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 from Escherichia coli which acts specificially upon UV-irradiated DNA (correndonuclease II) and is absent from the uvrA and uvrB mutants has been isolated and partially chacterized. The enzyme is present in normal amounts in the urvC mutant. It elutes from phosphocellulose at about 0.25 M potassium phosphate (pH 7.5) and passes through dialysis tubing. The enzyme binds tightly to UV-irradiated DNA but does not bind to unirradiated DNA. The enzyme incises irradiated DNA to the 5' side of a pyrimidine dimer and leaves a 5'-phosphoryl terminus which can be resealed with polynucleotide ligase. The Km of the enzyme is about 1.5 X 10(-8) M dimers. Endonucleolytic activity of the enzyme is inhibited by caffeine with a KI of about 10mM.
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PMID:The Escherichia coli UV endonuclease (correndonuclease II). 110 24

DNA molecules that have been damaged in both strands at the same level are not subject to repair by excision but instead can be repaired through recombination with homologous molecules. Examples of two-strand damage include postreplication gaps opposite pyrimidine dimers, two-strand breaks produced by X-rays, and chemically induced interstrand cross-links. In ultraviolet-irradiated bacteria, the newly synthesized DNA is of length equal to the interdimer spacing. With continued incubation, this low-molecular-weight DNA is joined into high-molecular-weight chains (postreplication repair), a process associated with sister exchanges in bacteria. Recombination is initiated by pyrimidine dimers opposite postreplication gaps and by interstrand cross-links that have been cut by excision enzymes. The free ends at the resulting gaps presumably initiate the exchanges. Postreplication repair in Escherichia coli occurs in recB- AND RECC but is greatly slowed in recF- mutants. RecB and recC are the structural genes for exonuclease V, which digests two-stranded DNA by releasing oligonucleotides first from one strand and then from the other. The postreplication sister exchanges in ultra-violet-irradiated bacteria result in the distribution of pyrimidine dimers between parental and daughter strands, indicating that long exchanges involving both strands of each duplex occur. The R1 restriction endonuclease from E. COli has been used to cut the DNA of a bacterial drug-resistance transfer factor with one nuclease-sensitive site, and also DNA from the frog Xenopus enriched for ribosomal 18S and 28S genes. The fragments were annealed with the cut plasmid DNA and ligated, producing a new larger plasmid carrying the eukaryotic rDNA and able to infect and replicate in E. coli.
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PMID:Repair by genetic recombination in bacteria: overview. 110 33

Previous studies have indicated that the ultraviolet endonuclease of bacteriophage T4 acts specifically at pyrimidine dimer sites in ultraviolet-irradiated DNA. At such sites the enzyme could conceivably catalyze endonucleolytic incision of the DNA either on the dimer-containing strand or on the strand directly opposite to the dimer. In the present work, a direct test of these alternatives was made. Substrate molecules containing one irradiated and one unirradiated strand were prepared from differentially isotopically labeled purified complementary strands of bacteriophage lambdaDNA. Following incubation with the enzyme, the sedimentation profiles of the DNA strands in alkaline sucrose density gradients were compared. The results show that the enzyme selectively nicks the irradiated strand.
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PMID:Action of bacteriophage T4 ultraviolet endonuclease on duplex DNA containing one ultraviolet-irradiated strand. 118 90

The action of an endonuclease from Micrococcus luteus that operates on UV damage in DNA overlaps with that of DNA photolyase from yeast: homo- and heterocyclobutane dipyrimidines in DNA are substrates for both enzymes, but pyrimidine adducts or the "spore photoproduct" in DNA are not. As expected from this overlap, the action of the two enzymes is mutually interfering: single-strand nicks introduced by the endonuclease effectively preclude photoreactivation; conversely, formation of a photolyase-cyclobutane dipyrimidine complex can prevent nicking by the endonuclease.
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PMID:Substrate specificity of Micrococcus luteus UV endonuclease and its overlap with DNA photolyase activity. 119 Nov 74

Human cells prelabeled with [32P]phosphate were exposed to UV and then pulse-labeled with [3H]thymidine. The DNA from these cells was subsequently treated with T4 endonuclease V, an enzyme which specifically nicks DNA at positions adjacent to pyrimidine dimers. Sedimentation in alkaline sucrose gradients revealed that both the DNA made before and that made after irradiation contained nuclease-sensitive sites, indicating that a recombinational process between these DNAs might be occurring during postirradiation incubation. Sedimentation in neutral sucros gradients showed that the molecular weight of native DNA remained unchanged for both DNAs upon endonuclease treatment, indicating that gaps opposite dimers are not necessarily formed after irradiation.
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PMID:Postreplication repair in human cells: on the presence of gaps opposite dimers and recombination. 119 Nov 86

A sensitive enzymatic assay has been utilized to monitor repair of UV-induced damage to DNA in primary human and embryonic chick cells and in multinucleate heterokaryons artificially derived from both. The assay exploits the unique ability of a purified repair endonuclease to attack UV-irradiated DNA at sites containing pyrimidine dimers. These nuclease-susceptible sites are subsequently observed as single-strand scissions by velocity sedimentation in alkaline sucrose gradients. Incubation of UV-damaged cultures followed by extraction and enzymatic analysis of the radioactively labeled DNA enables one to trace the disappearance of such sites in vivo and hence to monitor endogenous repair activity. When UV-irradiated human cells are incubated in the dark, the curve for site removal exhibits a two-phase exponetial decline; i.e. there exists a fast component responsible for elimination of 60% of the initial damage and a second one approximately 7 times slower in rate. The removal of sites is not further enhanced by exposing cells to blacklight during post-UV incubation. Conversely, UV-damaged chick cells rid their DNA of all nuclease-susceptible sites rapidly (i.e. at an exponential rate approximately 13 times faster than the fast component of site removal in human cells) when incubated under blacklight but not when kept in the dark. These data indicate the presence in human and embryonic chick cells of distinct enzymatic mechanisms for the elimination of dimer-containing sites. Wheneras human fibroblasts rely heavily on a light-independent process, excision-repair, chick fibroblasts possess a light-dependent mechanism, presumably photoenzymatic repair. Advantage has been taken of the contrasting repair properties of the human and embryonic chick fibroblasts to evaluate the extent to which each can assist the other in the removal of UV-induced damage from its DNA. The two cell types were fused to form giant human/chick heterokaryons containing a number of intact nuclei from both strains. Experimental conditions were selected so that UV-induced damage resided only in DNA foreign to the repair enzymes under study. Our results strongly suggest that repair enzyme(s) coded for by either fusion partner can remove dimer-containing sites from the DNA of the other with an efficiency comparable to that attained when acting on its own DNA in unfused, parental cells. Further, the light-requiring repair process supplied by the chick is more proficient at operating on these sites in human DNA than is excision-repair, the parallel mechanism available to human cells for this purpose.
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PMID:Use of enzymatic assay to evaluate UV-induced DNA repair in human and embryonic chick fibroblasts and multinucleate heterokaryons derived from both. 123 81

Human diploid cells (WI38) were pre-labeled with 32Pi, exposed to ultraviolet irradiation and then pulse labeled with [3H]thymidine. The extracted DNA from these cells was subsequently treated with the T4-endonuclease V, an enzyme which specifically nicks DNA strands at positions adjacent to pyrimidine dimers. Sedimentation in alkaline sucrose gradients revealed that the DNA synthesized after irradiation, as well as that made before, contained endonuclease-sensitive sites. Our results suggest that pyrimidine dimers are transferred from parental to daughter DNA strands during post-irradiation incubation. Sedimentation in neutral sucrose gradients showed that the molecular weight of native DNA was not affected by the endonuclease treatment, suggesting that the gaps appearing in daughter strands after irradiation are not opposite dimers or that the enzyme cannot recognize dimers in the gap regions.
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PMID:T4-endonuclease V-sensitive sites in DNA from ultraviolet-irradiated human cells. 125 81

A DNA-binding protein specific for ultraviolet irradiated DNA has been purified extensively from human placenta. The binding preparation is free of exonuclease, polymerase, endonuclease, and N-glycosidase activity. The binding activity is salt dependent and is specific for double-stranded irradiated DNA. DNA from which the pyrimidine dimers have been monomerized by the action of photolyase (photoreactivating enzyme) remains an effective substrate for the binding protein, suggesting that the protein recognizes photoproducts other than pyrimidine dimers. This is supported by the finding that DNA irradiated under conditions which introduce only pyrimidine dimers is not a substrate for the binding protein. Examination of three of the xeroderma pigmentosum complementation groups has revealed no deficiency in this binding activity.
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PMID:A DNA binding protein from human placenta specific for ultraviolet damaged DNA. 127 48

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

T4 endonuclease V catalyzes the hydrolysis of the glycosyl bond of a thymine dimer in a DNA duplex and the cleavage of the 3'-phosphate by beta-elimination. We have previously identified a catalytic site for the first reaction (pyrimidine dimer-glycosylase activity) by systematic mutagenesis (Doi et al. Proc. Natl. Acad. Sci. USA 1992 in press) and by x-ray crystallography (Morikawa et al. Science, 256: 523-526, 1992). The results showed that replacement of Glu23 with either glutamine or aspartic acid completely abolished the glycosylase activity. We describe the investigation of the second reaction (apurinic/apyrimidinic endonuclease activity), using twenty two mutants of T4 endonuclease V plus a DNA mini duplex containing an abasic site. Replacement of Glu23 by glutamine abolished the second reaction, but replacement with aspartic acid did not. The pH optima of the mutant (23 Asp) and the wild type were found to be 5.0 and 5.5, respectively. We conclude that the carboxylate anion in position 23 may act as a general base in the beta-elimination reaction of the endonuclease.
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PMID:Participation of glutamic acid 23 of T4 endonuclease V in the beta-elimination reaction of an abasic site in a synthetic duplex DNA. 135 29


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