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)

The influence of oxidative stress by hydrogen peroxide (H2O2) was examined in mouse primary cultured hepatocytes. A change in morphology was observed in hepatocytes incubated for 30 min in saline A containing H2O2. The percentage of dead cells, as measured by the fluorescence method, was increased in a dose-dependent manner. In addition, a ladder-like DNA fragmentation pattern was detected by agarose gel electrophoresis 1 h after exposure to 3 mM H2O2. This phenomenon was prolonged for 24 h. Hydrogen peroxide-induced cell viability reduction and DNA fragmentation were dose-dependently protected by the addition of antioxidants (N-acetylcysteine, L-ascorbic acid), a metal-chelator (1,10-phenanthroline), iron-chelator (deferoxamine) and intracellular calcium ion chelator (quin 2-AM). No influence, however, was detected by endonuclease inhibitors (zinc, aurintricarboxylic acid) and poly (ADP-ribose) polymerase inhibitors (3-aminobenzamide, theophylline). These results following H2O2-induced cell viability reduction suggested that oxidative stress by H2O2 itself or H2O2-derived changes involved in ferrous or intracellular calcium ions resulted in apoptosis in mouse primary cultured hepatocytes. These phenomena are not likely to be associated with endonuclease or poly (ADP-ribose) polymerase.
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PMID:Characterization of hydrogen peroxide-induced apoptosis in mouse primary cultured hepatocytes. 1070 8

Direct exposure of human hepatoma cell line SMMC-7721 to hydrogen peroxide (H2O2) can induce apoptosis. Apoptosis induced by H2O2 was inhibited by cycloheximide, actinomycin D, 3-aminobenzamide, EGTA or Zn2+. H2O2 can increase the level of intracellular Ca2+, downregulate GSH levels, slightly induce lipid peroxidation, and lead to change in the ratio of reduced ion components to oxidized ion components of cells. Analysis of flow cytometry indicates that H2O2 decreases the level of Bcl-2. The data indicate that H2O2-induced apoptosis requires new mRNA and protein syntheses; H2O2 can activate Ca2+/Mg2+-dependent endonuclease leading to internucleosomal DNA fragmentation and activation of poly (ADP-ribose) polymerase interfering with the energy metabolism of the cell. The H2O2 downregulation of GSH may be more important for apoptosis than H2O2 induction of lipid peroxidation, and the H2O2 induced changes in redox status of the cell may be among the original events which lead up to other biochemical changes.
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PMID:Hydrogen peroxide induces apoptosis in human hepatoma cells and alters cell redox status. 1082 69

High molecular weight (HMW) fragmentation of nuclear chromatin was studied in cultured rat oligodendrocytes (OL) exposed to hydrogen peroxide (H2O2). Intact genomic DNA was isolated by agarose embedding, and analyzed by field inversion gel electrophoresis, with and without S1 endonuclease digestion to detect and discriminate between single and double stranded fragmentations, respectively. The exposure of OL to H2O2 resulted in a very rapid degradation of chromosomal DNA into HMW fragments that reflect native chromatin structure. Hence, within 10 min after the addition of 1 mM H2O2, a discrete pool representing approximately 45% of the nuclear chromatin underwent single strand digestion into >400 kb fragments likely at AT-rich matrix attachment regions. Subsequent accumulation of single stand breaks at these regions led to bifilar scission. Ultimately, chromatin within this susceptible pool was cleaved at remaining matrix attachment regions into 50-200 kb fragments. Chromatin digestion could be elicited with H2O2 concentrations as low as 50 microM. After the removal of H2O2, most >400 kb fragments were religated within 2 h; however, digestion into 50-200 kb fragments was irreversible. The DNA digestion was not accompanied by the degradation of nuclear proteins, i.e., lamins A/C and poly (ADP-ribose) polymerase indicating that chromatin fragmentation is unlikely to be mediated by proteolysis. In conclusion, H2O2 at pathologically relevant concentrations induces a very rapid and extensive digestion of OL chromatin into HMW fragments. Because the chromatin fragmentation is only partly reversible, it may be a decisive factor in committing oxidatively stressed OL to degeneration and/or death.
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PMID:Hydrogen peroxide induces rapid digestion of oligodendrocyte chromatin into high molecular weight fragments. 1091 83

Bacteria producing endonuclease colicins are protected against their cytotoxic activity by virtue of a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. DNase binding by the immunity protein occurs through a "dual recognition" mechanism in which conserved residues from helix III act as the binding-site anchor, while variable residues from helix II define specificity. We now report the 1.7 A crystal structure of the 24.5 kDa complex formed between the endonuclease domain of colicin E9 and its cognate immunity protein Im9, which provides a molecular rationale for this mechanism. Conserved residues of Im9 form a binding-energy hotspot through a combination of backbone hydrogen bonds to the endonuclease, many via buried solvent molecules, and hydrophobic interactions at the core of the interface, while the specificity-determining residues interact with corresponding specificity side-chains on the enzyme. Comparison between the present structure and that reported recently for the colicin E7 endonuclease domain in complex with Im7 highlights how specificity is achieved by very different interactions in the two complexes, predominantly hydrophobic in nature in the E9-Im9 complex but charged in the E7-Im7 complex. A key feature of both complexes is the contact between a conserved tyrosine residue from the immunity proteins (Im9 Tyr54) with a specificity residue on the endonuclease directing it toward the specificity sites of the immunity protein. Remarkably, this tyrosine residue and its neighbour (Im9 Tyr55) are the pivots of a 19 degrees rigid-body rotation that relates the positions of Im7 and Im9 in the two complexes. This rotation does not affect conserved immunity protein interactions with the endonuclease but results in different regions of the specificity helix being presented to the enzyme.
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PMID:Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes. 1096 13

The results of a 3-ns molecular dynamics simulation of the dodecamer duplex d(TATGGATCCATA)(2) recognized by the BamHI endonuclease are presented here. The DNA has been simulated as a flexible molecule using an AMBER force field and the Ewald summation method, which eliminates the undesired effects of truncation and permits evaluation of the full effects of electrostatic forces. The starting B conformation evolves toward a configuration quite close to that observed through x-ray diffraction in its complex with BamHI. This configuration is fairly stable and the Watson-Crick hydrogen bonds are well maintained over the simulation trajectory. Hydration analysis indicates a preferential hydration for the phosphate rather than for the ester oxygens. Hydration shells in both the major and minor groove were observed. In both grooves the C-G pairs were found to be more hydrated than A-T pairs. The "spine of hydration" in the minor groove was clear. Water residence times are longer in the minor groove than in the major groove, although relatively short in both cases. No special long values are observed for sites where water molecules were observed by x-ray diffraction, indicating that water molecules having a high probability of being located in a specific site are also fast-exchanging.
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PMID:Structure and hydration of BamHI DNA recognition site: a molecular dynamics investigation. 1096 90

Cells harvested from Fanconi anemia (FA) patients show an increased hypersensitivity to the multifunctional DNA damaging agent mitomycin C (MMC), which causes cross-links in DNA as well as 7,8-dihydro-8-oxoguanine (8-oxoG) adducts indicative of escalated oxidative DNA damage. We show here that the Drosophila multifunctional S3 cDNA, which encodes an N-glycosylase/apurinic/apyrimidinic (AP) lyase activity was found to correct the FA Group A (FA(A)) and FA Group C (FA(C)) sensitivity to MMC and hydrogen peroxide (H2O2). Furthermore, the Drosophila S3 cDNA was shown to protect AP endonuclease deficient E. coli cells against H(2)O(2) and MMC, and also protect 8-oxoG repair deficient mutM E. coli strains against MMC and H2O2 cell toxicity. Conversely, the human S3 protein failed to complement the AP endonuclease deficient E. coli strain, most likely because it lacks N-glycosylase activity for the repair of oxidatively-damaged DNA bases. Although the human S3 gene is clearly not the genetic alteration in FA cells, our results suggest that oxidative DNA damage is intimately involved in the overall FA phenotype, and the cytotoxic effect of selective DNA damaging agents in FA cells can be overcome by trans-complementation with specific DNA repair cDNAs. Based on these findings, we would predict other oxidative repair proteins, or oxidative scavengers, could serve as protective agents against the oxidative DNA damage that occurs in FA.
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PMID:The Drosophila S3 multifunctional DNA repair/ribosomal protein protects Fanconi anemia cells against oxidative DNA damaging agents. 1118 42

Antibiotic WS-5995A (code name J4) and two of its synthetic analogs, o-quinone J1 and model p-quinone J7, which show some structural similarity with both ellagic acid (EA) and genistein (GEN), were compared for their antileukemic activity in L1210 cells in vitro. Overall, J4 is more cytostatic and cytotoxic than J1 and J7, suggesting that methyl and methoxy substitutions, a p-quinone moiety, and a hydrogen bonding phenolic group may enhance the antitumor potential of these naphthoquinone lactones, which are all more potent than EA and GEN. For instance, the lead compound J4 inhibits tumor cell proliferation and viability at day 4 (IC(50): 0.24--0.65 microM) more effectively than EA (IC(50): 5--6 microM) and GEN (IC(50): 7 microM). Since J4 does not increase but rather decreases the mitotic index of L1210 cells at 24 h, it is not an antitubulin drug but might arrest early stages of cell cycle progression like EA and GEN. A 1.5- to 3-h pretreatment with J4 is sufficient to inhibit the rates of DNA, RNA and protein syntheses (IC(50): 2.0--2.5 microM) determined over 30- to 60-min periods of pulse-labeling in L1210 cells in vitro, whereas EA (IC(50): 20-130 microM) and GEN (IC(50): 40--115 microM) are less effective against macromolecule synthesis. In contrast to 156 microM EA, which is inactive, a 15-min pretreatment with 10--25 microM J4 has the advantage of also inhibiting the cellular transport of both purine and pyrimidine nucleosides over a 30 s period in vitro, an effect which can be mimicked by 156 microM GEN. Hence, the WS-5995 analogs and GEN may prevent the incorporation of [(3)H]adenosine and [(3)H]thymidine into DNA because they rapidly block the uptake of these nucleosides by the tumor cells. After 24 h, the concentration-dependent induction of DNA cleavage by J4 peaks at 10 microM and declines at 25 microM, whereas EA and GEN are ineffective at 10 microM but maximally stimulate DNA cleavage at 62.5 microM. Like EA and GEN, the mechanism by which J4 induces DNA fragmentation is inhibited by actinomycin D, cycloheximide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone and ZnSO(4), suggesting that J4 triggers apoptosis by caspase and endonuclease activation. Because they are more potent than EA and GEN, and affect both nucleoside transport and DNA cleavage, the WS-5995 antitumor antibiotics might be valuable in polychemotherapy to potentiate the action of antimetabolites and sensitize multidrug-resistant tumor cells.
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PMID:Quinone isomers of the WS-5995 antibiotics: synthetic antitumor agents that inhibit macromolecule synthesis, block nucleoside transport, induce DNA fragmentation, and decrease the growth and viability of L1210 leukemic cells more effectively than ellagic acid and genistein in vitro. 1139 69

Higher order chromatin degradation (HOCD), i.e. the scission of nuclear chromatin loops at the matrix attachment regions (MARs), is a hallmark of programmed cell death. We have previously demonstrated that hydrogen peroxide (H(2)O(2)) induces rapid HOCD in cultured oligodendrocytes generating two subpopulations of DNA fragments of >or=400 and 50-200 kb. In the present study, we examined the involvement of calcium in this process. HOCD was induced in primary rat oligodendrocytes by exposure to 1 mM H(2)O(2) and assessed by field inversion gel electrophoresis with and without S1 endonuclease digestion, to detect single and double stranded fragmentation, respectively. Chelating intracellular calcium with BAPTA/AM prior to H(2)O(2) exposure inhibited HOCD in a dose-dependent manner. Complete inhibition of HOCD was attained with 50 muM BAPTA/AM. The pretreatment of cells with desferroxamine mesylate, which may lower intracellular calcium levels, also resulted in a profound inhibition of HOCD, but the initial chromatin digestion into >or=400 kb single stranded DNA fragments was unaffected. Neither removing extracellular calcium nor blocking calcium release from intracellular stores with TMB-8 affected HOCD. Moreover, increasing intracellular calcium with A23187 calcium ionophore did not induce HOCD. Subsequent study in nuclei purified from C6 glioma cells revealed that the endonuclease responsible for HOCD is calcium-independent, but is magnesium-dependent. Magnesium-induced HOCD was not affected by the removal of calcium from nuclei with EGTA, but was practically abrogated in nuclei prepared from BAPTA/AM-pretreated cells. These results indicate that although H(2)O(2)-induced HOCD is not directly mediated by an increase of intracellular calcium concentration, normal resting levels of intracellular calcium are required for the maintenance of MAR-associated endonuclease in an active form.
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PMID:Higher order chromatin degradation in glial cells: the role of calcium. 1143 75

I-TevI is a site-specific, sequence-tolerant intron endonuclease. The crystal structure of the DNA-binding domain of I-TevI complexed with the 20 bp primary binding region of its DNA target reveals an unusually extended structure composed of three subdomains: a Zn finger, an elongated segment containing a minor groove-binding alpha-helix, and a helix-turn-helix. The protein wraps around the DNA, mostly following the minor groove, contacting the phosphate backbone along the full length of the duplex. Surprisingly, while the minor groove-binding helix and the helix-turn- helix subdomain make hydrophobic contacts, the few base-specific hydrogen bonds occur in segments that lack secondary structure and flank the intron insertion site. The multiple base-specific interactions over a long segment of the substrate are consistent with the observed high site specificity in spite of sequence tolerance, while the modular composition of the domain is pertinent to the evolution of homing endonucleases.
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PMID:Intertwined structure of the DNA-binding domain of intron endonuclease I-TevI with its substrate. 1144 4

Hydroxyurea is a chemotherapeutic agent used for the treatment of myeloproliferative disorders (MPD) and solid tumors. The mutagenic and carcinogenic potential of hydroxyurea has not been established, although hydroxyurea has been associated with an increased risk of leukemia in MPD patients. To clarify whether hydroxyurea has potential carcinogenicity, we examined site-specific DNA damage induced by hydroxyurea using (32)P-5'-end-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c-Ha-ras-1 protooncogene. Hydroxyurea caused Cu(II)-mediated DNA damage especially at thymine and cytosine residues. NADH efficiently enhanced hydroxyurea-induced DNA damage. The DNA damage was almost entirely inhibited by catalase and bathocuproine, a Cu(I)-specific chelator, suggesting the involvement of hydrogen peroxide (H(2)O(2)) and Cu(I). Typical free hydroxyl radical scavengers did not inhibit DNA damage by hydroxyurea, but methional did. These results suggest that crypto-hydroxyl radicals such as Cu(I)-hydroperoxo complex (Cu(I)-OOH) cause DNA damage. Formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was induced by hydroxyurea in the presence of Cu(II). An electron spin resonance spectroscopic study using N-(dithiocarboxy)sarcosine as a nitric oxide (NO)-trapping reagent demonstrated that NO was generated from hydroxyurea in the presence and absence of catalase. In addition, the generation of formamide was detected by both gas chromatography-mass spectrometry (GC-MS) and time-of-flight-mass spectrometry (TOF-MS). A high concentration of hydroxyurea induced depurination at DNA bases in an H(2)O(2)-independent manner, and endonuclease IV treatment led to chain cleavages. These results suggest that hydroxyurea could induce base oxidation as the major pathway of DNA modification and depurination as a minor pathway. Therefore, it is considered that DNA damage by hydroxyurea participates in not only anti-cancer activity, but also carcinogenesis.
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PMID:Hydroxyurea induces site-specific DNA damage via formation of hydrogen peroxide and nitric oxide. 1171 40


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