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 terminal differentiation on UV-induced DNA damage and its repair in transcriptionally active and inactive genomic sequences was investigated using the murine 3T3-T proadipocyte cell culture system. Actively cycling 3T3-T cells terminally differentiate into adipocytes after exposure to media containing platelet-depleted human plasma. Suitable DNA fragments were analyzed from four genes: beta-actin, adenosine deaminase, dihydrofolate reductase, and lipoprotein lipase. As a result of 3T3-T cell differentiation, lipoprotein lipase and beta-actin expression was modified, whereas adenosine deaminase and dihydrofolate reductase expression was not affected. A DNA fragment representing the transcriptionally inactive locus 70-38 was also evaluated. UV-induced cyclobutane pyrimidine dimers, detected as UV-specific endonuclease-sensitive sites, in each fragment increased linearly as a function of UV dose (0-20 J/m2) independently of gene expression or differentiation. Sequence-specific repair of dimers was measured in stem and terminally differentiated 3T3-T cells after UV irradiation (10 J/m2). For undifferentiated stem cells, the rate and extent of dimer repair was higher in the actively transcribed adenosine deaminase and dihydrofolate reductase genes than in the inactive lipoprotein lipase or 70-38 fragments, the greater difference being observed in the first 8 h post-UV irradiation. In contrast, similar dimer repair rates were found for each DNA fragment in terminally differentiated 3T3-T cells. These data suggest that cellular differentiation is accompanied by a loss of heterogeneity in intragenomic DNA repair.
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PMID:Loss of intragenomic DNA repair heterogeneity with cellular differentiation. 193 6

The contribution of DNA damage to the effects of 193-nm excimer laser radiation on mammalian cells in culture was studied in order to evaluate the mutagenic potential of this UV wavelength in vivo. Two approaches were taken: measurement of pyrimidine dimer-specific endonuclease-sensitive sites/megabase and comparison of the 193-nm radiation-induced cytotoxicity in normal versus DNA repair-deficient cells. The formation of pyrimidine dimer-specific endonuclease-sensitive sites/megabase was inversely related to the thickness of the cytoplasm overlying the nuclei of normal human fibroblasts (NHF) and Chinese hamster ovary cells. The results of these measurements and a calculation of the absorption coefficient of cytoplasm indicate that each 1 micron of cytoplasm attenuates the incident radiation by greater than 90% and, therefore, the nuclear DNA in tissue will be highly protected from 193-nm radiation by overlying cytoplasm. The reduction in colony-forming ability induced by 254-nm, 193-nm, and X-ray radiation was measured in NHF, xeroderma pigmentosum (group A) cells, and ataxia telangiectasia cells. Xeroderma pigmentosum (group A) cells were 16.5 times more sensitive to 254-nm radiation but only 3.5 times more sensitive to 193-nm radiation than NHF cells, indicating that cyclobutylpyrimidine dimers were not the major lethal lesion formed at 193 nm. AT cells were 3.4 times more sensitive to X-rays than NHF cells, but these cell types were almost equally sensitive to 193-nm radiation, indicating that 193 nm did not induce the same type of lethal lesions as X-rays.
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PMID:DNA damage induced by 193-nm radiation in mammalian cells. 198 91

Fluorescent light (FL) illumination of RBCF-1 cells, derived from a goldfish, prior to 254 nm UV-irradiation enhanced their ability to photorepair. The cells were illuminated with FL for 1 h (29 W/M2) and incubated for 8 h in the dark before being irradiated with 10 J/m2 UV. The surviving fraction of FL-treated cells after UV-irradiation rose about 7-fold (from 3 to 20%) by 20 min photorepair treatment with the same FL source, whereas 4-fold (from 1.6 to 6%) in the FL non-treated cells. Flow cytometric analysis showed that FL treatment did not affect the distribution of cell cycle phase at the time of UV-irradiation (8 h after FL treatment). Pyrimidine dimers induced by UV were measured by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. Initial yields of dimers after exposure to 10 J/m2 UV were almost the same (about 0.11 dimer/kb) between FL treated and non-treated cells. But after 20 min photorepair treatment, about 70% of dimers were removed in the FL treated samples, while less than 20% were removed in the non FL-treated ones.
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PMID:Enhancement of photorepair of ultraviolet-damage by preillumination with fluorescent light in cultured fish cells. 201 25

T4 endonuclease V is a pyrimidine dimer-specific DNA repair enzyme which has been previously shown not to require metal ions for either of its two catalytic activities or its DNA binding function by virtue of its ability to function in the presence of metal-chelating agents. However, we have investigated whether the single cysteine within the enzyme was able to bind metal salts and influence the various activities of this repair enzyme. A series of metals (Hg2+, Ag+, Cu+) were shown to inactivate both endonuclease Vs pyrimidine dimer-specific DNA glycosylase activity and the subsequent apurinic nicking activity. The binding of metal to endonuclease V did not interfere with nontarget DNA scanning or pyrimidine dimer-specific binding. The Cys-78 codon within the endonuclease V gene was changed by oligonucleotide site-directed mutagenesis to Thr-78 and Ser-78 in order to determine whether the native cysteine was directly involved in the enzyme's DNA catalytic activities and whether the cysteine was primarily responsible for the metal binding. The mutant enzymes were able to confer enhanced ultraviolet light (UV) resistance to DNA repair-deficient Escherichia coli at levels equal to that conferred by the wild type enzyme. The C78T mutant enzyme was purified to homogeneity and shown to be catalytically active on pyrimidine dimer-containing DNA. The catalytic activities of the C78T mutant enzyme were demonstrated to be unaffected by the addition of Hg2+ or Ag+ at concentrations 1000-fold greater than that required to inhibit the wild type enzyme. These data suggest that the cysteine is not required for enzyme activity but that the binding of certain metals to that amino acid block DNA incision by either preventing a conformational change in the enzyme after it has bound to a pyrimidine dimer or sterically interfering with the active site residue's accessibility to the pyrimidine dimer.
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PMID:Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding. 203 8

The time courses of excision repair and photoreactivation of pyrimidine dimers induced by 254-nm UV were examined in the genome overall and in the c-ras sequence of RBCF-1 cells derived from a goldfish, by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. Excision repair was more efficient in the ras sequence than in the genome overall, whereas no differences in efficiency of photoreactivation were detected. These results suggest that excision repair is affected by the accessibility of chromatin, while photoreactivation is not.
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PMID:Preferential excision repair and non-preferential photoreactivation of pyrimidine dimers in the c-ras sequence of cultured goldfish cells. 205 10

We have previously demonstrated preferential DNA repair of active genes in mammalian cells. The methodology involves the use of a specific endonuclease or other more direct approaches to create nicks at sites of damage followed by quantitative Southern analysis and probing for specific genes. Initially, we used pyrimidine dimer specific endonuclease to detect pyrimidine dimers after UV irradiation. We now also use the bacterial enzyme ABC excinuclease to examine the DNA damage and repair of a number of adducts other than pyrimidine dimers in specific genes. We can detect gene specific alkylation damage by creating nicks via depurination and alkaline hydrolysis. In our assay for preferential repair, we compare the efficiency of repair in the DHFR gene to that in the 3' flanking, non-coding region to the gene. In CHO cells, UV induced pyrimidine dimers are efficiently repaired from the active DHFR gene, but not from the inactive region. We have demonstrated that the 6-4 photoproducts are also preferentially repaired and that they are removed faster from the regions studied than pyrimidine dimers. Using similar approaches, we find that DNA adducts and crosslinks caused by cisplatinum are preferentially repaired in the active gene compared to the inactive regions and to the inactive c-fos oncogene. Also, nitrogen mustard and methylnitrosurea damage is preferentially repaired whereas dimethylsulphate damage is not. NAAAF adducts do not appear to be preferentially repaired in this system.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Gene specific damage and repair after treatment of cells with UV and chemotherapeutical agents. 206 87

1,2-Dioxetanes, efficient chemical sources of triplet excited carbonyl compounds, were observed to be genotoxic in isolated DNA, bacteria, and cultured mammalian cells. In superhelical DNA of bacteriophage PM2, various alkyl- and hydroxyalkyl-substituted dioxetanes (1) induced predominantly endonuclease-sensitive base modifications and only few single strand breaks. With a specific endonuclease a small fraction of the base modifications was identified as pyrimidine dimers. The psoralen dioxetane (2a) or PsD bound photochemically to calf thymus DNA at the alpha-pyrone ring of psoralen (fluorescence measurements). Photobinding was also observed when calf thymus DNA was incubated with psoralen and 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane. In Syrian hamster embryo fibroblasts and HL-60 cells, dioxetanes induced DNA single strand breaks. The alkyl- and hydroxyalkyl-substituted dioxetanes 1 and 2 were efficiently inactivated by cysteine, glutathione, ascorbic acid, tocopherol, NADH and FADH2. While dioxetanes 1 and 2 were not mutagenic in Salmonella typhimurium strain TA100, benzofuran dioxetanes 3 exhibited substantial effects. Further data imply that presumably a mutagenic intermediate with a lifetime of a few minutes is produced from the benzofuran dioxetane.
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PMID:Photobiological studies with dioxetanes in isolated DNA, bacteria, and mammalian cells. 212 62

Holliday junctions are intermediate structures that are formed and resolved during the process of genetic recombination. To investigate the interaction of junction-resolving nucleases with synthetic Holliday junctions that contain homologous arm sequences, we constructed substrates in which the junction point was free to branch migrate through 26 base-pairs of homology. In the absence of divalent cations, we found that both phage T4 endonuclease VII and phage T7 endonuclease I bound the synthetic junctions to form specific protein-DNA complexes. Such complexes were not observed in the presence of Mg2+, since the Holliday junctions were resolved by the introduction of symmetrical cuts in strands of like polarity. The major sites of cleavage were identified and found to occur within the boundaries of homology. T4 endonuclease VII showed a cleavage preference for the 3' side of thymine bases, whereas T7 endonuclease I preferentially cut the DNA between two pyrimidine residues. However, cleavage was not observed at all the available sites, indicating that in addition to their structural requirements, the endonucleases show strong site preferences.
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PMID:Cleavage specificity of bacteriophage T4 endonuclease VII and bacteriophage T7 endonuclease I on synthetic branch migratable Holliday junctions. 215 65

Irradiation of DNA with UV light induces pyrimidine dimers and (6-4) photoproducts. The presence of one of these photolesions in the restriction site of a given endonuclease inhibits DNA cleavage and induces the formation of fragments by incomplete DNA digestion which appear as additional, facultative bands in Southern hybridization autoradiograms. The number and size of these fragments show a positive correlation with the UV dose. The response to UV light of immunoglobulin light-chain constant kappa and heavy-chain constant mu genes was analyzed with 2 specific probes. Constant kappa and mu genes when irradiated as part of the chromatin of living lymphocytes showed a UV sensitivity similar to that of naked DNA. The same genes from granulocytes had 50-60 times lower UV sensitivity. When cells were allowed to repair photolesions for 24 h the facultative bands from granulocytes disappeared indicating that these cells were able to remove photolesions from constant kappa and mu genes. Facultative bands from lymphocytes showed a smaller decrease of density after 24 h repair. This suggests that lymphocytes are less efficient than granulocytes in removing UV damage from constant kappa and mu genes.
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PMID:Assessment by Southern blot analysis of UV-induced damage and repair in human immunoglobulin genes. 216 48

We studied DNA repair by injecting plasmids containing random pyrimidine dimers into Xenopus oocytes. We demonstrated excision repair by recovering plasmids and analyzing them with T4 UV endonuclease treatment and alkaline agarose gel electrophoresis. The mechanism for excision repair of these plasmids appears to be processive, rather than distributive, since repair occurs in 'all or none' fashion. At less than 4-5 dimers/plasmid, nearly all repair occurs within 4-6 hours (approximately 10(10) dimers repaired per oocyte); the oocyte, therefore, has abundant repair activity. Specific antibodies and inhibitors were used to determine enzymes involved in repair. We conclude that DNA polymerase alpha (and/or delta) is required because repair is inhibited by antibodies to human DNA polymerase alpha, as well as by aphidicolin, an inhibitor of polymerases alpha (and/or delta). Repair was not inhibited by hydroxyurea, cytosine beta-D-arabinofuranoside, or inhibitors of topoisomerase II (novobiocin). Oocyte repair does not activate semi-conservative DNA replication, nor is protein synthesis required. Photoreactivation cannot account for repair because dimer removal is independent of exogenous light.
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PMID:Excision repair of UV-damaged plasmid DNA in Xenopus oocytes is mediated by DNA polymerase alpha (and/or delta). 217 36


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