Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

We have previously described the isolation and characterization of an intact multiprotein complex for DNA replication, designated the DNA synthesome, from human breast cancer cells and biopsied human breast tumor tissue. The purified DNA synthesome was observed to fully support DNA replication in vitro. We had also proposed a model for the breast cell DNA synthesome, in which DNA polymerases alpha, delta, and epsilon, DNA primase, and replication factor C (RF-C) represent members of the core component, or tightly associated, proteins of the complex. This model was based on the observed fractionation, chromatographic, and sedimentation profiles for these proteins. We report here that poly(ADP-ribose)polymerase (PARP) and DNA ligase 1 are also members of the breast cell DNA synthesome core component. More importantly, in this report we present the results of coimmunoprecipitation studies that were designed to map the protein-protein interactions between several members of the core component of the DNA synthesome. Consistent with our proposed model for the breast cell DNA synthesome, our data indicate that DNA polymerases alpha and delta, DNA primase, RF-C, as well as proliferating cell nuclear antigen (PCNA), tightly associate with each other in the complex, whereas DNA polymerase epsilon, PARP, and several other components were found to interact with the synthesome via a direct contact with only PCNA or DNA polymerase alpha. The association of PARP with the synthesome core suggests that this protein may serve a regulatory function in the complex. Also, the coimmunoprecipitation studies suggest that the three DNA polymerases alpha, delta, and epsilon all participate in the replication of breast cell DNA. To our knowledge this is the first report ever to describe the close physical association of polypeptides constituting the intact human breast cell DNA replication apparatus.
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PMID:Mapping specific protein-protein interactions within the core component of the breast cell DNA synthesome. 956 11

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is present in tobacco smoke and is hepatocarcinogenic in rats. Its bioactivation in rat hepatocytes leads to methylation and pyridyloxobutylation of DNA. Rat hepatocytes were cultured in serum-free William medium E on collagen-coated dishes. We demonstrated that some enzymes of the base and/or excision-repair pathways were involved in repair of NNK-induced DNA damage, measured by [methyl-3H] thymidine incorporation. Unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU), NNK, N'-nitrosonornicotine (NNN) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) increased 2.9-, 2.8-, 1.5- and 3.5-fold, respectively, suggesting that methylated and/or pyridyloxobutylated-DNA by these four nitroso compounds is repaired by the excision pathway. Moreover, levels of NNK-induced UDS were dose (1-3 mM) and time (1-18 h) dependent. Enzymes involved in the excision repair pathways were selectively inhibited. Inhibitors of DNA topoisomerase I (camptothecin) and topoisomerase II (etoposide, nalidixic acid) did not decrease the induction of UDS, suggesting that topoisomerases are not involved in the repair of NNK-induced damage. While aphidicolin and arabinocytidine (DNA polymerase alpha, delta, epsilon inhibitors) totally inhibited NNK- and NNKOAc-induced UDS, dideoxythymidine (DNA polymerase beta inhibitor) inhibited NNK- and NNKOAc-induced UDS by 40 and 33%, respectively. We conclude that DNA polymerase alpha, delta or epsilon and to a lesser degree polymerase beta are involved in the repair of pyridyloxobutylated DNA. Previous studies showed that inhibition of poly(ADP-ribosyl) polymerase (PARP) by 3-aminobenzamide (3-ab) facilitated DNA ligation. Our results demonstrate that 3-ab increased NNK-induced UDS, but does not affect NNKOAc-induced UDS. These observations suggest that the ligation step is rate limiting in the repair of methylated DNA but not of pyridyloxobutylated DNA.
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PMID:Modulation of DNA repair by various inhibitors of DNA synthesis following 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced DNA damage. 956 22

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a zinc-finger DNA-binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents. In response to these breaks, the immediate poly(ADP-ribosyl)ation of nuclear proteins involved in chromatin architecture and DNA metabolism converts DNA damage into intracellular signals that can activate DNA repair programs or cell death options. To have greater insight into the physiological function of this enzyme, we have used the two-hybrid system to find genes encoding proteins putatively interacting with PARP. We have identified a physical association between PARP and the base excision repair (BER) protein XRCC1 (X-ray repair cross-complementing 1) in the Saccharomyces cerevisiae system, which was further confirmed to exist in mammalian cells. XRCC1 interacts with PARP by its central region (amino acids 301 to 402), which contains a BRCT (BRCA1 C terminus) module, a widespread motif in DNA repair and DNA damage-responsive cell cycle checkpoint proteins. Overexpression of XRCC1 in Cos-7 or HeLa cells dramatically decreases PARP activity in vivo, reinforcing the potential protective function of PARP at DNA breaks. Given that XRCC1 is also associated with DNA ligase III via a second BRCT module and with DNA polymerase beta, our results provide strong evidence that PARP is a member of a BER multiprotein complex involved in the detection of DNA interruptions and possibly in the recruitment of XRCC1 and its partners for efficient processing of these breaks in a coordinated manner. The modular organizations of these interactors, associated with small conserved domains, may contribute to increasing the efficiency of the overall pathway.
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PMID:XRCC1 is specifically associated with poly(ADP-ribose) polymerase and negatively regulates its activity following DNA damage. 958 96

Poly(ADP-ribose) polymerase (PARP) is a component of the multiprotein DNA replication complex (MRC, DNA synthesome) that catalyzes replication of viral DNA in vitro. PARP poly(ADP-ribosyl)ates 15 of the approximately 40 proteins of the MRC, including DNA polymerase alpha (DNA pol alpha), DNA topoisomerase I (topo I), and proliferating-cell nuclear antigen (PCNA). Although about equal amounts of MRC-complexed and free forms of PCNA were detected by immunoblot analysis of HeLa cell extracts, only the complexed form was poly(ADP-ribosyl)ated, suggesting that poly(ADP-ribosyl)ation of PCNA may regulate its function within the MRC. NAD inhibited the activity of DNA pol delta in the MRC in a dose-dependent manner, whereas the PARP inhibitor, 3-AB, reversed this inhibitory effect. The roles of PARP in modulating the composition and enzyme activities of the DNA synthesome were further investigated by characterizing the complex purified from 3T3-L1 cells before and 24 h after induction of a round of DNA replication required for differentiation of these cells; at the latter time point, approximately 95% of the cells are in S phase and exhibit a transient peak of PARP expression. The MRC was also purified from similarly treated 3T3-L1 cells depleted of PARP by antisense RNA expression; these cells do not undergo DNA replication nor terminal differentiation. Both PARP protein and activity and essentially all of the DNA pol alpha and delta activities exclusively cosedimented with the MRC fractions from S phase control cells, and were not detected in the MRC fractions from PARP-antisense or uninduced control cells. Immunoblot analysis further revealed that, although PCNA and topo I were present in total extracts from both control and PARP-antisense cells, they were present in the MRC fraction only from induced control cells, indicating that PARP may play a role in their assembly into an active DNA synthesome. In contrast, expression of DNA pol alpha, DNA primase, and RPA was down-regulated in PARP-antisense cells, suggesting that PARP may be involved in the expression of these proteins. Depletion of PARP also prevented induction of the expression of the transcription factor E2F-1, which positively regulates transcription of the DNA pol alpha and PCNA genes; thus, PARP may be necessary for expression of these genes when quiescent cells are stimulated to proliferate.
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PMID:Regulation of the expression or recruitment of components of the DNA synthesome by poly(ADP-ribose) polymerase. 964 17

We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP-ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37-fold at 1 h (p < 0.05 vs. sham) and 1.73-fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly(ADP-ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3-aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single-strand breaks assessed as DNA polymerase I-mediated biotin-dATP nick-translation (PANT)-positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3-aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain.
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PMID:Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia. 1073 22

Poly (ADP-ribose) polymerase (PARP) is an abundant chromatin associated protein important in DNA repair, maintenance of chromosomal stability and programmed cell death. Here we report that an increase in caspase 3-activity and cleavage of PARP serves as an early execution phase signal in human neuroblastoma. Human neuroblastoma SK-N-SH cells were exposed to a protein kinase inhibitor, staurosporine, or a topoisomerase II inhibitor, etoposide, at various concentrations and time points. Cells exposed to staurosporine (0.1 microM) for 30 min showed an increase in caspase 3-activity and by 1 h an increase in PARP 116-kDa band and an 85-kDa cleavage product, which further increased in density with time after treatment. Quantitative analysis for condensed chromatin material using bisbenzimide, and DNA fragmentation enzyme immunoassays showed a significant increase in apoptosis 5 h after staurosporine treatment. This was further confirmed with a Klenow fragment of DNA polymerase I assay which primarily detects single-stranded DNA breaks. A significant decrease in mitochondrial metabolism occurred within 8-12 h after treatment. Studies using Trypan Blue exclusion, and lactic dehydrogenase (LDH) release revealed a significant increase in membrane permeability 8 h after staurosporine (0.1 microM) or etoposide (10 microM) treatments. Cleavage of lamin B1, a protein important in maintaining the nuclear envelope integrity was observed 12 h after staurosporine treatment. Our results show that activation of caspase 3 followed by PARP cleavage occur at much earlier time point than any other morphological or biochemical parameters of apoptosis or cytotoxicity.
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PMID:Poly (ADP-ribose) polymerase induction is an early signal of apoptosis in human neuroblastoma. 1076 13

In mammalian cells, damaged bases in DNA are corrected by the base excision repair pathway which is divided into two distinct pathways depending on the length of the resynthesized patch, replacement of one nucleotide for short-patch repair, and resynthesis of several nucleotides for long-patch repair. The involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in both pathways has been investigated by using PARP-1-deficient cell extracts to repair single abasic sites derived from uracil or 8-oxoguanine located in a double-stranded circular plasmid. For both lesions, PARP-1-deficient cell extracts were about half as efficient as wild-type cells at the polymerization step of the short-patch repair synthesis, but were highly inefficient at the long-patch repair. We provided evidence that PARP-1 constitutively interacts with DNA polymerase beta. Using cell-free extracts from mouse embryonic cells deficient in DNA polymerase beta, we demonstrated that DNA polymerase beta is involved in the repair of uracil-derived AP sites via both the short and the long-patch repair pathways. When both PARP-1 and DNA polymerase beta were absent, the two repair pathways were dramatically affected, indicating that base excision repair was highly inefficient. These results show that PARP-1 is an active player in DNA base excision repair.
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PMID:Base excision repair is impaired in mammalian cells lacking Poly(ADP-ribose) polymerase-1. 1085 6

Poly(ADP-ribose) is formed in possibly all multicellular organisms by a familiy of poly(ADP-ribose) polymerases (PARPs). PARP-1, the best understood and until recently the only known member of this family, is a DNA damage signal protein catalyzing its automodification with multiple, variably sized ADP-ribose polymers that may contain up to 200 residues and several branching points. Through these polymers, PARP-1 can interact noncovalently with other proteins and alter their functions. Here we report the discovery of a poly(ADP-ribose)-binding sequence motif in several important DNA damage checkpoint proteins. The 20-amino acid motif contains two conserved regions: (i) a cluster rich in basic amino acids and (ii) a pattern of hydrophobic amino acids interspersed with basic residues. Using a combination of alanine scanning, polymer blot analysis, and photoaffinity labeling, we have identified poly(ADP-ribose)-binding sites in the following proteins: p53, p21(CIP1/WAF1), xeroderma pigmentosum group A complementing protein, MSH6, DNA ligase III, XRCC1, DNA polymerase epsilon, DNA-PK(CS), Ku70, NF-kappaB, inducible nitric-oxide synthase, caspase-activated DNase, and telomerase. The poly(ADP-ribose)-binding motif was found to overlap with five important functional domains responsible for (i) protein-protein interactions, (ii) DNA binding, (iii) nuclear localization, (iv) nuclear export, and (v) protein degradation. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions.
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PMID:Poly(ADP-ribose) binds to specific domains in DNA damage checkpoint proteins. 1101 34

Normal somatic cells have a defined number of divisions, a limited capacity to proliferative. The telomeres, sequences of TTAGGG repeats at the ends of chromosomes, are considered the direct responsible of the control of the cellular cycle. In fact, the progressive shortening of telomere length at each cellular division, causes the entrance of the cells in a phase of senescence and than apoptosis. The maintenance of the length of telomeres is carried out through: the telomerase, a DNA polymerase reverse transcriptase that extends sequence TTAGGG repeats, or the alternative lengthening of telomeres (ALT), between which the adaptive mechanisms, inactivation of TRF1, a protein bound to the telomeres with the functions of inhibiting the telomerase activity and Tankirase-PARP, an enzymatic complex that ADP-ribosylate TRF1 and reduce its binding to DNA. The alteration of the mechanism of maintenance of the telomeres length (Telomerase, TRF1, Tankirase-PARP) may represent a first step toward the cell immortalization and cancerogenesis. Together with the alteration of the control mechanisms of the telomere length, also the cell genic contest should be considered. In fact, the oncogene activation and/or oncosuppressor gene inactivation (p53, Rb, ras) may allow or reduce the cancerogenesis. From this point of view, the telomerase, the TRF1, Tanchirase-PARP and other proteins involved in telomere length could be, in a near future, used as new indicators of prognosis and as markers for new anti-cancer therapies.
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PMID:[The role of telomere-binding proteins in carcinogenesis]. 1125 11

To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by apurinic/apyrimidinic endonuclease creating a nick with 3'-hydroxyl and 5'-reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was added to the 3'-hydroxyl group. With near-UV light (312 nm) exposure of the extract/probe mixture, six proteins were strongly labeled. Four of these include poly(ADP-ribose) polymerase-1 (PARP-1) and the BER participants flap endonuclease-1, DNA polymerase beta, and apurinic/apyrimidinic endonuclease. The amount of the probe cross-linked to PARP-1 was greater than that cross-linked to the other proteins. The specificity of PARP-1 labeling was examined using various competitor oligonucleotides and DNA probes with alternate structures. PARP-1 labeling was stronger with a DNA representing a BER intermediate than with a nick in double-stranded DNA. These results indicate that proteins interacting preferentially with a photoreactive BER intermediate can be selected from the crude cellular extract.
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PMID:Photoaffinity labeling of mouse fibroblast enzymes by a base excision repair intermediate. Evidence for the role of poly(ADP-ribose) polymerase-1 in DNA repair. 1134 72


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