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)

1,2-Dioxetanes are efficient sources of triplet excited carbonyl compounds, into which they decompose on thermal or photochemical activation. In the presence of DNA, the decomposition of dioxetanes gives rise to DNA modifications, which have been studied by means of specific repair endonucleases. Cyclobutane pyrimidine dimers, which are generated by triplet-triplet energy transfer, were detected by a UV endonuclease; they made up between 2% and 30% of the total modifications recognized by a crude repair endonuclease preparation from Micrococcus luteus. For various 1,2-dioxetanes, the yield of pyrimidine dimers was proportional to their triplet excitation flux. DNA strand breaks, sites of base loss (AP sites; recognized by exonuclease III and endonuclease IV) and dihydropyrimidines (recognized by endonuclease III) were found to represent only a small fraction of the modifications. The majority of the modifications detected were recognized by formamidopyrimidine-DNA glycosylase (FPG protein) and represent 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) residues or other yet not defined base modifications which are recognized by this enzyme. The modifications were generated in similar relative yields by thermal and photo-induced decomposition of the 1,2-dioxetanes and therefore emanate under both conditions from the excited carbonyl compounds. The formation of the FPG protein-sensitive modifications was efficiently quenched by azide anions; the Stern-Volmer quenching of these modifications was 150-fold more effective than that of the pyrimidine dimers. The relative amounts of the two types of modifications were strongly dependent on the structure of the 1,2-dioxetanes and on the concentration of molecular oxygen. Singlet oxygen appears to be involved only to some extent in the generation of the FPG protein-sensitive base modifications as their yield was only moderately (approximately 2-fold) increased in D2O as solvent. A mechanism is suggested in which oxidized guanine is predominantly formed by a single-electron-transfer reaction of the triplet excited carbonyl product derived from the 1,2-dioxetane, followed by unknown secondary oxidations, which involve molecular oxygen and/or undecomposed 1,2-dioxetane.
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PMID:Photochemical DNA modifications induced by 1,2-dioxetanes. 133 14

Homogeneous Fpg protein of Escherichia coli has DNA glycosylase activity which excises some purine bases with damaged imidazole rings, and an activity excising deoxyribose (dR) from DNA at abasic (AP) sites leaving a gap bordered by 5'- and 3'-phosphoryl groups. In addition to these two reported activities, we show that the Fpg protein also catalyzes the excision of 5'-terminal deoxyribose phosphate (dRp) from DNA, which is the principal product formed by the incision of AP endonucleases at abasic sites. Moreover, the rate of the Fpg protein catalysis for the 2,6-diamino-4-hydroxy-5-formamidopyrimidine-DNA glycosylase activity is slower than the activities excising dR from abasic sites and dRp from abasic sites preincised by endonucleases. The product released by the Fpg protein in the excision of 5'-terminal dRp from an abasic site preincised by an AP endonuclease is a single base-free unsaturated dRp, suggesting that the excision results from beta-elimination. The release of 5'-terminal dRp by crude extracts of E. coli from wild type and fpg-mutant strains shows that the Fpg protein is one of the major EDTA-resistant activities catalyzing this reaction.
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PMID:Excision of 5'-terminal deoxyribose phosphate from damaged DNA is catalyzed by the Fpg protein of Escherichia coli. 137 43

The mutational potency of apurinic/apyrimidinic (AP) sites induced by heat-treatment under acidic conditions has been studied in mammalian cells. Abasic sites were induced on a single-stranded DNA shuttle vector carrying the supF tRNA gene, eliminating, therefore, any ambiguity concerning the damaged strand. This vector was able to replicate both in mammalian cells and in bacteria where the mutations induced in animal cells on the supF tRNA gene were screened by the white/blue beta-galactosidase assay in the presence of isopropyl-1-thio-beta-D-galactopyranoside and 5-bromo-4-chloro-3-indoyl-beta-D-galactoside. All white colonies contained plasmid with a mutation on the target gene which was directly sequenced. Our results show that one AP site was induced/22 min of heating as measured by sensitivity of DNA to alkali denaturation or treatment with the AP-endonuclease activity of the FPG protein (Fapy-DNA glycosylase). Putative AP sites decrease survival of the plasmid with a lethal hit of one AP site/single-stranded molecule. Mutation frequency was increased by a factor of approximately six after 2 h at 70 degrees C. Most of the induced mutations were point mutations not distributed at random and clustered in the gene region which will give rise to the mature tRNA. Mutations were abolished by treatments that eliminated AP sites such as alkali treatment or incubation with the Fapy-DNA glycosylase protein. Under our experimental conditions, when only single mutations were taken into account, the order of base insertion opposite AP sites was G greater than A greater than T greater than C.
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PMID:Mutation spectrum of heat-induced abasic sites on a single-stranded shuttle vector replicated in mammalian cells. 152 92

DNA modifications induced either by photosensitization (illumination in the presence of methylene blue) or by chemically generated singlet oxygen (thermal decomposition of an 1,4-etheno-2,3-benzodioxin) are recognized and incised by repair endonucleases present in crude bacterial cell extracts. Only a small fraction of the incised modifications are sites of base loss (AP-sites) sensitive to exonuclease III, endonuclease IV from E. coli or to the UV-endonuclease from M. luteus. Cell extracts from E. coli strains overproducing or defective in endonuclease III recognize the modifications induced by illumination in the presence of methylene blue just as well as do those from wild-type E. coli strains. This indicates that dihydropyrimidine derivatives, which are characteristic of hydroxyl radical-induced DNA modifications, are absent. In contrast, most of the modifications induced are not recognized by a cell extract from a fpg strain defective in formamidopyrimidine-DNA glycosylase FPG protein). Furthermore, incision by a cell extract from an E. coli strain overproducing FPG protein takes place at much lower protein concentration than with the wild-type strain. Experiments with purified FPG protein confirm that this enzyme is responsible for the recognition of singlet oxygen-induced DNA base modifications.
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PMID:Enzymatic recognition of DNA modifications induced by singlet oxygen and photosensitizers. 170 Mar 66

pBR322 plasmid DNA was treated with methylene blue plus visible light (MB-light) and tested for transformation efficiency in Escherichia coli mutants defective in either formamidopyrimidine-DNA glycosylase (Fpg protein) and/or UvrABC endonuclease. The survival of pBR322 DNA treated with MB-light was not significantly reduced when transformed into either fpg-1 or uvrA single mutants compared with that in the wild-type strain. In contrast, the survival of MB-light-treated pBR322 DNA was greatly reduced in the fpg-1 uvrA double mutant. The synergistic effect of these two mutations was not observed in transformation experiments using pBR322 DNA treated with methyl methanesulfonate, UV light at 254 nm, or ionizing radiation. In vitro experiments showed that MB-light-treated pBR322 DNA is a substrate for the Fpg protein and UvrABC endonuclease. The number of sites sensitive to cleavage by either Fpg protein or UvrABC endonuclease was 10-fold greater than the number of apurinic-apyrimidinic sites indicated as Nfo protein (endonuclease IR)-sensitive sites. Seven Fpg protein-sensitive sites per PBR322 molecule were required to produce a lethal hit when transformed into the uvrA fpg-1 mutant. These results suggest that MB-light induces DNA base modifications which are lethal and that these modifications are repaired by Fpg protein and UvrABC endonuclease in vivo and in vitro. Therefore, one of the physiological functions of Fpg protein might be to repair DNA base damage induced by photosensitizers and light.
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PMID:Escherichia coli Fpg protein and UvrABC endonuclease repair DNA damage induced by methylene blue plus visible light in vivo and in vitro. 171 Jun 17

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

Repair endonucleases, viz. endonuclease III, formamidopyrimidine-DNA glycosylase (FPG protein), endonuclease IV, exonuclease III and UV endonuclease, were used to analyse the modifications induced in bacteriophage PM2 DNA by 333 nm laser irradiation in the presence of acetone or acetophenone. In addition to pyrimidine dimers sensitive to UV endonuclease, 5,6-dihydropyrimidines (sensitive to endonuclease III) and base modifications sensitive to FPG protein were generated. The level of the last in the case of acetone was 50% and in the case of acetophenone 9% of the level of pyrimidine dimers. HPLC analysis of the bases excised by FPG protein revealed that least some of them were 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). In the damage induced by direct excitation of DNA at 254 nm, which was analysed for comparison, the number of FPG protein-sensitive base modifications was only 0.6% of that of the pyrimidine dimers. Mechanistic studies demonstrated that the formation of FPG protein-sensitive modifications did not involve singlet oxygen, as the damage was not increased in D2O as solvent. Hydroxyl radicals, superoxide and H2O2 were also not involved, since the relative number of single strand breaks and of sites of base loss (AP sites) was much lower than in the case of DNA damage induced by hydroxyl radicals and since the presence of SOD or catalase had no effect on the extent of the damage. However, the mechanism did involve an intermediate that was much more efficiently quenched by azide ions than the triplet excited carbonyl compounds and which was possibly a purine radical. Together, the data indicate that excited triplet carbonyl compounds react with DNA not only by triplet-triplet energy transfer yielding pyrimidine dimers, but also by electron transfer yielding preferentially base modifications sensitive to FPG protein, which include 8-hydroxyguanine.
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PMID:Endonuclease-sensitive DNA modifications induced by acetone and acetophenone as photosensitizers. 838 42

A number of repair endonuclease, viz. endonuclease III, formamidopyrimidine-DNA glycosylase (FPG protein), endonuclease IV, exonuclease III and UV endonuclease, is used to simultaneously quantify various types of DNA modifications, which were induced by agents that generate reactive oxygen species. Under cell-free conditions, two types of DNA damage profiles are obtained. The profiles induced by chemically generated singlet oxygen and by various photosensitizers (acridine orange, methylene blue, riboflavin, hematoporphyrin) plus light are dominated by base modifications sensitive to FPG protein, while 5,6-dihydropyrimidines (recognized by endonuclease III), sites of base loss (AP sites, recognized by endonuclease IV and exonuclease III) and strand breaks are minor lesions. In contrast, the DNA damage profile induced by hydroxyl radicals (gamma-rays) consists of approx. equal levels of base modifications. AP sites and strand breaks. The damage profiles induced by Fe(III)-EDTA in the presence of superoxide and by Fe(III)-nitrilotriacetate in the presence of H2O2 do not differ from that by hydroxyl radicals. The damage profile induced by Cu(II)-phenanthroline deviates by high levels of AP sites that are recognized by endonuclease IV and exonuclease III-but not by those AP endonucleases which cleave at the 3' site-and probably represent AP sites oxidized at C-1'. The damage induced by Fe(III)-bleomycin plus H2O2 deviates by an increased level of double strand breaks and the absence of endonuclease-sensitive base modifications. Cellular DNA damage profiles are obtained from bacteria, cultured mammalian cells and mammalian mitochondria after exposure to acridine orange plus visible light. A comparison with the cell-free profiles reveals that the damage in all three systems is not induced indirectly by hydroxyl radicals or an activation of cellular nucleases, but by the same mechanism that is responsible for the cell-free DNA damage.
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PMID:Use of repair endonucleases to characterize DNA damage induced by reactive oxygen species in cellular and cell-free systems. 838 92

Singlet molecular oxygen (1O2) has been implicated in several biological processes that may lead to genetic damage. The relevance of various repair pathways in plasmid inactivation mediated by 1O2 was investigated. Plasmid treated with 1O2, chemically generated, was transfected into Escherichia coli strains deficient in genes implicated in the DNA repair of oxidative damage. The ability to transform bacteria is significantly reduced in the double mutant xth,nfo, deficient in both exonuclease III and endonuclease IV, although it was similar to wild-type cells in single mutants. The products of these two genes are able to cleave DNA damaged by 1O2 and to remove DNA polymerization blocks from 3'-termini generated either directly by 1O2 treatment or after the action of the formamidopyrimidine-DNA-N-glycosylase (Fpg protein). The results indicate that the exonuclease III and endonuclease IV participate in the excision of lethal lesions induced in DNA by 1O2.
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PMID:Involvement of Escherichia coli exonuclease III and endonuclease IV in the repair of singlet oxygen-induced DNA damage. 864 Sep 34

N-Hydroxypyridine-2-thione (2-HPT), known to release hydroxyl radicals on irradiation with visible light, and two related compounds, viz. N-hydroxypyridine-4-thione (4-HPT) and N-hydroxyacridine-9-thione (HAT), were tested for their potency to induce DNA damage in L1210 mouse leukemia cells and in isolated DNA from bacteriophage PM2. DNA single-strand breaks and modifications sensitive to various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) were quantified. Illumination of cell-free DNA in the presence of 2-HPT and 4-HPT gave rise to damage profiles characteristic for hydroxyl radicals, i.e. single-strand breaks and the various endonuclease-sensitive modifications were formed in the same ratios as after exposure to established hydroxyl radical sources. In contrast, HAT plus light gave rise to a completely different DNA damage profile, namely that characteristic for singlet oxygen. Experiments with various scavengers (t-butanol, catalase, superoxide dismutase) and in D2O as solvent confirmed that hydroxyl radicals are directly responsible for the DNA damage caused by photoexcited 2-HPT and 4-HPT, while the damage by HAT plus light is mediated by singlet oxygen and type I reactions. The type of DNA damage characteristic of hydroxyl radicals was also observed in L1210 mouse leukemia cells when treated with 2-HPT plus light or with H2O2 at 0 degrees C. t-Butanol (2%) inhibited the cellular DNA damage by approximately 50%. A dose of 2-HPT plus light that generated single-strand breaks at a frequency of 5 x 10(-7)/bp was associated with 50% cell survival. No DNA damage and cytotoxicity was observed after treatment with 2-HPT in the dark. We propose that 2-HTP and 4-HTP may serve as new agents to study the consequences of DNA damage induced by hydroxyl radicals in cells. In addition, the data provide direct evidence that hydroxyl radicals are ultimately responsible for the genotoxic effects caused by H2O2 in the dark.
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PMID:Photolysis of N-hydroxpyridinethiones: a new source of hydroxyl radicals for the direct damage of cell-free and cellular DNA. 864 78


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