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)

Uracil-DNA-glycosidase that releases free uracil from single-stranded or double-stranded deaminated DNA and poly d(A-U) has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 16,000 and can be separated from uracil-endonuclease and endonucleases (AP-endonucleases) specific for apurinic and apyrimidinic sites. Uracil-DNA-glycosidase does not act on guanine residues opposite uracil in double-stranded DNA and on xanthine in deaminated DNA. The glycosidase generates apyrimidinic sites which can serve as substrate sites for different AP-endonucleases from M. luteus.
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PMID:Enzymes from Micrococcus luteus involved in the initial steps of excision repair of spontaneous DNA lesions: uracil-DNA-glycosidase and apurinic-endonucleases. 65 27

Ultraviolet irradiation of poly(dG-dC) and poly(dA-dU) in solution produces pyrimidine hydrates that are repaired by bacterial and mammalian DNA glycosylases [Boorstein et al. (1989) Biochemistry 28, 6164-6170]. Escherichia coli endonuclease III was used to quantitate the formation and stability of these hydrates in the double-stranded alternating copolymers poly(dG-dC) and poly(dA-dU). When poly(dG-dC) was irradiated with 100 kJ/m2 of 254-nm light at pH 8.0, 2.2% of the cytosine residues were converted to cytosine hydrate (6-hydroxy-5,6-dihydrocytosine) while 0.09% were converted to uracil hydrate (6-hydroxy-5,6-dihydrouracil). To measure the stability of these products, poly(dG-dC) was incubated in solution for up to 24 h after UV irradiation. Cytosine hydrate was stable at 4 degrees C and decayed at 25, 37, and 55 degrees C with half-lives of 75, 25, and 6 h. Uracil hydrate produced in irradiated poly(dA-dU) was stable at 4 degrees C and at 25 degrees C and decayed with a half-life of 6 h at 37 degrees C and less than 0.5 h at 55 degrees C. Uracil hydrate and uracil were also formed in irradiated poly(dG-dC). These experiments demonstrate that UV-induced cytosine hydrate may persist in DNA for prolonged time periods and also undergo deamination to uracil hydrate, which in turn undergoes dehydration to yield uracil. The formation and stability of these photoproducts in DNA may have promoted the evolutionary development of the repair enzyme endonuclease III and analogous DNA glycosylase/endonuclease activities of higher organisms, as well as the development of uracil-DNA glycosylase.
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PMID:Formation and stability of repairable pyrimidine photohydrates in DNA. 227 56

Although multiple chromosomes occur in bacteria, much remains to be learned about their structural and functional interrelationships. To study the structure-function relationships of chromosomes I and II of the facultative photosynthetic bacterium Rhodobacter sphaeroides 2.4.1T, auxotrophic mutants were isolated. Five strains having transposon insertions in chromosome II showed requirements for p-aminobenzoic acid (pABA)-dihydroxybenzoic acid (dHBA), serine, thymine, uracil, or histidine. The His, Thy, and pABA-dHBA mutants reverted to prototrophy at low frequency and concordantly lost their transposon insertions from the genome. The Ser, Ura, and pABA-dHBA mutants were complemented by cosmids that carried the region of chromosome II where the transposon insertions were located. The cosmids used for complementation analysis were selected, on the basis of map position, from a set of overlapping clones that had been ordered by a combination of hybridization and restriction endonuclease mapping. These experiments provide the basis for detailed studies of the structure, function, and interaction between each chromosome, and they demonstrate at this early stage of investigation that no fundamental differences exist between each chromosome.
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PMID:Multiple chromosomes in bacteria: structure and function of chromosome II of Rhodobacter sphaeroides 2.4.1T. 800 95

Genetic manipulation of Candida albicans is constrained by its diploid genome and asexual life cycle. Recessive mutations are not expressed when heterozygous and undesired mutations introduced in the course of random mutagenesis cannot be removed by genetic back-crossing. To circumvent these problems, we developed a genotypic screen that permitted identification of a heterozygous recessive mutation at the URA3 locus. The mutation was introduced by targeted mutagenesis, homologous integration of transforming DNA, to avoid introduction of extraneous mutations. The ura3 mutation was rendered homozygous by a second round of transformation resulting in a Ura- strain otherwise isogenic with the parental clinical isolate. Subsequent mutation of the Ura- strain was achieved by targeted mutagenesis using the URA3 gene as a selectable marker. URA3 selection was used repeatedly for the sequential introduction of mutations by flanking the URA3 gene with direct repeats of the Salmonella typhimurium hisG gene. Spontaneous intrachromosomal recombination between the flanking repeats excised the URA3 gene restoring a Ura- phenotype. These Ura- segregants were selected on 5-fluoroorotic acid-containing medium and used in the next round of mutagenesis. To permit the physical mapping of disrupted genes, the 18-bp recognition sequence of the endonuclease I-SceI was incorporated into the hisG repeats. Site-specific cleavage of the chromosome with I-SceI revealed the position of the integrated sequences.
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PMID:Isogenic strain construction and gene mapping in Candida albicans. 834 5

Previously, we reported that histone H1 binding to nucleosome cores results in the repression of binding of the basic helix-loop-helix upstream stimulatory factor (USF) (Juan, L.-J., Utley, R. T., Adams, C. C., Vettese-Dadey, M., and Workman, J. L. (1994) EMBO J. 13, 6031-6040). We have tested whether this inhibition resulted from H1-mediated changes in nucleosome positioning (Ura, K., Hayes, J. J., and Wolffe, A. P. (1995) EMBO J. 14, 3752-3765) forcing the USF recognition sequence into less accessible locations within the nucleosome. Nucleosome boundaries were determined by assays combining micrococcal nuclease and restriction endonuclease digestion. A unique pair of boundaries were observed, indicating a single nucleosome translational position. This nucleosome position did not change on H1 or USF binding. Thus, H1 repression of USF binding was independent of nucleosome mobility, indicating an alternative mechanism of H1 repression. H1 repressed USF binding at a site 35 base pairs into the nucleosome core more effectively than at a site near the "linker" DNA, suggesting that inhibition by H1 was not simply due to steric occlusion. Instead, these data are consistent with a model by which H1 binding reduces transient dynamic exposure of the DNA from the histone octamer surface (Polach, K. L., and Widom, J. (1995) J. Mol. Biol. 254, 130-149).
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PMID:H1-mediated repression of transcription factor binding to a stably positioned nucleosome. 901 16

Uracil can arise in DNA by misincorporation of dUTP into nascent DNA and/or by cytosine deamination in established DNA. Based on recent findings, both pathways appear to be promoted in the methyl-deficient model of hepatocarcinogenesis. A chronic increase in the ratio dUTP:dTTP with folate/methyl deficiency can result in a futile cycle of excision and reiterative uracil misincorporation leading to premutagenic apyrimidinic (AP) sites, DNA strand breaks, DNA fragmentation and apoptotic cell death. The progressive accumulation of unmethylated cytosines with chronic methyl deficiency will increase the potential for cytosine deamination to uracil and further stress uracil mismatch repair mechanisms. Uracil is removed by a highly specific uracil-DNA glycosylase (UDG) leaving an AP site that is subsequently repaired by sequential action of AP endonuclease, 5'-phosphodiesterase, a DNA polymerase and DNA ligase. Since the DNA polymerases cannot distinguish between dUTP and dTTP, an increase in dUTP:dTTP ratio will promote uracil misincorporation during both DNA replication and repair synthesis. The misincorporation of uracil for thymine (5-methyluracil) may constitute a genetically significant form of DNA hypomethylation distinct from cytosine hypomethylation. In the present study a significant increase in the level of uracil in liver DNA as early as 3 weeks after initiation of folate/methyl deficiency was accompanied by parallel increases in DNA strand breaks, AP sites and increased levels of AP endonuclease mRNA. In addition, uracil was also detected within the p53 gene sequence using UDG PCR techniques. Increased levels of uracil in DNA implies that the capacity for uracil base excision repair is exceeded with chronic folate/methyl deficiency. It is possible that enzyme-induced extrahelical bases, AP sites and DNA strand breaks interact to negatively affect the stability of the DNA helix and stress the structural limits of permissible uracil base excision repair activity. Thus substitution of uracil for thymine induces repair-related premutagenic lesions and a novel form of DNA hypomethylation that may relate to tumor promotion in the methyl-deficient model of hepatocarcinogenesis.
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PMID:Presence and consequence of uracil in preneoplastic DNA from folate/methyl-deficient rats. 939 4

Endonuclease V (deoxyinosine 3' endonuclease), the product of the nfi gene, has a specificity that encompasses DNAs containing dIMP, abasic sites, base mismatches, uracil, and even untreated single-stranded DNA. To determine its importance in DNA repair pathways, nfi insertion mutants and overproducers (strains bearing nfi plasmids) were constructed. The mutants displayed a twofold increase in spontaneous mutations for several markers and an increased sensitivity to killing by bleomycin and nitrofurantoin. An nfi mutation increased both cellular resistance to and mutability by nitrous acid. This agent should generate potential cleavage sites for the enzyme by deaminating dAMP and dCMP in DNA to dIMP and dUMP, respectively. Relative to that of a wild-type strain, an nfi mutant displayed a 12- to 1,000-fold increase in the frequency of nitrite-induced mutations to streptomycin resistance, which are known to occur in A x T base pairs. An nfi mutation also enhanced the lethality caused by a combined deficiency of exonuclease III and dUTPase, which has been attributed to unrepaired abasic sites. However, neither the deficiency nor the overproduction of endonuclease V affected the growth of the single-stranded DNA phages M13 or phiX174 nor of Uracil-containing bacteriophage lambda. These results suggest that endonuclease V has a significant role in the repair of deaminated deoxyadenosine (deoxyinosine) and abasic sites in DNA, but there was no evidence for its cleavage in vivo of single-stranded or uracil-containing DNA.
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PMID:Endonuclease V (nfi) mutant of Escherichia coli K-12. 942 91

The error frequency of uracil-initiated base excision repair (BER) DNA synthesis in human and Escherichia coli cell-free extracts was determined by an M13mp2 lacZ alpha DNA-based reversion assay. Heteroduplex M13mp2 DNA was constructed that contained a site-specific uracil target located opposite the first nucleotide position of opal codon 14 in the lacZ alpha gene. Human glioblastoma U251 and colon adenocarcinoma LoVo whole-cell extracts repaired the uracil residue to produce form I DNA that was resistant to subsequent in vitro cleavage by E. coli uracil-DNA glycosylase (Ung) and endonuclease IV, indicating that complete uracil-initiated BER repair had occurred. Characterization of the BER reactions revealed that (1) the majority of uracil-DNA repair was initiated by a uracil-DNA glycosylase-sensitive to Ugi (uracil-DNA glycosylase inhibitor protein), (2) the addition of aphidicolin did not significantly inhibit BER DNA synthesis, and (3) the BER patch size ranged from 1 to 8 nucleotides. The misincorporation frequency of BER DNA synthesis at the target site was 5.2 x 10(-4) in U251 extracts and 5.4 x 10(-4) in LoVo extracts. The most frequent base substitution errors in the U251 and LoVo mutational spectrum were T to G > T to A >> T to C. Uracil-initiated BER DNA synthesis in extracts of E. coli BH156 (ung) BH157 (dug), and BH158 (ung, dug) was also examined. Efficient BER occurred in extracts of the BH157 strain with a misincorporation frequency of 5.6 x 10(-4). A reduced, but detectable level of BER was observed in extracts of E. coli BH156 cells; however, the mutation frequency of BER DNA synthesis was elevated 6.4-fold.
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PMID:Uracil-initiated base excision DNA repair synthesis fidelity in human colon adenocarcinoma LoVo and Escherichia coli cell extracts. 1155 95

Deamination of DNA bases can occur spontaneously, generating highly mutagenic lesions such as uracil, hypoxanthine, and xanthine. When cells are under oxidative stress that is induced either by oxidizing agents or by mitochondrial dysfunction, additional deamination products such as 5-hydroxymethyluracil (5-HMU) and 5-hydroxyuracil (5-OH-Ura) are formed. The cellular level of these highly mutagenic lesions is increased substantially when cells are exposed to DNA damaging agent, such as ionizing radiation, redox reagents, nitric oxide, and others. The cellular repair of deamination products is predominantly through the base excision repair (BER) pathway, a major cellular repair pathway that is initiated by lesion specific DNA glycosylases. In BER, the lesions are removed by the combined action of a DNA glycosylase and an AP endonuclease, leaving behind a one-base gap. The gapped product is then further repaired by the sequential action of DNA polymerase and DNA ligase. DNA glycosylases that recognize uracil, 5-OH-Ura, 5-HMU (derived from 5-methylcytosine) and a T/G mismatch (derived from a 5-methylcytosine/G pair) are present in most cells. Many of these glycosylases have been cloned and well characterized. In yeast and mammalian cells, hypoxanthine is efficiently removed by methylpurine N-glycosylase, and it is thought that BER might be an important pathway for the repair of hypoxanthine. In contrast, no glycosylase that can recognize xanthine has been identified in either yeast or mammalian cells. In Escherichia coli, the major enzyme activity that initiates the repair of hypoxanthine and xanthine is endonuclease V. Endonuclease V is an endonuclease that hydrolyzes the second phosphodiester bond 3' to the lesion. It is hypothesized that the cleaved DNA is further repaired through an alternative excision repair (AER) pathway that requires the participation of either a 5' endonuclease or a 3'-5' exonuclease to remove the damaged base. The repair process is then completed by the sequential actions of DNA polymerase and DNA ligase. Endonuclease V sequence homologs are present in all kingdoms, and it is conceivable that endonuclease V might also be a major enzyme that initiates the repair of hypoxanthine and xanthine in mammalian cells.
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PMID:Repair of deaminated bases in DNA. 1236

Uracil DNA glycosylase (UDG), a ubiquitous and highly specific enzyme, commences the uracil excision repair pathway. Structural studies have shown that the tyrosine in a highly conserved GQDPY water-activating loop of UDGs blocks the entry of thymine or purines into the active site pocket. To further understand the role of this tyrosine (Y66 in Escherichia coli UDG), we have overproduced and characterized Y66F, Y66H, Y66L and Y66W mutants. The complexes of the wild-type, Y66F, Y66H and Y66L UDGs with uracil DNA glycosylase inhibitor (Ugi) (a proteinaceous substrate mimic) were stable to 8 M urea. However, some dissociation of the complex involving the Y66W UDG occurred at this concentration of urea. The catalytic efficiencies (V(max) / K(m)) of the Y66L and Y66F mutants were similar to those of the wild-type UDG. However, the Y66W and Y66H mutants were approximately 7- and approximately 173-fold compromised, respectively, in their activities. Interestingly, the Y66W mutation has resulted in an enzyme which is resistant to product inhibition. Preferential utilization of a substrate enabling a long range contact between the -5 phosphate (upstream to the scissile uracil) and the enzyme, and the results of modeling studies showing that the uracil-binding cavity of Y66W is wider than those of the wild type and other mutant UDGs, suggest a weaker interaction between uracil and the Y66W mutant. Furthermore, the fluorescence spectroscopy of UDGs and their complexes with Ugi, in the presence of uracil or its analog, 5-bromouracil, suggests compromised binding of uracil in the active site pocket of the Y66W mutant. Lack of inhibition of the Y66W UDG by apyrimidinic DNA (AP-DNA) is discussed to highlight a potential additional role of Y66 in shielding the toxic effects of AP-DNA, by lowering the rate of its release for subsequent recognition by an AP endonuclease.
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PMID:Substitutions at tyrosine 66 of Escherichia coli uracil DNA glycosylase lead to characterization of an efficient enzyme that is recalcitrant to product inhibition. 1465 97


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