Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The antiviral distamycin A and its phenyl mustard derivative FCE24517 possessing antitumor activity were tested for their ability to inhibit macromolecular synthesis in three human and one murine cell line. While distamycin A was poorly active in these systems, FCE24517 inhibited DNA synthesis efficiently, RNA synthesis to a lower extent and had little effect on protein synthesis. These findings suggest that the in vivo activity of FCE24517 derives from the specific inhibition of DNA synthesis. When the two drugs were tested on several enzymes involved in human DNA metabolism a strikingly similar pattern of inhibition appeared, with distamycin A being the more potent. Both drugs showed: A), no inhibitory activity against thymidine kinase and DNA primase; B), low activity against DNA topoisomerases I and II and the 3'-5' exonuclease associated with the DNA polymerase epsilon; C), high activity against DNA polymerases alpha and epsilon, uracil-DNA glycosylase and the joining activity of the replicative DNA ligase; D), the highest inhibitory activity against the AMP-dependent DNA relaxing activity of DNA ligase. The strong in vitro inhibition of several DNA enzymatic activities, including DNA ligase, do not match with the in vivo activities of the two drugs. However a unique difference was observed: only FCE24517 inhibited the DNA-independent reaction of adenylation of human DNA ligase while the adenylation reaction of T4 and E. coli DNA ligase was unaffected by either drug. It is still unclear whether these properties are relevant for modulating the killing activity of FCE24517 against proliferating cells both in culture and in vivo. Nevertheless FCE24517 is the first known molecule capable of interacting directly and specifically with human DNA ligase.
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PMID:Specific inhibition of human DNA ligase adenylation by a distamycin derivative possessing antitumor activity. 170 93

The DNA repair enzyme uracil-DNA glycosylase from Mycoplasma lactucae (831-C4) was purified 1,657-fold by using affinity chromatography and chromatofocusing techniques. The only substrate for the enzyme was DNA that contained uracil residues, and the Km of the enzyme was 1.05 +/- 0.12 microM for dUMP containing DNA. The product of the reaction was uracil, and it acted as a noncompetitive inhibitor of the uracil-DNA glycosylase with a Ki of 5.2 mM. The activity of the enzyme was insensitive to Mg2+, Mn2+, Zn2+, Ca2+, and Co2+ over the concentration range tested, and the activity was not inhibited by EDTA. The enzyme activity exhibited a biphasic response to monovalent cations and to polyamines. The enzyme had a pI of 6.4 and existed as a nonspherical monomeric protein with a molecular weight of 28,500 +/- 1,200. The uracil-DNA glycosylase from M. lactucae was inhibited by the uracil-DNA glycosylase inhibitor from bacteriophage PBS-2, but the amount of inhibitor required for 50% inhibition of the mycoplasmal enzyme was 2.2 and 8 times greater than that required to cause 50% inhibition of the uracil-DNA glycosylases from Escherichia coli and Bacillus subtilis, respectively. Previous studies have reported that some mollicutes lack uracil-DNA glycosylase activity, and the results of this study demonstrate that the uracil-DNA glycosylase from M. lactucae has a higher Km for uracil-containing DNA than those of the glycosylases of other procaryotic organisms. Thus, the low G + C content of the DNA from some mollicutes and the A.T-biased mutation pressure observed in these organisms may be related to their decreased capacity to remove uracil residues from DNA.
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PMID:A mollicute (mycoplasma) DNA repair enzyme: purification and characterization of uracil-DNA glycosylase. 234 31

Uracil-DNA glycosylase is the DNA repair enzyme responsible for the removal of uracil from DNA, and it is present in all organisms investigated. Here we report on the cloning and sequencing of a cDNA encoding the human uracil-DNA glycosylase. The sequences of uracil-DNA glycosylases from yeast, Escherichia coli, herpes simplex virus type 1 and 2, and homologous genes from varicella-zoster and Epstein-Barr viruses are known. It is shown in this report that the predicted amino acid sequence of the human uracil-DNA glycosylase shows a striking similarity to the other uracil-DNA glycosylases, ranging from 40.3 to 55.7% identical residues. The proteins of human and bacterial origin were unexpectedly found to be most closely related, 73.3% similarity when conservative amino acid substitutions were included. The similarity between the different uracil-DNA glycosylase genes is confined to several discrete boxes. These findings strongly indicate that uracil-DNA glycosylases from phylogenetically distant species are highly conserved.
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PMID:Molecular cloning of human uracil-DNA glycosylase, a highly conserved DNA repair enzyme. 255 54

Cells from patients with Bloom's syndrome, a rare human disease with autosomal recessive mode of inheritance, exhibit cytological abnormalities involving DNA metabolism. Bloom's syndrome is characterized by a greatly increased cancer frequency which may reflect a specific defect in DNA repair and replication. Evidence has recently been presented of the existence in Bloom's syndrome of an abnormality of the DNA ligase involved in semiconservative DNA replication. Another abnormality, in the excision-repair pathway of Bloom's syndrome cells, is reportedly due to an aberrant immunological reactivity of the DNA-repair enzyme uracil-DNA glycosylase. In this investigation we show, however, that the catalytic activity of uracil-DNA glycosylase appears to be normal in Bloom's syndrome lymphoblastoid cells.
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PMID:Normal uracil-DNA glycosylase activity in Bloom's syndrome cells. 290 71

A number of enzymes thought to be involved in DNA replication have been identified in the brain. These include single-stranded DNA-binding proteins, topoisomerases I and II, DNA polymerase alpha, a protein that binds Ap4A and might be classified as a DNA polymerase alpha accessory protein, RNase H, DNA polymerase beta, DNA ligase, an endo- and an exonuclease of unknown function, DNA methyl transferase and poly(ADPR) synthase. In contrast, little is known about the enzymology of DNA repair in brain. The few enzymes identified comprise uracil-DNA glycosylase, DNA polymerase beta, DNA polymerase alpha (which in neurons is present only at immature stages), DNA ligase, poly(ADPR) synthase, and O6-alkylguanine-DNA alkyltransferase. In addition, an exonuclease acting on depurinated single-stranded DNA (tentatively listed here as 3'----5' exonuclease), an endonuclease of unknown function as well as ill-defined acid and alkaline deoxyribonucleases also occur in brain.
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PMID:Enzymology of DNA replication and repair in the brain. 300 64

Activity of the DNA repair enzyme uracil-DNA glycosylase has been shown to increase in herpes simplex virus type 2 (HSV-2)-infected cells. When mRNA derived from either HSV-1- or HSV-2-infected HeLa S3 cells was translated in an in vitro translation system, significant uracil-DNA glycosylase activity could be detected in the lysate. This activity was specific for the removal of uracil from DNA. Lysates from in vitro translation of mRNA derived from uninfected HeLa cells did not contain measurable glycosylase activity. A cDNA library was constructed with mRNA derived from HSV-2-infected cells 10 h postinfection. Pooled isolates from this library were used in hybrid-arrest and in vitro translation reactions to isolate a uracil-DNA glycosylase-specific cDNA. In vitro translation of hybrid-selected RNA, by using this cDNA, produced glycosylase activity in the lysate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled products from this translation reaction showed a protein component with a molecular weight of 39,000. This is consistent with the molecular weight determinations of the purified glycosylase enzyme derived from either uninfected or HSV-infected HeLa cells. Northern (RNA blot) analysis of HSV-derived RNA, by using the glycosylase cDNA as a probe, revealed five overlapping transcripts of 3.4, 2.8, 2.4, 1.7, and 1.0 kilobases. Southern analysis indicated that the DNA sequence encoding the HSV-specific uracil-DNA glycosylase was located between 0.065 and 0.08 map units on the prototypic arrangement of the HSV genome.
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PMID:Isolation of a herpes simplex virus cDNA encoding the DNA repair enzyme uracil-DNA glycosylase. 304 Oct 25

The biosynthesis of the human DNA repair enzyme uracil-DNA glycosylase has been characterized by the reaction of in vitro- and in vivo-produced protein with an anti-human placental uracil-DNA glycosylase monoclonal antibody. In vitro synthesis of the DNA repair enzyme was examined after the translation of human placental polyadenylated [poly(A)+] RNA by immunoprecipitation of the [35S]methionine-labeled translation products. As defined by sucrose density analysis, immunoprecipitable in vitro products were translated from 16S poly(A)+ RNA and 11S poly(A)+ RNA. While the products of the 11S poly(A)+ RNA were smaller than purified uracil-DNA glycosylase, the product of the 16 S poly(A)+ RNA had a molecular weight of 37,000, identical to the size previously observed for purified human placental uracil-DNA glycosylase. Immunoblot analysis of human placental cell extracts and of normal human fibroblast cell extracts demonstrated the recognition of one Mr 37,000 protein. Immunoprecipitation of [35S]methionine-labeled normal human cell extracts with the anti-glycosylase monoclonal antibody specifically detected only the Mr 37,000 uracil-DNA glycosylase protein. Pulse-chase analysis demonstrated that the 35S radioactivity in the Mr 37,000 uracil-DNA glycosylase decreased over a 5-h interval. These results show that immunoreactive human uracil-DNA glycosylase protein was synthesized at its enzymatically active molecular weight of 37,000 as the primary translation product of a 16S polyadenylated messenger RNA.
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PMID:Biosynthesis of the human base excision repair enzyme uracil-DNA glycosylase. 379 Nov 99

Lipid peroxidation aldehydes of the 4-hydroxy-alpha, beta-unsaturated type, as well as the tobacco-smoke related alpha, beta-unsaturated aldehyde, acrolein, were highly cytotoxic and decreased the intracellular thiol content in cultured human bronchial fibroblasts after treatment with micromolar concentrations. In comparison, formaldehyde and acetaldehyde were less toxic and 100- to 300-fold higher doses were required to affect cell survival or thiol levels. The unsaturated aldehydes also markedly inhibited the DNA repair enzyme O6-methylguanine-DNA methyltransferase known to have a cysteine residue in its active site, but had no effect on the activity of uracil-DNA glycosylase. Our results indicate that reactive aldehydes of either exogenous or endogenous origin have direct cytotoxic effects and may also make cells more susceptible to other toxic chemicals due to an impairment in cellular defense mechanisms, e.g., DNA repair and detoxification by systems requiring glutathione.
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PMID:Cytotoxicity, thiol depletion and inhibition of O6-methylguanine-DNA methyltransferase by various aldehydes in cultured human bronchial fibroblasts. 406 50

Escherichia coli cells containing elevated levels of the DNA repair enzyme uracil-DNA glycosylase (the ung gene product) have been constructed by in vitro recombination methods. First, lambdanadB transducing phages were isolated from two E. coli DNA libraries by selection of nicotinate-independent lysogens. lambdanadB phage from one of the libraries were also ung+ and carried the ung-nadB genes on an 8.3-kb HindIII restriction fragment. The ung and nadB genes were subcloned into plasmids and a restriction map of the ung region of the E. coli chromosome was constructed. The uracil glycosylase gene was localized to a 1.4-kb restriction fragment by subcloning the gene into pBR322. Uracil glycosylase was overproduced (relative to the specific activity of wild type cells) by about two-fold in lambdaung lysogens and by 15- to 20-fold in cells containing pBR322ung derivatives. When the ung gene and its promoter were placed downstream from the bacteriophage lambdaPL promoter in the plasmid pKC30, uracil glycosylase production was heat-induced to more than 100-fold above the levels of a wild-type cell. By relating the insertion orientation of the lambdaung gene in the plasmid pKC30 to its orientation in lambdaung-nadB transducing phages, the transcription direction of the ung gene on the E. coli linkage map was found to be clockwise.
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PMID:The cloning and overproduction of Escherichia coli uracil-DNA glycosylase. 623 5

A rapid assay of O6-MeG-DNA methyltransferase activity is described. Following incubation of cell extracts with O6-[3H]MeG-containing DNA, remaining radioactive DNA was hydrolyzed in trichloroacetic acid and separated from methylated radioactive protein by filtration or centrifugation. Transfer of radioactive methyl from DNA to protein was proportional to the amount of protein added, and was not linear with time. More than 90% of the radioactivity precipitated after acid hydrolyses was in S-methyl cysteine residues. The method was used to measure O6-MeG-DNA methyltransferase activity in extracts of 24 neoplastic tissues from human organs. Although five tumor tissues had 28-84% lower activity of O6-MeG-DNA methyltransferase than the corresponding normal tissue from the same patient, higher or similar levels of activity were found more frequently. Thus, a lack of O6-MeG-DNA methyltransferase activity in human tumours appears not to be a frequent event. The DNA repair enzyme uracil-DNA glycosylase was also measured in the same extracts. Most frequently the level of uracil-DNA glycosylase activity was essentially similar in tumors and normal tissues but significantly higher or lower levels were also observed.
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PMID:A simplified assay for O6-methylguanine-DNA methyltransferase activity and its application to human neoplastic and non-neoplastic tissues. 674 14


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