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 aim of this study was to compare the sensitivity of chromatin from representative cellular stages of spermiogenesis to a single-strandeded nuclease after heat denaturation. Thermal denaturation of chromatin was assayed in situ in fixed round, elongating and elongated spermatids and in testicular sperm from mice. Production of single-stranded deoxyribonucleic acid (DNA) at elevated temperatures was monitored by digesting chromatin with endonuclease specific for single-stranded DNA (S1 nuclease), staining the residual DNA with gallocyanin-chrome alum (GAC) and measuring the stain content by absorption cytophotometry. Changes in GCA staining were minimal over the temperature range of 22-90 degrees C in each cell type not exposed to nuclease. Staining of undigested cells decreased progressively with advancing cell maturity. Nuclease had no effect on the GCA content of round spermatids below 60 degrees C, but above this temperature there was a progressive decrease in GCA-stainable chromatin. Both round and elongating spermatid stages showed a significantly greater sensitivity to nuclease digestion than did more mature stages; sperm showed no effects of nuclease action below 80 degrees C. Progressive chromatin condensation and a concomitant decrease in the number of available DNA phosphate groups during spermiogenic cell maturation may be responsible for the observed decline in sensitivity to nuclease and decreased GCA staining. Thermal denaturation of round spermatids labeled with 3H-thymidine produced no change in autoradiographic mean nuclear grain counts, indicating no loss of thymidine-labeled DNA from the slides during denaturation. When round spermatids and sperm were hydrolyzed with hot tricholoroacetic acid before staining, both nuclear GCA content and autoradiograph grain count were partially reduced, indicating incomplete DNA removal. Almost complete loss of Feulgen-stainable material occurred in these cells and may be due to depurination and elimination of Feulgren-reactant aldehyde groups.
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PMID:Single-strand nuclease action on heat-denatured spermiogenic chromatin. 6 Apr 38

Nucleoprotein complexes containing viral DNA and cellular histones were extracted from nuclei of permissive cells infected with polyoma virus or simian virus 40 (SV40) and examined by electron microscopy. Polyoma and SV40 nucleoprotein complexes are almost identical. They appear as relaxed circular molecules consisting of 20 to 21 globular particles interconnected by thin filaments. Their contour length in 0.02 M salt is 2.7 times shorter than that of viral DNA form I obtained after dissociation of the proteins in 1 M NaCl. The nucleosomes have an average diameter of 12.5 nm. Each nucleosome contains 175 to 205 DNA base pairs condensed fivefold in length. The nucleosomes are regularly spaced on the circular molecule. The internucleosomal filaments are made of naked DNA, and each filament contains about 55 base pairs. The partial sensitivity of the nucleoprotein complex to cleavage by EcoR1 endonuclease suggests that the nucleosomes are not formed at specific sites on the viral genome. Faster sedimenting nucleoprotein complexes containing replicative intermediates were studied. Isopycnic centrifugation in metrizamide gradients in the absence of aldehyde fixation showed that these molecules conserved the same DNA-to-protein ratio as the form I DNA-containing complexes.
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PMID:Chromatin-like structures in polyoma virus and simian virus 10 lytic cycle. 17 84

The inactivation efficiency and repair of single-strand breaks was investigated using model strand breaks created by endonucleolytic incision of damaged DNA. Phi X-174 duplex transfecting DNA containing either thymine glycols, urea residues, or abasic (AP) sites was incubated with AP endonucleases that produce breaks on the 3' side, the 5' side, or both sides of the lesion. For each lesion, incubation with Escherichia coli endonuclease III results in a single-strand break containing a 3' alpha, beta-unsaturated aldehyde (4-hydroxy-2-pentenal), while treatment of AP- or urea-containing DNA with E. coli endonuclease IV results in a single-strand break containing a 5' deoxyribose or a 5' deoxyribosylurea moiety, respectively. Incubation of lesion-containing DNA with both enzymes results in a base gap. Ligatable nicks containing 3' hydroxyl and 5' phosphate moieties were produced by subjecting undamaged DNA to DNase I. When the biological activity of these DNAs was assessed in wild-type cells, ligatable nicks were not lethal, but each of the other strand breaks tested was lethal, having inactivation efficiencies between 0.12 and 0.14. These inactivation efficiencies are similar to those of the base lesions from which the strand breaks were derived. In keeping with the current model of base excision repair, when phi X duplex DNA containing strand breaks with a blocked 3' terminus was transfected into an E. coli double mutant lacking the major 5' cellular AP endonucleases, a greater than twofold decrease in survival was observed. Moreover, when this DNA was treated with a 5' AP endonuclease prior to transfection, the survival returned to that of wild type. As expected, when DNA containing strand breaks with a 5' blocked terminus or DNA containing base gaps was transfected into the double mutant lacking 5' AP endonucleases, the survival was the same as in wild-type cells. The decreased survival of transfecting DNA containing thymine glycols, urea, or AP sites observed in appropriate base excision repair-defective mutants was also obviated if the DNA was incubated with the homologous enzyme prior to transfection. Thus, in every case, with both base lesions and single-strand breaks, the lesion was repaired in the cell by the enzyme that recognizes it in vitro. Furthermore, the repair step in the cell could be eliminated if the appropriate enzyme was added in vitro prior to transfection.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Processing of model single-strand breaks in phi X-174 RF transfecting DNA by Escherichia coli. 185 23

Treatment of an end-labeled DNA restriction fragment with the nonprotein chromophore of neocarzinostatin induced lesions which, after treatment with endonuclease IV or putrescine, were expressed as site-specific double-strand breaks. Analysis of the termini at cleavage sites in each strand showed that the neocarzinostatin-induced lesions consisted of an apurinic/apyrimidinic site plus a closely opposed break in the complementary strand. The break always occurred opposite the base two positions upstream from the apurinic/apyrimidinic site and had the 3'-phosphate and 5'-aldehyde termini characteristic of neocarzinostatin-induced breaks. This positioning suggests that neocarzinostatin simultaneously attacks two DNA sugars on opposite edges of the minor groove. The sequence specificity for formation of apurinic/apyrimidinic sites with closely opposed breaks reflected that of neocarzinostatin-induced mutagenesis. The potent mutagenicity of these lesions may be attributable to the presence of closely opposed damage in both DNA strands.
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PMID:Neocarzinostatin-induced DNA base release accompanied by staggered oxidative cleavage of the complementary strand. 297 36

A clone of DNA, obtained from the luminescent bacterium Vibrio fischeri ATCC 7744 and inserted into pBR322, was found to express luminescence in Escherichia coli. Polypeptides involved in biosynthesis of the fatty aldehyde substrate for the light reaction were identified by fatty acid acylation of proteins synthesized in E. coli from the recombinant plasmid. The cloned region was similar to that reported for the V. fischeri MJ1 luminescence system (Engebrecht et al., Cell 32:773-781), except for some differences in endonuclease restriction sites and the requirement of a lower temperature for the expression of light in our cloned system. Fatty acid reductase activity could be detected in extracts of E. coli harboring the recombinant plasmid but not in extracts of the parental V. fischeri strain. Using in vivo labeling with [3H]tetradecanoic acid, we showed that the acylated polypeptides synthesized in the cloned system corresponded to the labeled polypeptides in V. fischeri (34, 42, and 54 kilodaltons) and that they could only be detected after induction of luminescence. These results provide direct evidence that the genes coding for the fatty acid reductase polypeptides are an integral part of the luminescence operon in the V. fischeri luminescence system.
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PMID:Functional identification of the fatty acid reductase components encoded in the luminescence operon of Vibrio fischeri. 299 37

The specific interaction between left-handed Z DNA sequences in negatively supercoiled bacteriophage phi X174 replicative form I (RFI) DNA and anti-Z DNA immunoglobulin G (IgG) was investigated by high resolution darkfield immuno-electron microscopy. DNA-antibody complexes were formed and maintained under optimal binding conditions, purified by column chromatography, and visualized after uranyl acetate staining without using aldehyde fixation, shadowing, or second antibody. Bivalent anti-Z DNA IgGs bound to RFI molecules, thus forming intramolecular bridges. They could also oligomerize separate molecules by intermolecular linking of Z DNA sequences. At relatively low ionic strength and low temperature, high affinity anti-Z IgG was retained at certain loci even after restriction endonuclease cleavage of the DNA. In these cleaved molecules some superhelices could be visualized in the loops generated by the bivalent IgG. To our knowledge this is the first example of polypeptide stabilization of local superhelical strain in a cut molecule. Z DNA sequences in phi X174 RFI DNA were mapped. Alternating tracts of purines and pyrimidines starting at nucleotides 763, 1027, 1714, 2146, 2363, 3504, 4161, 4911 and 5345 occur within the nine different anti-Z IgG binding sites which were expressed with varying frequencies (53-3%) on the molecules. Usually, a limited number of sites (generally less than or equal to 2) exists on any one molecule. The formation of multiple Z sites (at the extracted superhelix density) in a given molecule is probably non-cooperative due to relaxation of torsional stress by the B----Z transition. Z sites occur in several different genes, including regions where transcription is attenuated and, in one case, in front of a promoter of transcription.
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PMID:Different Z DNA forming sequences are revealed in phi X174 RFI by high resolution darkfield immuno-electron microscopy. 624 Nov 50

The formation of apurinic/apyrimidinic sites (AP-sites) and single strand breaks (SSB) by chromate and ascorbate (AsA) in isolated DNA was investigated using a number of agents that cleave DNA at AP-sites (putrescine, exonuclease III, the tripeptide Lys-Trp-Lys, and an AP-endonuclease containing fraction isolated from human fibroblasts). Relative to the number of SSB caused by chromate and AsA alone, all these agents induced additional nicking, indicating the induction of AP-sites. Chromate/AsA-induced AP-sites contain aldehyde groups, as cleavage by putrescine could be prevented by treatment with borohydride which reduced the aldehyde. The time course for the formation of both DNA lesions was very similar, and there was a 1:1 ratio of the number of SSB to the number of AP-sites. The addition of catalase to incubation mixtures containing chromate/AsA led to an almost complete suppression of AP-sites and SSB. In systems containing lower concentrations of chromate/AsA, the exclusion of oxygen inhibited the formation of both lesions. It is suggested that AP-sites and SSB arise from attack by reactive species deriving from chromate/AsA on one single site at DNA, probably the sugar moiety. In view of the known mutagenicity of AP-sites, these results could aid an understanding of the mechanisms underlying chromium(VI) carcinogenicity.
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PMID:The reductive conversion of chromium (VI) by ascorbate gives rise to apurinic/apyrimidinic sites in isolated DNA. 749 38

Ethanol-inducible cytochrome P4502E1 is the main pathway in the non-alcohol dehydrogenase oxidation of ethanol. Its coding gene, CYP2E1, is polymorphic at the Rsa I restriction site in the 5'-flanking region. The mutant genotype c2c2 has a higher transcriptional activity than the genotype c1c1 or c1c2. Heavy drinkers carrying the c2 allele might be at a higher risk of alcoholic cirrhosis since they might synthesize greater amounts of acetaldehyde, the compound believed responsible for hepatotoxicity of ethanol. With the aim of establishing if the c2 allele increases the risk of cirrhosis in heavy drinkers, we studied 58 (6 female) chronic heavy drinkers with liver cirrhosis and 137 healthy normal controls of the same ethnic (white Spaniards) origin. After extraction of DNA from white blood cells, alleles c1 and c2 of CYP2E1 were identified by restriction fragment length polymorphism (RFLP) with endonuclease Rsa I. Fifty-six patients and 130 controls were classified as homozygous c1c1 and two and seven, respectively, as heterozygous c1c2. No homozygous c2c2 were detected. The c2 allele frequencies were 0.017 in patients and 0.026 in controls (non-significant differences). We conclude that the Rsa I RFLP polymorphism is probably not related to the risk of cirrhosis in Spanish heavy drinkers.
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PMID:Rsa I polymorphism at the cytochrome P4502E1 locus is not related to the risk of alcohol-related severe liver disease. 902 17

Escherichia coli exonuclease III and endonuclease III are two distinct DNA-repair enzymes that can cleave apurinic/apyrimidinic (AP) sites by different mechanisms. While the AP endonuclease activity of exonuclease III generates a 3'-hydroxyl group at AP sites, the AP lyase activity of endonuclease III produces a 3'-alpha,beta unsaturated aldehyde that prevents DNA-repair synthesis. Saccharomyces cerevisiae Apn1 is the major AP endonuclease/3'-diesterase that also produces a 3'-hydroxyl group at the AP site, but it is unrelated to either exonuclease III or endonuclease III. apn1 deletion mutants are unable to repair AP sites generated by the alkylating agent methyl methane sulphonate and display a spontaneous mutator phenotype. This work shows that either exonuclease III or endonuclease III can functionally replace yeast Apn1 in the repair of AP sites. Two conclusions can be derived from these findings. The first of these conclusions is that yeast cells can complete the repair of AP sites even though they are cleaved by AP lyase. This implies that AP lyase can contribute significantly to the repair of AP sites and that yeast cells have the ability to process the alpha,beta unsaturated aldehyde produced by endonuclease III. The second of these conclusions is that unrepaired AP sites are strictly the cause of the high spontaneous mutation rate in the apn1 deletion mutant.
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PMID:Normal processing of AP sites in Apn1-deficient Saccharomyces cerevisiae is restored by Escherichia coli genes expressing either exonuclease III or endonuclease III. 919 99

One of the most prevalent lesions in DNA is the apurinic/apyrimidinic (AP) site, which is derived from the cleavage of the N-glycosyl bond by DNA glycosylase or by spontaneous depurination. AP sites are repaired by AP endonucleases during the process of base excision repair; however, an imbalance in this DNA repair system may cause mutations as well as cell death. We have established a sensitive and convenient slot-blot method to detect AP sites in genomic DNA using a novel aldehyde reactive probe (ARP), which reacts with the aldehydic group of ring-opened AP sites. The reaction of 1 mM of ARP with 15 microg of genomic DNA containing AP sites at 37 degrees C was completed within 1 min. The AP site-ARP complex was remarkably stable during incubation in TE buffer, even at 100 degrees C for 60 min. The sensitivity of this assay enables detection of 2.4 AP sites per 10(7) bases. By using this ARP-slot-blot assay, the rate of spontaneous depurination of calf thymus DNA was determined. Under physiological conditions, AP sites were increased at 1.54 AP sites/10(6) nucleotides/day (9000 AP sites/cell/day). This highly sensitive assay allows us to determine the endogenous level of AP sites in genomic DNA, as well as to investigate whether DNA-damaging agents cause imbalances of base excision/AP endonuclease repair in vivo and in vitro.
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PMID:Highly sensitive apurinic/apyrimidinic site assay can detect spontaneous and chemically induced depurination under physiological conditions. 944 96


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