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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

There is compelling evidence for the central role of oxidative damage in the aging process and for the participation of reactive oxygen species in tumor initiation and promotion. Caloric restriction (CR) or energy restriction retards age-associated increases in mitochondrial free-radical production and reduces the accumulation of oxidatively damaged cell components. CR has also been shown to slow down age-related declines in various repair capabilities, including some types of DNA repair. It is proposed that inhibitors of mitochondrial electron transport and/or uncouplers of oxidative phosphorylation (rotenone, amytal, amiodarone, valinomycin, etc.), when used at extremely low doses, could mimic the effects of CR in model systems. The objective is to lower mitochondrial free-radical production by decreasing the fraction of electron carriers in the reduced state. In addition to a variety of other effects, CR has been shown to increase the rate of apoptosis, particularly in preneoplastic cells, and in general, to promote elevated levels of free glucocorticoids (GCs). GCs are known to induce tissue-specific apoptosis and to upregulate gap-junction-mediated intercellular communication (GJIC). Tumor promoters like phorbol esters have the opposite effect, in that they inhibit both the process of apoptosis and GJIC. The enzyme poly (ADP-ribose) polymerase (PARP) is thought to play a central role in apoptosis, in a manner that has been highly conserved in evolution. There is good evidence that the apoptosis-associated Ca/Mg-dependent DNA endonuclease is maintained in a latent form by being poly (ADP-ribosylated). Apoptosis would require the removal of this polymer from the endonuclease, and, most likely, its removal from topoisomerase II and histone H1 as well. The role of poly (ADP-ribose) in apoptosis, carcinogenesis, and aging could be studied by the use of modulators of PARP activity (3-aminobenzamide, 3-nitrosobenzamide, 1% ethanol, etc.), inhibitors of poly ADP-ribose) glycohydrolase activity (ethacridine, 43 degrees C, etc.), and inhibitors of the PARP-specific protease (interleukin-1 beta converting enzyme (ICE)-like protease). Also, it would be of interest to determine if CR can decrease the half-life of poly (ADP-ribose), upregulate GJIC, and modulate the activities of PARP, the glycohydrolase, and the PARP-specific protease, factors potentially important in these processes.
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PMID:The beneficial effects of dietary restriction: reduced oxidative damage and enhanced apoptosis. 865 88

Weanling male F344 rats were fed either a semi-purified diet low in methionine and lacking in choline and folic acid (folate/methyl deficient) or a supplemented control diet for periods of 2, 5, 7 days, 3 weeks, and 9 weeks. Two days after initiating the folate/methyl deficient diet in weanling F344 rats, the incidence of apoptotic bodies, identified by in situ end-labeling of 3'-OH DNA strand breaks, was significantly increased in liver sections from the deficient rats. Apoptotic cell death was confirmed biochemically by an increase in nuclear Ca2+/Mg2+-dependent endonuclease activity that paralleled the increase in apoptotic bodies over the 9-week feeding period. There was no morphologic evidence of necrotic foci or necrosis-associated inflammatory response over the 9-week period. Confirming that cell turnover is chronically elevated in this model, the increase in apoptotic rate was accompanied by a sustained increase in the mitotic index (MI). The DNA repair-associated enzyme, poly(ADPribose) polymerase (PARP), was similarly elevated and was associated with significant decreases in the substrate for ADPribose polymer synthesis, nicotinamide adenine dinucleotide (NAD). Because folate metabolites are essential for de novo purine and thymidine biosynthesis, prolonged deficiency in folic acid can induce an imbalance in the deoxynucleotide precursors for DNA replication/repair and negatively affect the fidelity of DNA synthesis. Using an HPLC method, hepatic deoxyuridine triphosphate (dUTP) levels were increased at 3 and 9 weeks after initiation of the deficient diet and levels of thymidine triphosphate (dTTP) were reduced. An increase in dUTP/ dTTP ratio is consistent with a block in folate-dependent de novo thymidylate biosynthesis and may predispose to uracil misincorporation and DNA repair-related DNA strand breaks.
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PMID:Apoptosis and proliferation under conditions of deoxynucleotide pool imbalance in liver of folate/methyl deficient rats. 905 20

The synthesis and rapid turnover of ADP-ribose polymers is an immediate cellular response to DNA damage. We report here the isolation and characterization of cDNA encoding poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for polymer turnover. PARG was isolated from bovine thymus, yielding a protein of approximately 59 kDa. Based on the sequence of oligopeptides derived from the enzyme, polymerase chain reaction products and partial cDNA clones were isolated and used to construct a putative full-length cDNA. The cDNA of approximately 4.1 kilobase pairs predicted expression of a protein of approximately 111 kDa, nearly twice the size of the isolated protein. A single transcript of approximately 4. 3 kilobase pairs was detected in bovine kidney poly(A)+ RNA, consistent with expression of a protein of 111 kDa. Expression of the cDNA in Escherichia coli resulted in an enzymatically active protein of 111 kDa and an active fragment of 59 kDa. Analysis of restriction endonuclease fragments from bovine DNA by Southern hybridization indicated that PARG is encoded by a single copy gene. Taken together, the results indicate that previous reports of multiple PARGs can be explained by proteolysis of an 111-kDa enzyme. The deduced amino acid sequence of the bovine PARG shares little or no homology with other known proteins. However, it contains a putative bipartite nuclear location signal as would be predicted for a nuclear protein. The availability of cDNA clones for PARG should facilitate structure-function studies of the enzyme and its involvement in cellular responses to genomic damage.
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PMID:Isolation and characterization of the cDNA encoding bovine poly(ADP-ribose) glycohydrolase. 911 50

tRNA splicing is essential in yeast and humans and presumably all eukaryotes. The first two steps of yeast tRNA splicing, excision of the intron by endonuclease and joining of the exons by tRNA ligase, leave a splice junction bearing a 2'-phosphate. Biochemical analysis suggests that removal of this phosphate in yeast is catalyzed by a highly specific 2'-phosphotransferase that transfers the phosphate to NAD to form ADP-ribose 1"-2" cyclic phosphate. 2'-Phosphotransferase catalytic activity is encoded by a single essential gene, TPT1, in the yeast Saccharomyces cerevisiae. We show here that Tpt1 protein is responsible for the dephosphorylation step of tRNA splicing in vivo because, during nonpermissive growth, conditional lethal tpt1 mutants accumulate 2'-phosphorylated tRNAs from eight different tRNA species that are known to be spliced. We show also that several of these tRNAs are undermodified at the splice junction residue, which is always located at the hypermodified position one base 3' of the anticodon. This result is consistent with previous results indicating that modification of the hypermodified position occurs after intron excision in the tRNA processing pathway, and implies that modification normally follows the dephosphorylation step of tRNA splicing in vivo.
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PMID:A conditional lethal yeast phosphotransferase (tpt1) mutant accumulates tRNAs with a 2'-phosphate and an undermodified base at the splice junction. 940 90

Ultraviolet light (UV) induced rapid apoptosis of U937 leukemia cells, concurrent with DNA fragmentation and cleavage of poly(ADP-ribose)polymerase (PARP) by activated caspase-3. The in vitro reconstitution of intact HeLa S3 nuclei and apoptotic U937 cytosolic extract (CE) revealed that (i) Ca2+/Mg(2+)-dependent, Zn(2+)-sensitive endonuclease activated in the apoptotic CE induced DNA ladder in HeLa nuclei at pH 6.8-7.4, (ii) activated caspase-3 cleaved PARP in HeLa nuclei, and (iii) when the apoptotic CE was treated with the caspase-3 inhibitor (1 microM Ac-DEVD-CHO) or the caspase-1 inhibitor (10 microM Ac-YVAD-CHO), the former, but not the latter, caused a 50% inhibition of DNA fragmentation and the complete inhibition of PARP cleavage in HeLa nuclei. Similarly, Ac-DEVD-CHO (100 microM) inhibited apoptosis and DNA ladder by 50% and PARP cleavage completely in UV-irradiated U937 cells, but Ac-YVAD-CHO (100 microM) did not. Thus, UV-induced apoptosis of U937 cells involves the Ca2+/Mg(2+)-dependent endonuclease pathway and the caspase-3-PARP cleavage-Ca2+/Mg(2+)-dependent endonuclease pathway. The former pathway produced directly 50% of apoptotic DNA ladder, and the latter involved activated caspase-3 and PARP cleavage, followed by formation of the remaining 50% DNA ladder by the activated endonuclease. In UV-irradiated B-cell lines, further, p53-dependent increase of Bax resulted in a greater caspase-3 activation compared to its absence. However, UV-induced activation of JNK1 and p38 was not affected by the caspase-1 and -3 inhibitors in U937 cells, so that caspases-1 and -3 do not function upstream of JNK1 and p38.
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PMID:Mechanism of UV-induced apoptosis in human leukemia cells: roles of Ca2+/Mg(2+)-dependent endonuclease, caspase-3, and stress-activated protein kinases. 952 59

Oxidative stress induced by tert-butyl hydroperoxide (tBOOH) in freshly isolated rat hepatocytes caused DNA damage and loss of membrane integrity. Such DNA lesions are likely to be single strand breaks since neither caryolysis nor chromatine condensation was seen in electron micrographs from tBOOH-treated cells. In addition, pulsed field gel electrophoresis of genomic DNA from both control and tBOOH-treated hepatocytes showed similar profiles, indicating the absence of internucleosomal DNA cleavage, a classical reflection of apoptotic endonuclease activity. The activation of the repair enzyme poly(ADP-ribose)polymerase (PARP) following DNA damage by tBOOH induced a dramatic drop in both NAD(+) and ATP. The inhibition of PARP by 3-aminobenzamide enhanced DNA damage by tBOOH, restored NAD(+) and ATP levels, but did not result in better survival against cell killing by tBOOH. The lack of the protective effect of PARP inhibitor, therefore, does not implicate PARP in the mechanism of tBOOH-induced cytotoxicity. Electron micrographs also show no mitochondrial swelling in cells under oxidative stress, but such organelles were mainly located around the nucleus, a picture already observed in autoschizis, a new suggested kind of cell death which shows both apoptotic and necrotic morphological characteristics.
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PMID:Activation of Poly(ADP-Ribose)Polymerase in rat hepatocytes does not contribute to their cell death by oxidative stress. 1062 77

In this study, we investigated the mechanism of apoptosis by 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in cocultures of parenchymal and nonparenchymal liver cells, since the liver consists of various cell types and they cooperatively respond to chemicals. It was found that cocultures were more susceptible to cell death by Trp-P-1 than culture of each cell type alone. In cocultures, Trp-P-1 induced DNA fragmentation accompanied by the activation of 18-kDa endonuclease. Trp-P-1 (30 microM) caused a rapid increase in Bid protein level in mitochondria and the leakage of cytochrome c from mitochondria into the cytosol 15 min after treatment. On the other hand, an increase in Bax protein and a decrease in Bcl-2 protein were detected in the mitochondrial fraction 2 h after treatment following the increases in p53 protein level and DNA binding activity of NF-kappa B. Caspase-8 was activated within 30 min followed by the activation of downstream caspases as measured using the corresponding peptide substrates. The activation of caspases was also confirmed by cleavage of caspase-3, poly(ADP-ribose)polymerase, and protein kinase C-delta as analyzed by Western blotting. A peptide inhibitor of caspase-8 diminished DNA ladder formation and the activation of downstream caspases, but a caspase-9 inhibitor and pyrrolidinedithiocarbamate as an inhibitor of NF-kappa B showed only partial inhibition, suggesting that caspase-8 is the apical caspase in the cascade. These results led to the conclusion that Trp-P-1 mainly drives the caspase-8-mediated pathway that involves Bid, accompanied by a delay in the p53/NF-kappa B-mediated side pathway that involves Bax, Bcl-2, and caspase-9.
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PMID:The heterocyclic amine, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole induces apoptosis in cocultures of rat parenchymal and nonparenchymal liver cells. 1170 1

DNA single-strand breaks (SSB) are one of the most frequent DNA lesions produced by reactive oxygen species and during DNA metabolism, but the analysis of cellular responses to SSB remains difficult due to the lack of an experimental method to produce SSB alone in cells. By using human cells expressing a foreign UV damage endonuclease (UVDE) and irradiating the cells with UV through tiny pores in membrane filters, we created SSB in restricted areas in the nucleus by the immediate action of UVDE on UV-induced DNA lesions. Cellular responses to the SSB were characterized by using antibodies and fluorescence microscopy. Upon UV irradiation, poly(ADP-ribose) synthesis occurred immediately in the irradiated area. Simultaneously, but dependent on poly(ADP-ribosyl)ation, XRCC1 was translocated from throughout the nucleus, including nucleoli, to the SSB. The BRCT1 domain of XRCC1 protein was indispensable for its poly(ADP-ribose)-dependent recruitment to the SSB. Proliferating cell nuclear antigen and the p150 subunit of chromatin assembly factor 1 also accumulated at the SSB in a detergent-resistant form, which was significantly reduced by inhibition of poly(ADP-ribose) synthesis. Our results show the importance of poly(ADP-ribosyl)ation in sequential cellular responses to SSB.
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PMID:Spatial and temporal cellular responses to single-strand breaks in human cells. 1274 98

We have evaluated the influence of protein poly(ADP-ribosyl)ation in the relaxation of chromatin by exposing a rat liver polynucleosomal extract to micrococcal nuclease (MNase) digestion. The kinetic susceptibility of polynucleosomes to endonuclease digestion was determined as a function of the time of incubation as well as endonuclease concentration. To validate our assay, we also ran control experiments with protein-free calf thymus DNA as the opposite of polynucleosomal DNA. Rat liver chromatin was also incubated in the absence or presence of exogenously added 200 microM betaNAD(+), the poly(ADP-ribosyl)ation substrate, before MNase digestion. For incubations in the presence of betaNAD(+), the synthesis of polynucleosomal poly(ADP-ribose) was stopped with 1 mM benzamide. After addition of MNase, endonuclease digestion was blocked with EDTA to chelate the Mg(2+) ions needed for enzymatic activation, and the samples were subjected to electrophoresis through 1.5% agarose gels. As expected, a faster degradation of chromatin into oligonucleosomal DNA ladders was observed upon protein poly(ADP-ribosyl)ation when the chromatin extract was preincubated with 200 microM betaNAD(+). Thus, our results are consistent with the conclusion that the covalent poly(ADP-ribosyl)ation of polynucleosomal proteins favors a more "relaxed" or "open" structure, which renders chromatin more susceptible to MNase digestion.
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PMID:Polynucleosomal synthesis of poly(ADP-ribose) causes chromatin unfolding as determined by micrococcal nuclease digestion. 1565 42

Zinc ions exert an inhibitory effect on Ca(2+)Mg(2+)-dependent endonuclease which is supposed to be responsible for the fragmentation of DNA during apoptosis. In the experimental system we used, that is HeLa cells treated with VP-16, the protection from internucleosomal DNA degradation is modulated by Zn concentration and appears to be dependent on the time after treatment. This effect does not prevent cell death or occurrence of apoptotic parameters, suggesting that DNA ladder appearance is not a crucial event in apoptosis. The activation of poly(ADP-ribose)polymerase following the administration of VP-16, is not observed in cells in which DNA fragmentation has been abolished by zinc, supporting the hypothesis that this event is regulated by the appearance of small-sized DNA fragments.
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PMID:Dose-dependent zinc inhibition of DNA ladder in apoptotic HeLa cells regulates the activity of poly(ADP-ribose) polymerase and does not protect from death induced by VP-16. 1718 23


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