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)

Abasic sites in DNA are generated either spontaneously or after removal of altered bases during the base excision repair process. These as well as 3' damaged ends of DNA at single-strand breaks induced by reactive oxygen species are repaired by AP-endonucleases. The major human AP-endonuclease (named APE-1) has two unrelated activities. It may function as an activator of c-Fos and c-Jun transcription factors and as a repressor of the parathyroid hormone (PTH) gene by binding to the negative Ca(2+)-response elements (nCaRE) in its promoter. Preliminary studies indicate that the h-APE-1 gene is highly regulated. Analysis of its promoter activity by transient expression of the luciferase reporter gene in human, HeLa and TK6 cells suggested the presence of a negative regulatory element in the promoter. Two nCaRE-like sequences were identified in the promoter segment responsible for inhibiting reporter gene expression. Competitive electrophoretic mobility shift assay with HeLa nuclear extract indicated that the nCaRE sequences of the APE-1 and PTH genes are recognized by the APE-1 polypeptide. These results suggest that the APE-1 gene may be down-regulated by its own product.
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PMID:Negative regulation of the major human AP-endonuclease, a multifunctional protein. 894 27

The mutagenic and lethal effects of abasic sites in DNA are averted by repair initiated by 'class II' apurinic (AP) endonucleases, which cleave immediately 5'to abasic sites. We examined substrate binding by the human AP endonuclease, Ape protein (also called Hap1, Apex or Ref-1). In electrophoretic mobility-shift experiments, Ape bound synthetic DNA substrates containing single AP sites or tetrahydrofuran (F) residues. No complexes were detected with single-stranded substrates or unmodified duplex DNA. In EDTA, the concentration of Ape required to shift 50% of duplex F-DNA was approximately 50 nM, while the addition of 10 mM MgCl2 nearly eliminated detectable F-DNA@Ape complexes. Filter-binding studies demonstrated a half-life of approximately 50 s at 0 degrees C for F-DNA@Ape complexes in the presence of EDTA, and <15 s after the addition of Mg2+. The DNA recovered from F-DNA@Ape complexes was intact but was rapidly cleaved upon addition of Mg2+, which suggests that these protein-DNA complexes are on the catalytic pathway for incision. Methylation and ethylation interference experiments identified DNA contacts critical for Ape binding, and Cu-1, 10-phenanthroline footprinting suggested an Ape-induced structural distortion at the abasic site prior to cleavage.
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PMID:Abasic site binding by the human apurinic endonuclease, Ape, and determination of the DNA contact sites. 902 1

Thioredoxin (TRX) is a pleiotropic cellular factor that has thiol-mediated redox activity and is important in regulation of cellular processes, including proliferation, apoptosis, and gene expression. The activity of several transcription factors is posttranslationally altered by redox modification(s) of specific cysteine residue(s). One such factor is nuclear factor (NF)-kappa B, whose DNA-binding activity is markedly augmented by TRX treatment in vitro. Similarly, the DNA-binding activity of activator protein 1 (AP-1) is modified by a DNA repair enzyme, redox factor 1 (Ref-1), which is identical to a DNA repair enzyme, AP endonuclease. Ref-1 activity is in turn modulated by various redox-active compounds, including TRX. We here report the molecular cascade of redox regulation of AP-1 mediated by TRX and Ref-1. Phorbol 12-myristate 13 acetate efficiently translocated TRX into the HeLa cell nucleus where Ref-1 preexists. This process seems to be essential for AP-1 activation by redox modification because co-overexpression of TRX and Ref-1 in COS-7 cells potentiated AP-1 activity only after TRX was transported into the nucleus by phorbol 12-myristate 13 acetate treatment. To prove the direct active site-mediated association between TRX and Ref-1, we generated a series of substitution-mutant cysteine residues of TRX. In both an in vitro diamide-induced cross-linking study and an in vivo mammalian two-hybrid assay we proved that TRX can associate directly with Ref-1 in the nucleus; also, we demonstrated the requirement of cysteine residues in the TRX catalytic center for the potentiation of AP-1 activity. This report presents an example of a cascade in cellular redox regulation.
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PMID:AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. 910 29

p53 can be isolated from cells in a form that is inert for binding to DNA but that can be stimulated dramatically by phosphorylation, antibody binding, or short single strands of DNA. This suggests that upon genotoxic stress, cells can convert latent p53 to one that is active for DNA binding. Surprisingly, we observed that latent p53 is as effective in activating transcription in vitro as is active p53. We found that HeLa nuclear extracts can stimulate DNA binding by latent p53 and have purified from them a p53-stimulating protein that we have determined to be the product of the Ref-1 gene. Interestingly, Ref-1 is a dual function protein that can both regulate the redox state of a number of proteins and function as a DNA repair (A/P) endonuclease. We observed that oxidized forms of full-length and carboxy-terminally truncated p53 (p53 delta30), which are inactive for DNA binding, are both stimulated by the Ref-1 protein. However, in the presence of reducing agent, Ref-1 is an extremely potent stimulator of full-length p53 but not p53 delta30. These and additional data indicate that Ref-1 protein stimulates p53 by both redox-dependent and -independent means and imply a key role for it in p53 regulation. Importantly, we have also determined that Ref-1 can stimulate p53 transactivation in vivo. This is the first example of a noncovalent protein modifier of p53 function identified in cells.
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PMID:Identification of redox/repair protein Ref-1 as a potent activator of p53. 911 21

Earlier work indicates that the major DNA repair phosphodiesterase (PDE) in yeast cells is the well-characterized Apn1 protein. Apn1 demonstrates both Mg2+-independent PDE activity and Mg2+-independent class II apurinic/apyrimidinic (AP) endonuclease activity and represents greater than 90% of the activity detected in crude extracts from wild-type yeast cells. Apn1 is related to Echerichia coli endonuclease IV, both in its enzymatic properties and its amino acid sequence. In this work, we report the partial purification of a novel yeast protein, Pde1, present in Apn1-deficient cells. Pde1 is purified by sequential BioRex-70, PBE118, and MonoS chromatography steps using a sensitive and highly specific 3'-phosphoglycolate-terminated oligonucleotide-based assay as a measure of PDE activity. Mg2+-stimulated PDE and Mg2+-stimulated class II AP endonuclease copurify during this procedure. These results indicate that yeast, like many other organisms studied to date, has enzymatic redundancy for the repair of 3'-blocking groups and abasic sites.
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PMID:Partial purification of Pde1 from Saccharomyces cerevisiae: enzymatic redundancy for the repair of 3'-terminal DNA lesions and abasic sites in yeast. 916 80

Several oxidative DNA-damaging agents, including ionizing radiation, can generate multiply damaged sites in DNA. Among the postulated lesions are those with abasic sites located in close proximity on opposite strands. The repair of an abasic site requires strand scission by a repair endonuclease such as human apurinic/apyrimidinic endonuclease (Ape) or exonuclease III in Escherichia coli. Therefore, a potential consequence of the "repair" of bistranded abasic sites is the formation of double-strand breaks. To test this possibility and to investigate the influence of the relative distance between the two abasic sites and their orientation to each other, we prepared a series of oligonucleotide duplexes containing abasic sites at defined positions either directly opposite each other or separated by 1, 3, or 5 base pairs in the 5'- or 3'-direction. Analysis following Ape and exonuclease III treatment of these substrates indicated a variety of responses. In general, cleavage at abasic sites was slower in duplexes with paired lesions than in control duplexes with single lesions. Double-strand breaks were, however, readily generated in duplexes with abasic sites positioned 3' to each other. With the duplex containing abasic sites set 1 base pair apart, 5' to each other, both Ape and exonuclease III slowly cleaved the abasic site on one strand only and were unable to incise the other strand. With the duplex containing abasic sites set 3 base pairs apart, 5' to each other, Ape protein was unable to cleave either strand. These data suggest that closely positioned abasic sites could have several deleterious consequences in the cell. In addition, this approach has allowed us to map bases that make significant contact with the enzymes when acting on an abasic site on the opposite strand.
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PMID:Reactivity of human apurinic/apyrimidinic endonuclease and Escherichia coli exonuclease III with bistranded abasic sites in DNA. 918 54

We purified a 44-kDa nuclear protein from salt-extract of permeable mouse ascites sarcoma cells in an effort to isolate factors involved in the repair of acid-depurinated DNA. It was copurified with a major AP endonuclease (APEX nuclease) by sequential column chromatography then further purified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis as a possible DNA repair support factor. Its partial amino acid sequences were determined, and a cDNA clone for the protein was isolated from a mouse T-cell cDNA library using long degenerate oligonucleotide probes deduced from the amino acid sequence. The complete nucleotide sequence of the cDNA (1.7 kilobases) was determined. Northern hybridization using this cDNA detected two transcripts: 1.8 kb being the major one and 2.6 kb being the minor one. The complete amino acid sequence for the protein predicted from the nucleotide sequence of the cDNA indicates that the 44-kDa protein consists of 394 amino acids with a calculated molecular weight of 43,698. In tests performed thus far, the recombinant 44-kDa protein expressed in Escherichia coli has not expressed any repair-support activity. It remains to be analyzed whether the protein attains this activity after appropriate posttranslational modifications. Most parts of the 44-kDa protein cDNA and the deduced amino acid sequence were found to be identical to those of the protein p38-2G4, recently reported as a cell cycle-specifically modulated nuclear protein of 38kDa. The p38-2G4 may be a truncated form of the present 44-kDa protein.
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PMID:cDNA cloning, sequence analysis and expression of a mouse 44-kDa nuclear protein copurified with DNA repair factors for acid-depurinated DNA. 928 67

We have used a 32P-postlabelling assay to examine the activity of purified Esherichia coli endonuclease IV, human apurinic/apyrimidinic endonuclease I and human cell-free extracts towards irradiated DNA. The assay can detect thymine glycols, 3'-phosphoglycolate groups and at least one other major lesion that has yet to be fully characterized. It was observed that endonuclease IV removed the phosphoglycolates and the uncharacterized lesion(s) suggesting that the latter are abasic sites with modified deoxyribose residues. The purified human enzyme acted only on the phosphoglycolate residues. Cell-free extract, prepared from A549 lung carcinoma cells by sonication or treatment with toluene, efficiently removed the phosphoglycolate and unknown lesions, but was less reactive towards thymine glycols. The extract was completely inactivated by heating at 60 degrees C for 10 min. Removal of the unknown product and phosphoglycolate did not require magnesium, but 1 mM EDTA did inhibit release of the latter. The cell-free extract exhibited substantially more activity towards native than heat-denatured DNA. A comparison of extracts prepared from 4 cell lines displaying a range of radiosensitivities, including an ataxia telangiectasia cell line, showed that all contained similar levels of repair activity towards the detectable lesions.
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PMID:Use of a postlabelling assay to examine the removal of radiation-induced DNA lesions by purified enzymes and human cell extracts. 928 91

A neutral Mg(2+)-dependent deoxyribonuclease from the Colorado potato beetle was isolated and characterized in physicochemical terms. An electrophoretically homogeneous preparation of the enzyme was obtained using salt fractionation, Sephadex G-100 gel filtration, and subsequent preparative isoelectrofocusing in an Ultrodex layer. The molecular weight of the purified DNase preparation (with a purification degree of 104) and its isoelectric point were 100 kD and 9.1, respectively. The enzyme activity was maximal at pH 7.2 and 46 degrees C in the presence of 10 mM Mg2+. The DNase of the Colorado beetle preferentially hydrolysed denatured DNA via the endonuclease pathway, degrading the substrate to oligonucleoside-3'-phosphates. As far as the physical and chemical properties are concerned, this Colorado beetle DNase seems different from previously investigated DNases of other insect species.
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PMID:Isolation, purification, and characterization of a neutral Mg(2+)-dependent deoxyribonuclease of the Colorado potato beetle Leptinotarsa decemlineata Say. 936 Mar 4

A dose-limiting toxicity of certain chemotherapeutic alkylating agents is their toxic effects on nontarget tissues such as the bone marrow. To overcome the myelosuppression observed by chemotherapeutic alkylating agents, one approach is to increase the level of DNA repair proteins in hematopoietic stem and progenitor cells. Toward this goal, we have constructed a human fusion protein consisting of O6-methylguanine DNA methyltransferase coupled with an apurinic endonuclease, resulting in a fully functional protein for both O6-methylguanine and apurinic/apyrimidinic (AP) site repair as determined by biochemical analysis. The chimeric protein protected AP endonuclease-deficient Escherichia coli cells against methyl methanesulfonate and hydrogen peroxide (H2O2) damage. A retroviral construct expressing the chimeric protein also protected HeLa cells against 1,3-bis(2-chloroethyl)-1-nitrosourea and methyl methanesulfonate cytotoxicity either when these agents were used separately or in combination. Moreover, as predicted from previous analysis, truncating the amino 150 amino acids of the apurinic endonuclease portion of the O6-methylguanine DNA methyltransferase-apurinic endonuclease protein resulted in the retention of O6-methylguanine DNA methyltransferase activity but loss of all AP endonuclease activity. These results demonstrate that the fusion of O6-methylguanine DNA methyltransferase and apurinic endonuclease proteins into a combined single repair protein can result in a fully functional protein retaining the repair activities of the individual repair proteins. These and other related constructs may be useful for protection of sensitive tissues and, therefore, are candidate constructs to be tested in preclinical models of chemotherapy toxicity.
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PMID:Creation of a fully functional human chimeric DNA repair protein. Combining O6-methylguanine DNA methyltransferase (MGMT) and AP endonuclease (APE/redox effector factor 1 (Ref 1)) DNA repair proteins. 942 28


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