Moreover, we have demonstrated that the expressed protein complements a S. cerevisiae yeast strain deficient for Ubc9. The protein encoded by the isolated cDNA is thus a new human counterpart of the ubiquitin-conjugating enzyme family and has been called hUbc9. The hubc9 gene locus has been assigned to the chromosomal location 16p13.2-p13.3. By means of two-hybrid analysis it was discovered that hUbc9 interacts with the automodification domain of PARP. This interaction was further confirmed using GST (glutathione-S-transferase) tagged fusion proteins: (i) in vivo, by transfecting cos7 cells with hUbc9 cloned in an eukaryotic expression vector, and (ii) in vitro, by mixing purified PARP with hUbc9 purified and expressed in bacteria. The possible significance and function of this interaction is discussed while taking into account the possible intracellular role of hUbc9.
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PMID:Poly(ADP-ribose) polymerase interacts with a novel human ubiquitin conjugating enzyme: hUbc9. 919 46

The acridine derivative m-AMCA (methyl-N-[4-(9-acridinylamino)-2-methoxyphenyl]carbamate hydrochloride), a carbamate analogue of the topoisomerase II poison amsacrine, is distinguished by its high cytotoxicity against non-cycling tumour cells. We compared the response of cultured Lewis lung carcinoma cells to m-AMCA, amsacrine and the topoisomerase I poison camptothecin. The DNA polymerase inhibitor aphidicolin reversed the cytotoxicity of camptothecin fully, that of amsacrine partially, and that of m-AMCA minimally. The ability of m-AMCA to induce the enzyme poly(ADP-ribose)polymerase (PARP) was markedly lower than that of camptothecin or amsacrine. Cell cycle responses to m-AMCA and amsacrine were similar, with slowing of progress through S-phase and arrest in G2-phase. These cell cycle changes were also observed when plateau phase cultures were exposed to drug for 1 h, washed free of drug and cultured in fresh medium, with m-AMCA having a more pronounced effect than amsacrine and camptothecin having no effect. We also examined the role of p53 protein in the response using cultured human H460 cells. Both m-AMCA and amsacrine induced p53 protein expression in proliferating but not in non-proliferating H460 cells, and induced p21WAF1 regardless of proliferation status. Both induced G1-phase cell cycle arrest. It is suggested that two cytotoxicity mechanisms can be distinguished using these drugs. The first is specific for S-phase cells, is reversed by aphidicolin and induces PARP activity. The second is cell cycle non-specific, does not induce PARP and is unaffected by aphidicolin. Camptothecin activates only the first, m-AMCA primarily the second and amsacrine activates both.
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PMID:Cellular responses to methyl-N-[4-9-acridinylamino)-2-methoxyphenyl] carbamate hydrochloride, an analogue of amsacrine active against non-proliferating cells. 938 32

Apoptosis induced by numerous cancer chemotherapeutic and other toxic agents has been shown to proceed through a cascade of proteases, now termed caspases, culminating in cleavage of a set of proteins. The ability of photodynamic treatment (PDT) with the phthalocyanine Pc 4 to activate cellular caspases has been assessed during the rapid apoptosis in murine lymphoma L5178Y-R cells. Cells were exposed to combinations of Pc 4 and activating red light that result in > or =90% cell death, as judged by a clonogenic assay. The rate of entry of cells into apoptosis was dose dependent. For 0.5 microM Pc 4 and either 2.1 or 3 kJ/m2, which kill 90 or 99.9% of the cells, oligonucleosomal fragmentation was visible on agarose gels as early as 60 or 30 min after PDT, respectively. To assess caspase activation, cells were harvested at various times after PDT, and cell proteins were subjected to electrophoresis and Western blot analysis, using an antibody to poly(ADP-ribose) polymerase (PARP). The cleavage of the normally Mr 116,000 PARP into fragments of Mr approximately 90,000 was observed at approximately the same time as the earliest DNA fragmentation. An antibody to the polymer, poly(ADP-ribose), did not recognize the Mr approximately 90,000 PARP cleavage products, in contrast to the parent enzyme. This analysis also revealed that levels of a poly(ADP-ribosylated) Mr 100,000 protein, tentatively identified as topoisomerase I, were maintained in cells after PARP was fully cleaved. Caspase-3 (and/or caspase-7) activity, as measured in cell lysates with the fluorogenic substrate DEVD-AMC, was elevated almost immediately after PDT. The cell-permeable, irreversible caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoro-methylketone, inhibited PDT-induced apoptosis and PARP cleavage, whereas the inactive peptide analogue, benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone, was without effect. The results indicate that PDT-induced apoptosis is mediated by activation of caspase-3 and/or other similar caspases.
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PMID:Protease activation and cleavage of poly(ADP-ribose) polymerase: an integral part of apoptosis in response to photodynamic treatment. 950 Apr 54

beta-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5, 6-dione) was previously shown to enhance the lethality of X-rays and radiomimetic agents and its radiosensitizing role in mammalian cells was attributed to a possible interference with topoisomerase I activity. Furthermore, beta-lapachone alone was found to induce chromosomal damage in Chinese hamster ovary (CHO) cells. The aim of the present study was to further elucidate the possible mechanisms by which beta-lapachone exerts its genotoxic action in cultured mammalian cells. Flow cytometry analysis of beta-lapachone-treated CHO cells indicated a selective cytotoxic effect upon S phase of the cell cycle. beta-lapachone produced DNA strand breaks as determined by alkaline elution assay; alkaline elution profiles from treated cells showed a bimodal dose-response pattern, with a threshold dose above which a massive dose-independent DNA degradation was observed. Furthermore, beta-lapachone increased the capacity of crude CHO cellular extracts to unwind supercoiled plasmid DNA, while significantly inhibiting in vitro poly(ADP-ribose) polymerase (PARP). These results suggest that damage induction is probably mediated by the interaction between beta-lapachone and cellular enzymatic function(s), rather than reflecting a direct action on the DNA. We suggest that the inhibition of PARP plays a central role in the complex biological effects induced by beta-lapachone in CHO cells.
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PMID:DNA damage and cytotoxicity induced by beta-lapachone: relation to poly(ADP-ribose) polymerase inhibition. 963 74

We have studied the apoptotic response of poly(ADP-ribose) polymerase (PARP)-/- cells to different inducers and the consequences of the expression of an uncleavable mutant of PARP on the apoptotic process. The absence of PARP drastically increases the sensitivity of primary bone marrow PARP-/- cells to apoptosis induced by an alkylating agent but not by a topoisomerase I inhibitor CPT-11 or by interleukin-3 removal. cDNA of wild type or of an uncleavable PARP mutant (D214A-PARP) has been introduced into PARP-/- fibroblasts, which were exposed to anti-CD95 or an alkylating agent to induce apoptosis. The expression of D214A-PARP results in a significant delay of cell death upon CD95 stimulation. Morphological analysis shows a retarded cell shrinkage and nuclear condensation. Upon treatment with an alkylating agent, expression of wild-type PARP cDNA into PARP-deficient mouse embryonic fibroblasts results in the restoration of the cell viability, and the D214A-PARP mutant had no further effect on cell recovery. In conclusion, PARP-/- cells are extremely sensitive to apoptosis induced by triggers (like alkylating agents), which activates the base excision repair pathway of DNA, and the cleavage of PARP during apoptosis facilitates cellular disassembly and ensures the completion and irreversibility of the process.
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PMID:Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant. 983 34

We have previously demonstrated that calpain is responsible for the cleavage of Bax, a proapoptotic protein, during drug-induced apoptosis of HL-60 cells (Wood, D. E., Thomas, A., Devi, L. A., Berman, Y., Beavis, R. C., Reed, J. C., and Newcomb, E. W. (1998) Oncogene 17, 1069-1078). Here we show the sequential activation of caspases and calpain during drug-induced apoptosis of HL-60 cells. Time course experiments using the topoisomerase I inhibitor 9-amino-20(S)-camptothecin revealed that cleavage of caspase-3 substrates poly(ADP-ribose) polymerase (PARP) and the retinoblastoma protein as well as DNA fragmentation occurred several hours before calpain activation and Bax cleavage. Pretreatment with the calpain inhibitor calpeptin blocked calpain activation and Bax cleavage but did not inhibit PARP cleavage, DNA fragmentation, or 9-amino-20(S)-camptothecin-induced morphological changes and cell death. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) inhibited PARP cleavage, DNA fragmentation, calpain activation, and Bax cleavage and increased cell survival by 40%. Interestingly, Z-VAD-fmk-treated cells died in a caspase- and calpain-independent manner that appeared morphologically distinct from apoptosis. Our results suggest that excessive or uncontrolled calpain activity may play a role downstream of and distinct from caspases in the degradation phase of apoptosis.
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PMID:Caspase-dependent activation of calpain during drug-induced apoptosis. 1007 37


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