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)

An endonuclease which is active with regard to depurinated, alkylated, arylated, and arylamidated DNA has been purified 500-fold from Micrococcus luteus. In this purification, separation from the pyrimidine-dimer-specific ultraviolet-endonuclease has been achieved. The enzyme has a molecular weight of 30000 on the basis of gel filtration; its activity is not absolutely dependent upon the presence of Mg2+, but 5--30 mM Mg2+ produces a five-fold stimulation. Potassium chloride concentrations of less than 100 mM are optimal, while concentrations exceeding 100 mM inhibit. The enzyme has no effect on native DNA, but introduces single-strand breaks into DNA containing apurinic/apyrimidinic sites produced by heating at an acidic pH. DNA treated with such carcinogens as N-alkyl-N-nitrosoureas, alkyl methanesulfonates, alkyl sulfates, nitrogen mustard, beta-propiolactone, 7-bromomethyl-benz[a]anthracene, N-acetoxy-2-acetylaminofluorene, and 7,12-dimethyl-benz[a]anthracene-5,6-oxide also becomes susceptible to enzymic action. The activity of the enzyme has been detected by making use of the difference in mobility between supercoiled closed-circular DNA of Pseudomonas phage PM2 and its nicked form in agarose gel elctrophoresis. Even depurinated or carcinogen-modified supercoiled PM2 DNA migrated faster than the respective relaxed nicked forms. A comparison of the number of enzyme-catalyzed single-strand breaks with the number of alkali-labile (i.e. apurinic) sites in carcinogen-modified PM2 DNA showed that the enzyme preparation introduced approximately twice as many breaks into the substrates as the number of apurinic sites present. We conclude that the enzyme preparation either recognizes both apurinic sites and DNA bases carrying carcinogenic residues or contains DNA glycosidase activity in addition to the endonuclease activity. Exposure of ultraviolet-irradiated PM2 DNA to the endonuclease preparation showed that pyrimidine dimers were not substrates. The yield of enzyme-catalyzed single-strand breaks found in ultraviolet-irradiated DNA was five times the number of alkali-labile sites present suggesting that minor photoproducts, possibly 5,6-saturated pyrimidine residues, were recognized in addition to apurinic sites.
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PMID:Purification and characterization of an endonuclease from Micrococcus luteus that acts on depurinated and carcinogen-modified DNA. 71 Apr 10

The mei-9(a) mutant of Drosophila melanogaster , which reduces meiotic recombination in females (Baker and Carpenter 1972), is deficient in the excision of UV-induced pyrimidine dimers in both sexes. Assays were performed in primary cultures and established cell lines derived from embryos. An endonuclease preparation from M. luteus , which is specific for pyrimidine dimers, was employed to monitor UV-induced dimers in cellular DNA. The rate of disappearance of endonuclease-sensitive sites from DNA of control cells is 10-20 times faster than that from mei-9(a) cells. The mutant mei-218, which is also deficient in meiotic recombination, removes nuclease-sensitive sites at control rates. The mei-9(a) cells exhibit control levels of photorepair, postreplication repair and repair of single strand breaks. In mei-9 cells DNA synthesis and possibly postreplication repair are weakly sensitive to caffeine. Larvae which are hemizygous for either of the two mutants that define the mei-9 locus are hypersensitive to killing by the mutagens methyl methanesulfonate, nitrogen mustard and 2-acetylaminofluorene. Larvae hemizygous for the mei-218 mutant are insensitive to each of these reagents. These data demonstrate that the mei-9 locus is active in DNA repair of somatic cells. Thus functions involved in meiotic recombination are also active in DNA repair in this higher eukaryote. The results are consistent with the earlier suggestions (Baker and Carpenter 1972; Carpenter and Sandler 1974) that the mei-9 locus functions in the exchange events of meiosis. The mei-218 mutation behaves differently in genetic tests and our data suggest its function may be restricted to meiosis. These studies demonstrate that currently recognized modes of DNA repair can be efficiently detected in primary cell cultures derived from Drosophila embryos.
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PMID:The mei-9 alpha mutant of Drosophila melanogaster increases mutagen sensitivity and decreases excision repair. 82 52

An endonuclease present in partially purified preparations of calf thymus DNA polymerase has been purified to homogeneity. It has a molecular weight of 53,000 +/- 2,500 as determined by sucrose gradient sedimentation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates the protein is composed of four subunits, each polypeptide possessing a molecular weight of 13,000. Its isoelectric point is 10.3 +/- 0.2. The endonuclease has a pH optimum at 6.6, requires Mg2+ or Mn2+ for activity, and does not attack RNA. The enzyme appears to be present in tissues other than calf thymus. The enzyme catalyzes the endonucleolytic cleavage of both denatured and native eukaryotic DNA. The enzyme introduces a limited number of single strand nicks into native DNA; hydrolysis of denatured DNA produces acid-soluble oligonucleotides. The average size of the limit product, sedimented in an alkaline sucrose gradient, is 1200 nucleotides for native DNA. The product contains 5'-phosphoryl and 3'-hydroxyl termini. While all four deoxynucleotides are found at the 5' termini, pyrimidine residues predominate. Calf thymus DNase V degrades closed circular duplex SV40 DNA and glucosylated T4DNA but not poly(dA-dT). The rate of hydrolysis of homopolymers is: poly(dT) greater than poly(dA) greater than poly(dC) greater than poly(dG) in the presence of Mg2+, and poly(dT) greater than poly(dC) greater than poly(dA) = poly(dG) in the presence of Mn2+.
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PMID:Mammalian endonuclease, DNase V. Purification and properties of enzyme of calf thymus. 83 11

The effects of substituents at position 5 in the pyrimidine ring of a variety of phage DNAs upon EcoRI endonuclease and methylase activities have been examined. The replacement of cytidine in DNA with glucosylated hydroxymethylcytidine confers resistance to cleavage by the EcoRI endonuclease. Substitution of thymidine in DNA by hydroxy-methyluridine(a change in the methyl at position 5 of thymidine for a hydroxymethyl) lowers the maximal velocity of endonucleolytic cleavage 20-fold, but has no detectable effect upon the Km. Substitution of thymidine in DNA by uridine (a change in the methyl at position 5 of thymidine for a hydrogen atom) has no effect upon either the maximal velocity or the Km. The effect of these modifications upon EcoRI methylase activity was markedly different. DNA containing glucosylated hydroxymethylcytidine is methylated as well as normal DNA. DNA containing uridine or hydroxy-methyluridine, in place of thymidine, is much more poorly methylated than normal DNA. These different sensitivities of the EcoRI endonuclease and methylase to modifications in the pyrimidine rings of DNA suggest there are significant differences in the manner by which these enzymes recognize and bind to the canonical EcoRI sequence.
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PMID:EcoRI cleavage and methylation of DNAs containing modified pyrimidines in the recogintion sequence. 86 78

The ultraviolet-light induction of DNA damage has been measured in the epidermis of hairless mice with the use of damage-specific endonucleases from Micrococcus luteus. The rates of induction of endonuclease-sensitive sites in HRS/J/Anl and Skh:hairless-1 mice were 6.1 +/- 0.5 X 10(-11) and 6.5 +/- 0.8 X 10(-11)/dalton/J/sq m from a FS40 fluorescent sun lamp (280 to 400 nm), respectively. Enzymatic photoreactivation with yeast photoreactivating enzyme showed that approximately 80% of the endonuclease-sensitive sites were cycloburyl pyrimidine dimers. In both strains of mice the pyrimidine dimers remained in high-molecular-weight DNA for 24 hr after irradiation. These data show that mouse epithelial cells in vivo have little or no capacity for the excision repair of pyrimidine dimers.
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PMID:Induction and persistence of pyrimidine dimers in the epidermal DNA of two strains of hairless mice. 88 73

Five peaks of endonuclease activity showing a preference for ultraviolet-damaged DNA have been chromatographically identified from extracts of Micrococcus luteus. They are numerically designated as I to V in order of their elution from phosphocellulose (Whatman P-11) columns. The first two of these peaks have been highly purified by a combination of gel filtration and affinity chromatography and are catalytically homogeneous judging from their effect on transforming DNAs. Peak I, which has an isoelectric point of 4.7, is heat-stable, requires high ionic strength for optimal activity, acts with equal facility on ultraviolet-irradiated native and denatured DNA, and has been designated as Py pyrimidine dimer Py correndonuclease I. Peak II which has a pI value of 8.7, is heat-labile, is inhibited by high ionic strength, acts on ultraviolet-irradiated native but not denatured DNA, and has been designated as Py pyrimidine dimer Py correndonuclease II. Both enzymes are inhibited by Ca2+ and Zn2+, do not show any cofactor or sulfhydryl requirement, act optimally between pH 7.0 and 7.4, and have molecular weights between 11,000 and 15,000. Py pyrimidine dimer Py correndonuclease I requires a dose about 1.6 times that for Py pyrimidine dimers Py correndonuclease II for incision saturation of irradiated phiX174 RFI DNA.
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PMID:Micrococcus luteus correndonucleases. I. resolution and purification of two endonucleases specific for DNA containing pyrimidine dimers. 89 8

A UV-specific endonuclease was used to detect ultraviolet light-induced pyrimidine dimers in chloroplast DNA of Chlamydomonas reinhardi that was specifically labeled with tritiated thymidine. All of the dimers induced by 100 J/m2 of 254 nm light are removed by photoreaction. Wild-type cells exposed to 50 J/m2 of UF light removed over 80% of the dimers from chloroplast DNA after 24 h of incubation in growth medium in the dark. A UV- sensitive mutant, UVS1, defective in the excision of pyrimidine dimers from nuclear DNA is capable of removing pyrimidine dimers from chloroplast DNA nearly as well as wild-type, suggesting that nuclear and chloroplast DNA dark-repair systems are under separate genetic control.
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PMID:Photoreactivation and dark repair of ultraviolet light-induced pyrimidine dimers in chloroplast DNA. 90 95

The ultraviolet-endonuclease isolated from Micrococcul luteus, specific for pyrimidine dimers, is able to attack not only ultraviolet-irradiated DNA (leading to 3'OH-5'PO4 single-strand breaks) but also superhelical covalently-closed circular DNA of phage lambda damaged by heating at 70 degrees C, pH 5.93. The number of endonuclease-sensitive defects in the DNA corresponds to the number of alkalilabile bonds (apurinic sites) induced by heating. Competition between ultraviolet-induced lesions and apurinic sites for ultraviolet-endonuclease is demonstrated; the affinity of the enzyme for pyrimidine dimers is about three times that for apurinic sites. Both activities of the ultraviolet-endonuclease are inactivated at 50 degrees C at the same rate. The ultraviolet-endonuclease is able to reduce the infectious activity of depurinated lambda DNA towards Ca2+-treated uvr+ and uvr A Escherichia coli cells. It is concluded that both pyrimidine dimers and apurinic sites can be recognized by one and the same enzyme (the ultraviolet-endonuclease).
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PMID:Substrate specificity of the ultraviolet-endonuclease from Micrococcus luteus. Endonucleolytic cleavage of depurinated DNA. 99 58

We have studied excision-repair of UV-irradiated phiX174 RFI DNA in vitro with UV-specific endonuclease from Micrococcus luteus (UV-endo), DNA polymerase I from Escherichia coli and DNA ligase from phage T4 infected E. coli. Excision-repair was measured a) by physico-chemical methods, i.e. by determination of the conversion of RF I DNA into RF II DNA by UV-endo and by the subsequent conversion of RF II DNA ligase, b) by biological methods i. e. by measuring the ability of the reaction product to form phages upon incubation with spheroplasts from the appropriate strains of E. coli. Using the first method, we have shown, that more than 90% of the pyrimidine dimers can be repaired in vitro; with the latter method we have shown, that the molecules which are repaired as defined by method a) have regained full biological activity. Exonuclease III was found to be not essential for excision-repair in vitro and also did not stimulate repair. From this result we conclude that UV-endo generates 3'OH endgroups, in agreement with results obtained by Hamilton et al. (1974). The usefulness of the method presented in this paper with regard to the study of excision-repair is discussed.
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PMID:Physico-chemical and biological study of excision-repair of UV--irradiated phiX174 RF DNA in vitro. 105 35

UV-endonuclease from Microcossuc luteus induces single-stranded breaks in UV-irradiated DNA of phage lambda and the average length of the fragments produced (after UV-doses to DNA of 135 and 675 erg/mm2) is equal to the average spacing between pyrimidine dimers. The plaque-forming ability of UV-irradiated lambda DNA used to infect Ca++-treated uvr A6, uvrB5 or uvrC34 recipient Escherichia coli cells (but not uve+ cells) may be significantly enhanced by treatment of lambda DNA with UV-endonuclease. This enzyme strongly decreases the reactivation of UV-irradiated lambda DNA caused by UV-irradiation of uvr+ or uvrA6 Ca++-treated cells and eliminates most clear-mutations especially if mutations are analysed using Ca++-treated uvr A6 recipient cells. It is concluded that UV-endonuclease switches a significant part of potentially mutagenic pyrimidine dimers from the UV-induced "error-prone" repair pathway to "error-free" excision repair pathway.
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PMID:Ultraviolet reactivation and ultraviolet mutagenesis of infectious lambda DNA: strong inhibition by treatment of DNA in vitro with UV-endonuclease from Micrococcus luteus. 109 9


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