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 cytotoxic action of the antitumor antibiotic mitomycin C occurs primarily at the level of DNA. Using highly sensitive fluorescence assays which depend on the enhancement of ethidium fluorescence only when it intercalates duplex regions of DNA, three aspects of mitomycin C action on DNA have been studied: (a) cross-linking events, (b) alkylation without necessarily cross-linking, and (c) strand breakage. Cross-linking of DNA is determined by the return of fluorescence after a heat denaturation step at alkaline pH's. Under these conditions denatured DNA gives no fluorescence. The cross-linking was independently confirmed by S1-endonuclease (EC 3.1.4.-) digestion. At relatively high concentrations of mitomycin the suppression of ethidium fluorescence enhancement was shown not to be due to depurination but rather to alkylation, as a result of losses in potential intercalation sites. A linear relationship exists between binding ratio for mitomycin and loss of fluorescence. The proportional decrease in fluorescence with pH strongly suggests that the alkylation is due to the aziridine moiety of the antibiotic under these conditions. A parallel increase in the rate and overall efficiency of covalent cross-linking of DNA with lower pH suggests that the cross-linking event, to which the primary cytotoxic action has been linked, occurs sequentially with alkylation by aziridine and then by carbamate. Mitomycin C, reduced chemically, was shown to induce single strand cleavage as well as monoaklylation and covalent cross-linking in PM2 covalently closed circular DNA. The inhibition of this cleavage by superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6), and by free radical scavengers suggests that the degradation of DNA observed to accompany the cytotoxic action of mitomycin C is largely due to the free radical O2. In contrast to the behavior of the antibiotic streptonigrin, mitomycin C does not inactivate the protective enzymes superoxide dismutase or catalase. Lastly, mitomycin C is able to cross-link DNA in the absence of reduction at pH 4. This is consistent with the postulated cross-linking mechansims.
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PMID:Studies related to antitumor antibiotics. Part V. Reactions of mitomycin C with DNA examined by ethidium fluorescence assay. 0 1

The nfo (endonuclease IV) gene of Escherichia coli is induced by superoxide generators such as paraquat (methyl viologen). An nfo'-lacZ operon fusion was used to isolate extragenic mutations affecting its expression. The mutations also affected the expression of glucose 6-phosphate dehydrogenase, Mn2(+)-superoxide dismutase (sodA), and three lacZ fusions to soi (superoxide-inducible) genes of unknown function. The mutations were located 2 kilobases clockwise of ssb at 92 min on the current linkage map. One set of mutations, in a new gene designated soxR, caused constitutive overexpression of nfo and the other genes. It included insertions or deletions affecting the carboxyl end of a 17-kilodalton polypeptide. In a soxR mutant, the expression of sodA, unlike that of nfo, was also regulated independently by oxygen tension. Two other mutants were isolated in which the target genes were noninducible; they had an increased sensitivity to killing by superoxide-generating compounds. One had a Tn10 insertion in or near soxR; the other had a multigene deletion encompassing soxR. Therefore, the region functions as a positive regulator because it encodes one or more products needed for the induction of nfo. Regulation is likely to be at the level of transcription because the mutations were able to affect the expression of an nfo'-lac operon fusion that contained the ribosome-binding site for lacZ. Some mutant plasmids that failed to suppress (or complement) constitutivity in trans had insertion mutations several hundred nucleotides upstream of soxR in the general region of a gene for a 13-kilodalton protein encoded by the opposite strand, raising the possibility of a second regulatory gene in this region. The result define a new regulon, controlled by soxR, mediating at least part of the global response to superoxide in E. coli.
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PMID:soxR, a locus governing a superoxide response regulon in Escherichia coli K-12. 169 93

Escherichia coli responds to superoxide-generating agents by inducing approximately 40 proteins. We have identified a genetic locus, soxR (superoxide response), that positively regulates 9 of these proteins during superoxide stress. Induction under soxR control is at the transcriptional level, as shown with lac fusions to five paraquat-inducible promoters. Members of the soxR regulon include at least three proteins with demonstrable antioxidant roles: Mn-containing superoxide dismutase (which destroys superoxide radicals), endonuclease IV (which repairs radical-induced damages in DNA), and glucose-6-phosphate dehydrogenase (which produces NADPH). Induction of the soxR regulon also leads to diminished levels of the major outer membrane protein OmpF and alteration of the small-subunit ribosomal protein S6. These latter changes confer resistance to a variety of antibiotics. The soxR regulon may thus operate as an inducible defense against xenobiotics in general.
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PMID:Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli. 169 18

soxR governs a superoxide response regulon that contains the genes for endonuclease IV, Mn2(+)-superoxide dismutase, and glucose 6-phosphate dehydrogenase. The soxR gene encodes a 17-kDa protein; some mutations of this gene cause constitutive overexpression of the regulon. Induction by paraquat (methyl viologen) requires both soxR and a new gene, soxS. soxS is adjacent to soxR, it encodes a 13-kDa protein, and it is required for paraquat resistance. These functions were revealed by studies in which the sequence of the 1.1-kb soxR-soxS region was determined, the 5' ends of the mRNAs were mapped, and complementation tests were performed with soxRS plasmids containing deletions of known sequence. The two genes are divergently transcribed, and the transcripts overlap. The soxS promoter is within the 85-nucleotide intergenic region, whereas the soxR promoter is within soxS. soxS mRNA increases after induction. Both protein products have possible DNA-binding (helix-turn-helix) domains. SoxR contains four cysteines (CX2CXCX5C) that might be part of a sensor region. SoxS shows 17 to 31% homology to the C-terminal portions of members of the AraC family of positive regulators.
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PMID:Two divergently transcribed genes, soxR and soxS, control a superoxide response regulon of Escherichia coli. 170 80

Sera from the majority of individuals that were positive in an enzyme-linked immunosorbent assay (ELISA) retrovirus (ARV), an isolate of the for antibodies to acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV), an isolate of the retrovirus identified as the etiologic agent of AIDS, were found to react with a 31,000-dalton protein (p31) in virus Western blot assays. To determine if this 31,000-dalton immunoreactive species originated from the putative endonuclease region of the polymerase (pol) gene of ARV, we cloned this portion of pol into bacterial expression vectors for direct expression and for expression as a fusion protein with human superoxide dismutase. Transformants from both constructions expressed immunoreactive protein detected in immunoblots with an AIDS patient's serum. Extracts from transformants expressing these sequences competed with the binding of antibodies from AIDS patients' sera to the 31,000-dalton protein in virus immunoblots, confirming that viral p31 originated from the endonuclease domain of the ARV polymerase gene. The superoxide dismutase-p31 fusion protein was purified, and an ELISA for detecting antibodies to p31 was developed. The majority (95%) of serum samples obtained from individuals seropositive in the virus ELISA were also positive in the p31 antibody ELISA.
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PMID:Recombinant polypeptide from the endonuclease region of the acquired immune deficiency syndrome retrovirus polymerase (pol) gene detects serum antibodies in most infected individuals. 241 93

The addition of paraquat (methyl viologen) to a growing culture of Escherichia coli K-12 led within 1 hr to a 10- to 20-fold increase in the level of endonuclease IV, a DNase for apurinic/apyrimidinic sites. The induction was blocked by chloramphenicol. Increases of 3-fold or more were also seen with plumbagin, menadione, and phenazine methosulfate. H2O2 produced no more than a 2-fold increase in endonuclease IV activity. The following agents had no significant effect: streptonigrin, nitrofurantoin, tert-butyl hydroperoxide, gamma rays, 260-nm UV radiation, methyl methanesulfonate, mitomycin C, and ascorbate. Paraquat, plumbagin, menadione, and phenazine methosulfate are known to generate superoxide radical anions via redox cycling in vivo. A mutant lacking superoxide dismutase was unusually sensitive to induction by paraquat. In addition, endonuclease IV could be induced by merely growing the mutant in pure O2. The levels of endonuclease IV in uninduced or paraquat-treated cells were unaffected by mutations of oxyR, a H2O2-inducible gene that governs an oxidative-stress regulon. The results indicate that endonuclease IV is an inducible DNA-repair enzyme and that its induction can be mediated via the production of superoxide radicals.
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PMID:Endonuclease IV of Escherichia coli is induced by paraquat. 243 76

Escherichia coli treated with nontoxic levels of the superoxide-generating redox-cycling agents menadione and paraquat showed dramatic changes in protein composition as monitored by two-dimensional gel analysis. The distribution of proteins synthesized after treatment with these agents overlapped significantly with that seen after hydrogen peroxide treatment, and it included all the proteins in the oxyR regulon. The redox-cycling agents also elicited the synthesis of at least 33 other proteins that were not seen with hydrogen peroxide, including three heat shock proteins, the Mn-containing superoxide dismutase, the DNA repair protein endonuclease IV, and glucose-6-phosphate dehydrogenase. At least some of these redox-inducible proteins appear to be part of a specific response to intracellular superoxide. E. coli is thus equipped with a network of inducible responses against oxidative damage, controlled in multiple regulatory pathways.
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PMID:A global response induced in Escherichia coli by redox-cycling agents overlaps with that induced by peroxide stress. 247 81

Two-dimensional gel analyses were made of proteins synthesized in Escherichia coli during various O2- -generating conditions. Nine proteins were constitutively synthesized over wild-type levels in superoxide dismutase (sodA sodB) double mutants. Addition of redox cycling agents such as paraquat and plumbagin at various concentrations induced up to 13 proteins in wild-type cells. Among these 13 were 5 of the 9 constitutively synthesized in the sodA sodB double mutants. Addition of these agents to the superoxide dismutase mutants in low micromolar concentrations induced an additional set of 14 proteins. The proteins induced included only five proteins that have been previously associated with stress responses, consisting of endonuclease IV (Nfo), three oxyR-regulated proteins, and one heat shock protein. O2- -mediated induction of the superoxide inducible (Soi) proteins in the wild type was independent of the oxyR+ gene for all but the three oxyR-regulated proteins. Analyses of proteins from three soi::lacZ gene fusions previously isolated (T. Kogoma, S. B. Farr, K. M. Joyce, and D. O. Natvig, Proc. Natl. Acad. Sci. USA 85:4799-4803, 1988) indicated the specific loss of one of these induced proteins in each fusion strain and the constitutive expression of some Soi proteins.
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PMID:Escherichia coli proteins inducible by oxidative stress mediated by the superoxide radical. 253 20

Exposure of the plasmid pBR 322 to the aerobic xanthine oxidase reaction introduced single strand scissions and endonuclease III-sensitive sites. The latter may be residues of thymine glycol. Both forms of DNA damage were completely prevented by superoxide dismutase or catalase, whereas bovine serum albumin was much less effective. Mannitol and benzoate, added as scavengers of HO., and desferrioxamine or diethylene triamine pentaacetate, added to sequester Fe(III), also protected. These results indicate a metal-catalyzed interaction of O2- with H2O2, which produces HO. which, in turn, causes DNA strand scission and oxidation of thymine residues to thymine glycol. Plasmid isolated from aerobically-incubated cells contained more strand scissions and endonuclease III-sensitive sites than did plasmid from anaerobically-incubated cells, and a low molecular weight scavenger of O2- prevented the damage seen with the aerobic cells. Genetic defects in AP endonucleases rendered E. coli more susceptible to the dioxygen-dependent lethality of plumbagin, which mediates O2- production. Similarly, plasmid DNA, within the endonuclease-deficient cells, exhibited more strand scissions and endonuclease III-sensitive sites upon aerobic exposure to plumbagin than did endonuclease-sufficient cells, and a low molecular weight scavenger of O2- was protective. These results are consistent with the conclusions that strand scissions and formation of endonuclease III-sensitive sites are among the consequences of exposure of DNA to O2- plus H2O2, both in vitro and in vivo.
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PMID:Formation of endonuclease III-sensitive sites as a consequence of oxygen radical attack on DNA. 254 64

The SOD-1 gene on chromosome 21 and approximately 100 kb of chromosomal DNA from the 21q22 region have been isolated and characterized. The gene which is present as a single copy per haploid genome spans 11 kb of chromosomal DNA. Heteroduplex analysis and DNA sequencing reveals five rather small exons and four introns that interrupt the coding region. The donor sequence at the first intron contains an unusual variant dinucleotide 5'-G-C, rather than the highly conserved 5'-GT. The unusual splice junction is functional in vivo since it was detected in both alleles of the SOD-1 gene, which were defined by differences in the length of restriction endonuclease fragments (RFLPs) that hybridize to the cDNA probe. Genomic blots of human DNA isolated from cells trisomic for chromosome 21 (Down's syndrome patients) show the normal pattern of bands. At the 5' end of gene there are the 'TATA' and 'CAT' promoter sequences as well as four copies of the -GGCGGG- hexanucleotide. Two of these -GC- elements are contained within a 13 nucleotide inverted repeat that could form a stem-loop structure with stability of -33 kcal. The 3'-non coding region of the gene contains five short open reading-frames starting with ATG and terminating with stop codons.
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PMID:Architecture and anatomy of the chromosomal locus in human chromosome 21 encoding the Cu/Zn superoxide dismutase. 316 May 82


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