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)

The formation of apurinic/apyrimidinic sites (AP-sites) and single strand breaks (SSB) by chromate and ascorbate (AsA) in isolated DNA was investigated using a number of agents that cleave DNA at AP-sites (putrescine, exonuclease III, the tripeptide Lys-Trp-Lys, and an AP-endonuclease containing fraction isolated from human fibroblasts). Relative to the number of SSB caused by chromate and AsA alone, all these agents induced additional nicking, indicating the induction of AP-sites. Chromate/AsA-induced AP-sites contain aldehyde groups, as cleavage by putrescine could be prevented by treatment with borohydride which reduced the aldehyde. The time course for the formation of both DNA lesions was very similar, and there was a 1:1 ratio of the number of SSB to the number of AP-sites. The addition of catalase to incubation mixtures containing chromate/AsA led to an almost complete suppression of AP-sites and SSB. In systems containing lower concentrations of chromate/AsA, the exclusion of oxygen inhibited the formation of both lesions. It is suggested that AP-sites and SSB arise from attack by reactive species deriving from chromate/AsA on one single site at DNA, probably the sugar moiety. In view of the known mutagenicity of AP-sites, these results could aid an understanding of the mechanisms underlying chromium(VI) carcinogenicity.
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PMID:The reductive conversion of chromium (VI) by ascorbate gives rise to apurinic/apyrimidinic sites in isolated DNA. 749 38

Exposure of human white blood cells to UICC crocidolite asbestos in vitro resulted in the formation of DNA strand breakage in a dose-dependent manner up to a fibre concentration of 100 micrograms/ml. Subsequent incubations with the iron chelator desferrioxamine or the intracellular Ca2+ chelator Quin-2 prevented DNA strand break formation above control incubations. Addition of aurintricarboxylic acid, an endonuclease inhibitor, similarly abolished crocidolite-induced DNA strand breaks in these cells. These results suggest that crocidolite-derived hydroxyl radicals do not directly induce DNA strand breakage in mammalian white blood cells. In order to assess Ca2+ mobilisation from intracellular stores in control and crocidolite-treated cells, the fullness of these stores was measured by treating with thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. On addition of thapsigargin to fura-2AM-loaded cells treated with crocidolite we demonstrated that the endoplasmic reticulum stores had been depleted as no further Ca2+ was released, unlike control cells. We suggest that strand breakage is caused by a complex set of events involving oxygen free radicals that may disturb intracellular Ca2+ homoeostasis and the breaks are produced by secondary reactions, involving Ca(2+)-mediated enzymes.
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PMID:Calcium chelator Quin-2 prevents crocidolite-induced DNA strand breakage in human white blood cells. 752 85

We previously demonstrated the UV-induced formation of cytosine hydrate in DNA and its deamination product, uracil hydrate, via their release from the DNA backbone by the DNA glycosylase activity of Escherichia coli endonuclease III. Subsequently, endonuclease III-mediated release of thymine hydrate from UV-irradiated poly(dA-dT) was reported. Therefore, we asked whether 5-methylcytosine residues in DNA underwent photohydration and deamination to thymine hydrate in analogy to UV-induced deamination of cytosine. An alternating DNA copolymer containing 5-methylcytosine was irradiated with UVC and incubated with endonuclease III. No 5-methylcytosine hydrate was released. Instead, UV-induced nonenzymatic release of 5-methylcytosine occurred. Similarly, incubation of UV-irradiated poly(dA-dT) with endonuclease III did not release thymine hydrate; nonenzymatic release of thymine occurred. Nonenzymatic release of 5-methylpyrimidines was oxygen dependent, enhanced by ferric ion and inhibited by free radical scavengers. In contrast, photohydration of cytosine was oxygen independent, and only small amounts of cytosine were nonenzymatically released. Thus, 5-methylpyrimidine residues within alternating Pu-Py sequences in DNA do not undergo photohydration, but instead undergo cleavage of their N-glycosyl bonds yielding abasic (AP) sites. The inability to repair such AP sites may explain the UV sensitivity of E. coli xthnfo mutants, which lack AP endonuclease activity. We suggest that N-glycosyl bond cleavage is mediated by radical species formed via transfer of an electron from UV-excited triplet 5-methylpyrimidines to ground state oxygen and/or ferric ions.
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PMID:Comparison of the effects of UV irradiation on 5-methyl-substituted and unsubstituted pyrimidines in alternating pyrimidine-purine sequences in DNA. 754 89

Cell death via apoptosis is an important event involved in a number of immunological processes. Recently, apoptosis has been associated with oxidative stress in a number of cell systems. Here we assessed the inhibitory capacity of different antioxidants on UV- and drug-induced apoptosis in the human leukemic cell line, HL-60. We found that the oxygen radical scavenger, BHA, the radioprotector cysteamine and the metal chelators, pyrrolidinedithiocarbamate (PDTC), diethyldithiocarbamate (DEDTC), and dimethyldithiocarbamate (DMDTC), were able to significantly inhibit nuclear fragmentation and reduce the formation of apoptotic bodies in UV-irradiated human leukemic cells. Both BHA and PDTC were found to reduce DNA fragmentation as assessed by in situ DNA nick-end labelling and quantification thereof using fluorescence flow cytometry. In addition to inhibiting UV-induced apoptosis, PDTC was also capable of reducing the amount of apoptosis induced by a range of cytotoxic drugs, such as actinomycin-D, camptothecin, etoposide, and melphalan, whereas BHA and cysteamine were not as effective in these cases after more than four hours in culture when compared to PDTC. To further elucidate the working mechanism of PDTC, we have looked at the effect of PDTC on DNA fragmentation in isolated nuclei, under conditions that promote activation of endogenous endonuclease involved in apoptosis. In contrast to ZnCl2, a potent inhibitor of endonuclease activity, PDTC was unable to inhibit DNA-ladder formation in this assay. Taken together, these results indicate that oxygen radicals may have a central role to play in the induction of apoptosis and that dithiocarbamates can serve as potent inhibitors of apoptosis induced by a wide variety of stimuli.
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PMID:Inhibition of apoptosis by antioxidants in the human HL-60 leukemia cell line. 757 57

Glucocorticoid hormones stimulate apoptosis in thymocytes via a mechanism that involves changes in intracellular Ca2+, and exogenous Ca2+ can also directly promote the nuclear alterations of apoptosis (lamin degradation and chromatin cleavage) in isolated nuclei. Here we report that glucocorticoid treatment resulted in the production of reactive oxygen species and the depletion of reduced glutathione. Separation of apoptotic cells on Percoll gradients demonstrated that both effects selectively occurred in thymocytes undergoing apoptosis. Moreover, glucocorticoid-induced endonuclease activation was partially blocked by the antioxidant N-acetyl-L-cysteine. Although abrogation of methylprednisolone-induced Ca2+ increases using the intracellular Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid resulted in inhibition of endonuclease activation, it failed to prevent GSH depletion. However, N-acetyl-L-cysteine almost completely blocked methylprednisolone-induced elevations in cytosolic calcium levels, indicating that oxidative stress was playing a role in the Ca2+ response. Our results support the idea that oxidative stress is a key component of the apoptotic effector pathway in thymocytes, and that it interacts, at least in part, with the Ca2+ response.
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PMID:Oxygen radical production and thiol depletion are required for Ca(2+)-mediated endogenous endonuclease activation in apoptotic thymocytes. 759 22

The specific recognition of DNA modifications by repair endonucleases was used to characterize the DNA damage induced by photosensitizers in the presence of visible light. Under cell-free conditions, chemically unrelated photosensitizers (methylene blue, acridine orange, proflavin, riboflavin, hematoporphyrin) induce the same type of DNA damage. It is characterized by a high number of base modifications sensitive to the repair endonuclease FPG protein (formamidopyrimidine-DNA glycosylase), while both the number of DNA strand breaks and the number of sites of base loss (sensitive to exonuclease III or endonuclease IV) is low. Therefore the damage is markedly different from that induced by hydroxyl radicals. Mechanistically, the generation of the base modifications sensitive to FPG protein involves singlet oxygen in some, but possibly not all cases, as substituting D2O for H2O increases the reaction yield six-fold in the case of methylene blue, but only 1.4-fold in the case of acridine orange. In plasmids from Salmonella typhimurium strains treated with methylene blue or acridine orange plus light and from Escherichia coli strains treated with acridine orange or proflavin plus light, the same type of damage was observed as under cell-free conditions. In L1210 mouse leukemia cells exposed to acridine orange plus light, the numbers of modifications sensitive to FPG protein and exonuclease III were quantified, in addition to strand breaks, by a modified alkaline elution assay. Again, the number of base modifications sensitive to FPG protein was found to be several-fold higher than the number of strand breaks and sites of base loss. It has to be concluded that the DNA damage in the intact cells is not mediated by hydroxyl radicals or cellular nucleases, but by the same mechanism as operates under cell-free conditions with these agents.
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PMID:DNA damage induced by photosensitizers in cellular and cell-free systems. 768 82

The ribozyme derived from the intron of Tetrahymena thermophila pre-rRNA catalyzes a site-specific endonuclease reaction with both RNA and DNA oligonucleotides. The total transition-state stabilization by the ribozyme, encompassing the binding and chemical steps, is 4.8 kcal/mol greater with a single ribose at the cleavage site relative to the all-deoxyribose substrate. Here we show that this effect is specific to the chemical transition state, with a contribution of only approximately 0.7 kcal/mol toward binding. Substrates with a series of 2'-substituents, -OH(ribo), -F2 (2',2'-difluoro-2'-deoxyribo), F(2'-fluoro-2'-deoxyribo), and -H(deoxyribo), follow a linear free energy relationship between the rate of the chemical step of the ribozyme-catalyzed reaction and the pK(a) of the leaving group, with slope beta leaving group approximately -0.8. Because proton donation to the 3'-oxygen atom from a general acid of the ribozyme would be expected to render the rate insensitive to the pK(a) of the leaving group, it is suggested that this ribozyme does not employ general acid catalysis. The 2'-OCH3 (2'-methoxy-2'-deoxyribo) substituent does not follow this correlation, apparently due to steric hindrance within the active site. The rate of cleavage of the 2'-substituted substrates by the ribozyme follows the order 2'-F2 > -F > -H, suggestive of an inductive effect, i.e., acceleration of the reaction by electron-withdrawing groups. The 2'-OH group provides the largest transition-state stabilization. Because of uncertainty in the relative effect of the 2'-OH and 2'-H substituents on the pK(a) of the neighboring 3'-oxygen leaving group, we do not discount the possibility of interactions between the 2'-hydroxyl group and the ribozyme that further enhance reactivity. Nevertheless, the 2'-OH effect can be explained at least partially by an intramolecular hydrogen bond to an incipient oxyanion at the neighboring 3'-position. This oxyanion is forming as the phosphodiester bond is breaking, explaining why the stabilization is specific to the transition state. Analogous differential hydrogen bonding might be widely used by enzymes to achieve selective transition-state stabilization.
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PMID:The importance of being ribose at the cleavage site in the Tetrahymena ribozyme reaction. 768 73

The Apn1 DNA repair enzyme of Saccharomyces cerevisiae acts on abasic sites and oxygen radical damages. Apn1 is homologous to the repair endonuclease IV of Escherichia coli, but the yeast protein is approximately 80 residues longer at the C terminus. The Apn1 C terminus is rich in basic amino acids and includes two lysine/arginine clusters related to the nuclear transport signals of some other proteins. We show here by indirect immunofluorescence that Apn1 is localized to the yeast nucleus. Mutant Apn1 proteins were engineered with progressive deletions inward from the C terminus. Elimination of just the last 12 residues from Apn1 (to yield Apn355) did not alter the stability in yeast cells or the in vitro activity of the enzyme. Greater truncation of Apn1 produced proteins of apparently lower (Apn334) or much lower (Apn315 and Apn293) in vivo stability. Both Apn355 and Apn334 failed to concentrate in the yeast nucleus and remained in the cytoplasm. These delocalized derivatives also failed to restore wild-type resistance to oxidative or alkylating agents in a delta apn1 strain. Apn355 and Apn334 complemented repair-deficient E. coli as effectively as did wild-type Apn1. Resistance to these DNA-damaging agents in yeast was restored if Apn355 and Apn334 (but not Apn315 or Apn293) were overproduced approximately 20-fold, which suggests either weak active transport or passive diffusion of these derivatives into the nucleus. Replacement of the C-terminal 12 residues of Apn1 with the nuclear targeting sequence of SV40 T-antigen did not restore effective function or nuclear localization in yeast.
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PMID:Intracellular localization of the Apn1 DNA repair enzyme of Saccharomyces cerevisiae. Nuclear transport signals and biological role. 769 Jul 56

Ionizing radiation produces a variety of DNA damage through active oxygen species such as the superoxide radical (O2.-), the hydroxyl radical (OH.), and hydrogen peroxide (H2O2). The removal of alkylation-induced apurinic (AP) sites and 3'-blocking deoxyribose fragments by exonuclease III (xth) and endonuclease IV (nfo) has been well demonstrated in E. coli. Very little information on the repair of radiation-induced DNA damage by human apurinic endonuclease is available. We examined the biological roles of the human AP endonuclease in the repair of radiation-induced DNA damage. An expression vector was constructed with human APE cDNA and transformed into radiation-sensitive E. coli mutants (xth- and nfo-). The radiation cytotoxicity was assayed by cell survival curves. Expression of human AP endonuclease in E. coli confirmed that AP endonuclease could complement exonuclease III functionally to diminish radiation cytotoxicity. In contrast, AP endonuclease was not able to increase resistance to H2O2, owing to a poor 3'-termini repair. We also tested whether AP endonuclease is a limiting factor for radiation cytotoxicity by using a plasmid nicking assay. Cell extracts from mutant cells with or without AP endonuclease expression were added to irradiated supercoiled plasmid DNA. The inability to convert supercoiled plasmid DNA to relaxed or linear forms suggested that there were large accumulations of AP sites in the mutant cell extracts. The AP endonuclease activities estimated from the plasmid nicking assays are 20-fold lower in the cell extracts of AP endonuclease-deficient mutant than in AP endonuclease-expressing cells. Therefore, AP endonuclease is a limiting step of base excision repair for the radiation-sensitive E. coli mutant, BW528. Our results conclude that AP endonuclease is responsible for the removal of AP sites from gamma-radiation-induced base damage in E. coli.
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PMID:Reduction of radiation cytotoxicity by human apurinic endonuclease in a radiation-sensitive Escherichia coli mutant. 769 Sep 78

The type II restriction endonuclease EcoRV was crystallized as a complex with the substrate DNA undecamer AAAGATATCTT (recognition sequence underlined). These crystals diffract to much better resolution (2 A) than was the case for the previously reported complex with the decamer GGGATATCCC [Winkler, F. K., Banner, D. W., Oefner, C., Tsernoglou, D., Brown, R. S., Heathman, S. P., Bryan, R. K., Martin, P. D., Petratos, K., & Wilson, K. S. (1993) EMBO J. 12, 1781-1795]. The crystal structure contains one dimer complex in the asymmetric unit and was solved by molecular replacement. The same kinked DNA conformation characteristic for enzyme-bound cognate DNA is observed. Crystals, soaked with Mg2+, show the essential cofactor bound at only one active site of the dimer, and the DNA is not cleaved. The Mg2+ has one oxygen from the scissile phosphodiester group and two carboxylate oxygens, one form Asp74 and one from Asp90, in its octahedral ligand sphere. The scissile phosphodiester group is pulled by 1 A toward the Mg2+. After substrate cleavage in solution, isomorphous crystals containing the enzyme--product--Mg2+ complex were obtained. In this structure, each of the 5'-phosphate groups is bound to two Mg2+. The kinked DNA conformation is essentially maintained, but the two central adenines, 3' to the cleavage sites, form an unusual cross-strand base stacking. The structures have been refined to R factors of 0.16 at 2.1-2.0 A resolution maintaining very good stereochemistry. On the basis of these structures and inspired by recent kinetic data [Vipond, I. B., & Halford, S. E. (1994) Biochemistry (second paper of three in this issue)], we have constructed a transition state model with two metals bound to the scissile phosphorane group.
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PMID:Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 A resolution. 781 64


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