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)

Drosophila Rrp1 has several tightly associated enzymatic activities, including double-strand DNA 3'-exonuclease, apurinic/apyrimidinic endonuclease, 3'-phosphatase, and 3'-phosphodiesterase. The carboxyl-terminal third of Rrp1, homologous to Escherichia coli exonuclease III, is sufficient to repair oxidative and alkylation-induced DNA damage in vivo. Using a screen for partial complementation of repair-deficient E. coli, we isolated three mutants of the nuclease domain of Rrp1: T462A, K463Q, and L484P, that protect against methyl methanesulfonate (MMS)-induced but not t-BuO2H-induced DNA damage. Thr-462 and Lys-463 are highly conserved residues found in a cluster of 5 conserved amino acids (LQETK), while Leu-484 is poorly conserved. Gln-460 Glu-461, Thr-462, and Lys-463 and Leu-484 were altered by site-directed mutagenesis using a plasmid including the entire Rrp1 gene and mutant proteins were purified. Mutants of the three residues Glu-461, Thr-462, and Lys-463 demonstrate 8-200-fold lower phosphodiesterase specific activity than wild-type Rrp1. E461A has a 30-fold reduction in AP endonuclease and is MMS-sensitive, but all other mutants have near-normal AP endonuclease and are MMS-resistant. Glu-461 appears to be essential for the nuclease function for Rrp1. Lys-463 and, to a lesser extent, Thr-462 influence the substrate specificity of the Rrp1 nuclease.
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PMID:Single amino acid changes alter the repair specificity of Drosophila Rrp1. Isolation of mutants deficient in repair of oxidative DNA damage. 779 76

A deoxyribonuclease has been purified to electrophoretic homogeneity from young and old rat brain. The enzyme is an endonuclease, with an optimum pH 5.0. Divalent cations are not needed for the activity. The DNase showed highest activity towards Native DNA either as such or UV irradiated with little activity on denatured DNA, apurinic DNA or DNA pretreated with mitomycin C or actinomycin D. The enzyme hydrolyzes double stranded poly (dA-dT).(dA-dT) but not other homologous or heterologous synthetic polynucleotides. The enzyme does not excise pyrimidine dimers preferentially but acts at a site away from the dimer. The DNase was partially purified from nuclei also and both the nuclear and extra nuclear enzymes showed similar properties. The specific activity of brain DNase decreases markedly with age. DNase preparations from both young and old rats showed similar apparent molecular weight (62KD) and many other properties like elution profiles and the N-terminal amino acid. However the old enzyme was more susceptible to temperature and proteolytic digestion. These results are taken to indicate a possible role for this enzyme in recognizing conformational distortions in DNA and that altered molecules of this enzyme accumulate in aging brain.
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PMID:Purification and characterization of a deoxy-ribonuclease acting on native and UV irradiated DNA from young and aging rat brain. 784 85

Analyses of cleavage ends of DNA fragments in apoptotic rat thymocytes induced by gamma-ray irradiation or by treatment with dexamethasone revealed that in both cases the fragments produced had 3'-hydroxyl (OH) and 5'-phosphoryl (P) ends of DNA chains. Rat thymocyte nuclei contained at least three endonuclease activities (deoxyribonucleases alpha, beta and gamma) that were able to cleave chromatin to mononucleosomal and oligonucleosomal fragments. The nuclei of apoptotic rat thymocytes induced by gamma-ray irradiation or dexamethasone retained considerable deoxyribonuclease gamma activity, but not alpha or beta deoxyribonuclease activity. During the induction of apoptosis, treatment with cycloheximide, which suppressed apoptosis, resulted in marked decreases of deoxyribonucleases alpha and beta activities. After release of cycloheximide inhibition, DNA fragmentation associated with apoptosis occurred in the cycloheximide-treated thymocyte nuclei, in which deoxyribonuclease gamma activity was only observed. The purified deoxyribonucleases alpha and beta were divalent cation-independent acidic endonucleases, which were separated on a CM5PW column by HPLC. The molecular masses of deoxyribonucleases alpha and beta were 28 kDa and 30 kDa, respectively, as determined by TSK G-2000SW gel-filtration HPLC, and both were 32 kDa in molecular mass as determined by SDS/PAGE. In contrast, deoxyribonuclease gamma, a neutral endonuclease, required both Ca2+ and Mg2+ for full activity and was inhibited by Zn2+. The molecular mass of deoxyribonuclease gamma was 31 kDa and 33 kDa when measured by gel filtration and SDS/PAGE, respectively. Under these optimal conditions, deoxyribonuclease gamma was shown to produce 3'-OH/5'-P ends of nucleosomal DNA fragments, while deoxyribonucleases alpha and beta both formed DNA fragments with 3'-P/5'-OH ends. The ends formed by cleavage with deoxyribonuclease gamma were the same as those produced in apoptotic rat thymocytes. On the basis of these results, it seems likely that deoxyribonuclease gamma is responsible for internucleosomal cleavage of chromatin during thymic apoptosis.
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PMID:Identification of an endonuclease responsible for apoptosis in rat thymocytes. 795 53

1. Sequence analyses of APEX nuclease, a mammalian major apurinic/apyrimidinic (AP) endonuclease homologous to Escherichia coli exonuclease III, suggested that APEX nuclease is organized into two domains, a Mr 6000 N-terminal domain containing nuclear location signals and a Mr 29,000 C-terminal catalytic domain. 2. In order to study the enzyme structure further, vectors expressing APEX nuclease (pTAPXH1) and the Mr 29,000 C-terminal region (pTAPXH61) were constructed using cDNA (APX cDNA) for the human APEX nuclease and pTrc99A plasmid. The constructs were introduced into BW2001 strain (xth-11, nfo-2) cells of E. coli to produce transformants designated as BW2001/pTAPXH1 and BW2001/pTAPXH61, respectively. Both the APEX nuclease expressed in BW2001/pTAPXH1 and the Mr 29,000 C-terminal peptide expressed in BW2001/pTAPXH61 were partially purified by column chromatography and highly purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 3. The purified APEX nuclease and the Mr 29,000 C-terminal peptide both showed equally high AP endonuclease activity which indicates that the Mr 29,000 C-terminal region of the APEX nuclease is (or contains) the AP endonuclease domain.
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PMID:Expression of a putative catalytic domain of the human APEX nuclease (a major apurinic/apyrimidinic endonuclease) in Escherichia coli. 846 27

The multifunctional mammalian apurinic/apyrimidinic endonuclease (APE) is responsible for the repair of apurinic/apyrimidinic sites in DNA. In addition, this enzyme has been shown to function as a redox factor facilitating the DNA-binding capability of JUN and FOS, as well as numerous other transcription factors through the alteration of the transcription factor redox state. Biochemical studies of organ homogenates have shown that APE is present in the different tissues studied at similar concentrations. The present study examines the immunohistochemical distribution of APE in several organs and demonstrates new and unexpected patterns of cellular and subcellular localization of this enzyme. In the hippocampus, the APE protein was highly expressed in neurons of the dentate gyrus and regions CA3 and CA4, and unexpectedly, the staining was primarily cytoplasmic. AP endonuclease immunoreactivity in the cerebellum was found in the granule and Purkinje cells, both cytoplasmic and nuclear. APE staining of the hypoglossal nucleus of the brainstem, where motor neurons that control tongue movement reside, showed reactivity in the cytoplasmic Nissl substance. Skin, liver, and duodenum demonstrated nuclear staining; however, in the duodenum, only the enterocyte nuclei of the proximal villus and the crypts of Lieberkuhn were stained, with no staining of the distal villus. These results suggest that APE has different regulatory and functional roles in different cells and organs of the body. This study shows the importance of correlating in vitro findings in tissue culture cells with the organism as a whole. The cytoplasmic staining seen in parts of the brain and in liver suggests that there may be additional functions for the APE yet to be described.
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PMID:Differential cellular and subcellular expression of the human multifunctional apurinic/apyrimidinic endonuclease (APE/ref-1) DNA repair enzyme. 852 99

Abasic (AP) sites in DNA are potentially lethal and mutagenic. 'Class II' AP endonucleases initiate the repair of these and other DNA lesions. In yeast, the predominant enzyme of this type is Apn1, and its elimination sensitizes the cells to killing by simple alkylating agents or oxidants, and raises the rate of spontaneous mutation. We investigated the ability of the major human class II AP endonuclease, Ape, which is structurally unrelated to Apn1, to replace the yeast enzyme in vivo. Confocal immunomicroscopy studies indicate that approximately 25% of the Ape expressed in yeast is present in the nucleus. High-level Ape expression corresponding to approximately 7000 molecules per nucleus, equal to the normal Apn1 copy number, restored resistance to methyl methanesulfonate to near wild-type levels in Apn1-deficient (apn1-) yeast. Ape expression in apn1- yeast provided little protection against H2O2 challenges, consistent with the weak 3'-repair diesterase activity of the human enzyme. Ape expression at approximately 2000 molecules per nucleus reduced the spontaneous mutation rate of apn1- yeast to that seen for wild-type cells. Because Ape has a powerful AP endonuclease but weak 3'-diesterase activity, these findings indicate that endogenously generated AP sites can drive spontaneous mutagenesis.
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PMID:Trans-complementation by human apurinic endonuclease (Ape) of hypersensitivity to DNA damage and spontaneous mutator phenotype in apn1-yeast. 855 61

The multifunctional DNA repair enzyme (APEX nuclease) having apurinic/apyrimidinic (AP) endonuclease, 3'-5' exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities is thought to be involved in repair of AP sites and single-strand breaks with 3'-blocked termini. To investigate the biological role of the enzyme, we studied the correlation between APEX AP endonuclease activity in several human glioma cell lines having various degree of its expression and cellular susceptibility to cytotoxic agents such as methyl methanesulfonate (MMS), 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3- (2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), cis-diamminedichloroplatinum(II) (CDDP), etoposide (VP-16), hydrogen peroxide (H2O2), hyperthermia and X-ray. The cell lines having lower APEX expression showed higher sensitivity to MMS and H2O2 which are known to induce AP sites and single strand breaks on DNA, respectively. The cellular susceptibility to the other agents tested was not significantly correlated to the APEX expression. The present results are thought to support the notion that APEX nuclease plays an important role on repair of AP sites and single-strand DNA breaks with 3'-blocked termini in mammalian cells.
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PMID:Relationship between expression of a major apurinic/apyrimidinic endonuclease (APEX nuclease) and susceptibility to genotoxic agents in human glioma cell lines. 859 68

APEX nuclease is a mammalian DNA repair enzyme having apurinic/apyrimidinic (AP) endonuclease, 3'-5'-exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities. It is also a redox factor (Ref-1), stimulating DNA binding activity of AP-1 binding proteins such as Fos and Jun. In the present paper, a cDNA for the enzyme was isolated from a rat brain cDNA library using mouse Apex cDNA as a probe and sequenced. The rat Apex cDNA was 1221 nucleotides (nt) long, with a 951-nt coding region. The amino acid sequence of rat APEX nuclease has 98.4% identity with mouse APEX nuclease. Using the rat Apex cDNA as a probe for Northern blot analysis, the size of rat Apex mRNA was shown to be approximately 1.5 kb. Its expression was compared in 9 rat organs on postnatal days 7 and 28. Although Apex mRNA was expressed ubiquitously, the levels varied significantly, suggesting organ- or tissue-specific expression of the Apex gene. The highest level was observed in the testis, relatively high levels in the thymus, spleen, kidney and brain, and the lowest level in the liver. The level of expression at postnatal day 28, with the exception of the testis, was almost the same as or lower in respective organs than that at postnatal day 7. Postnatal developmental changes of Apex mRNA expression in the testis and thymus were further studied. The expression in testis was markedly increased on postnatal days 21 and 28. The expression in thymus increased once at postnatal day 14, and then decreased. The developmental changes of Apex mRNA expression in testis and thymus suggest that APEX nuclease is involved in processes such as recombinational events.
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PMID:cDNA cloning of rat major AP endonuclease (APEX nuclease) and analyses of its mRNA expression in rat tissues. 870 82

The purpose of this study was to determine the role of radiation-induced expression of c-jun and c-fos in radiation-induced apoptosis of cells of the Jurkat T-cell line. Doses of 10-20 Gy caused a massive number of cells to undergo apoptosis within the first 24 h. This was accompanied by extensive increases in c-jun mRNA levels and moderate increases in c-fos levels, both occurring at the time of onset of internucleosomal DNA fragmentation. Increased c-jun and c-fos expression was maximum at 8 h after irradiation with a 10-fold increase in c-jun and a 2-fold increase in c-fos mRNA levels. In comparison, stimulation of the Jurkat cells with PMA resulted in rapid induction of c-jun and c-fos within 1 h. The late induction of c-jun and c-fos was not preceded by induction of tumor necrosis factor-alpha (TNF-alpha) or the bifunctional repair endonuclease and nuclear redox factor Ref-1; rather a slow decrease in Ref-1 mRNA levels was found over the first 24 h. Our results showed that radiation-induced c-jun and c-fos expression is a late response in Jurkat cells, and is most likely a secondary effect not necessary for radiation-induced apoptosis. Furthermore, apoptosis was induced by the RNA synthesis inhibitor actinomycin D, which does not induce c-jun or c-fos expression. This demonstrates that massive late induction of c-jun and c-fos is not a general requirement for apoptosis in Jurkat cells.
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PMID:Apoptosis and delayed expression of c-jun and c-fos after gamma irradiation of Jurkat T cells. 875 5

We purified an apurinic/apyrimidinic (AP) endonuclease from mouse ascites sarcoma (SR-C3H/He) cells. The enzyme showed nicking activity on acid-depurinated DNA but not on untreated, intact DNA. It also showed priming activity for DNA polymerase on both acid-depurinated and bleomycin-damaged DNA. The priming activity on bleomycin-damaged DNA was two times higher than that on an acid-depurinated DNA. The enzymatic properties indicate that the enzyme is a class II AP endonuclease having DNA 3' repair diesterase activity. The purified enzyme has a molecular weight of 39,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal pH for AP endonuclease activity was 8.0 in 50 mM Tris-HCl buffer. The AP endonuclease activity depended on divalent cation such as Mg2+ and Co2+ ions, and was inhibited by 2 mM EDTA with no addition of the divalent cation. An appropriate concentration of sodium or potassium salt stimulated the activity. Partial digestion of the AP endonuclease with Staphylococcus aureus V8 protease produced 4 major peptide fragments which may be used for protein sequencing.
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PMID:Purification and characterization of a 39kDa apurinic/apyrimidinic endonuclease from mouse ascites sarcoma cells. 880 52


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