Immunological alteration of the Bloom's syndrome uracil DNA glycosylase in Epstein-Barr virus-transformed human lymphoblastoid cells

The immunological reactivity of the uracil DNA glycosylase was investigated in three Epstein-Barr virus-transformed human lymphoblastoid cell lines. Two were derived from normal human lymphocytes while the third was derived from a Bloom's syndrome patient. A panel of 3 anti-human placental uracil DNA glycosylase monoclonal antibodies (37.04.12, 40.10.09 and 42.08.07) was used. Immunological reactivity was determined in a double-blind enzyme-linked immunosorbent assay (ELISA); by inhibition of enzyme activity; and by immunoblot analysis. In the ELISA, the glycosylase from each lymphoblastoid cell line was recognized by glycosylase antibodies 37.04.12 and 42.08.07. In contrast, antibody 40.10.09 failed to recognize the glycosylase from the Bloom's syndrome cell line. Further analysis demonstrated that the 40.10.09 antibody was unable to inhibit catalysis by the Bloom's syndrome lymphoblast glycosylase. In contrast, the 40.10.09 antibody inhibited the activity of the two normal human lymphoblast enzymes. Denaturation of the Bloom's syndrome lymphoblast glycosylase rendered that protein immunoreactive with the 40.10.09 antibody. These results demonstrated that: (1) the immunological alteration in the Bloom's syndrome uracil DNA glycosylase was detected in hematopoietic cells; and (2) viral transformation did not affect the immunoreactivity of the enzyme from either normal human or Bloom's syndrome cells.     

Excision repair of uracil in higher plant cells: Uracil-DNA glycosylase and sister-chromatid exchanges

Uracil is not a normal constituent of DNA. Under natural conditions, it may appear either by deamination of cytosine residues or by incorporation of deoxyuridine monophosphate (dUMP). Visible light irradiation of BrdUrd-treated cells efficiently leads, under experimental situations, to the formation of dUMP residues in DNA. Plant cells, like other living organisms, can eliminate this potentially harmful base from DNA by an excision repair pathway, uracil-DNA glycosylase being the first enzyme acting during the incision process. Purified plant uracil-DNA glycosylase is a low molecular weight enzyme (27–29.5 kD) that specifically releases uracil present in DNA by splitting off the sugar-base bond. This enzyme is non-competitively inhibited by uracil and 6-aminouracil, but not by thymine, both in vitro and in vivo. However, other structurally related compounds do not show any inhibitory effect. This characteristic poses a number of unaswered questions regarding its mechanism of action. At the chromosome level, dUMP residues appear to be sister-chromatid exchange (SCE)-initiating events. This has been demonstrated for dUMP residue introduced either by visible light exposure of BrdUrd-treated cells or by dUMP mis-incorporation instead of dTMP in cells treated with inhibitors of thymidylate synthetase. The excision repair of uracil in plants appears to be finely regulated in different cell types depending on their proliferation rate and their development stage. Thus, high levels of uracil-DNA glycosylase do not seem to be necessarily associated with DNA replication, since non-proliferating cells, natural constituents of dormant meristems, contain enzyme levels comparable to those found in proliferating tissues, where it is modulated: the higher the cell cycle rate (and the DNA replication rate) the higher the uracil-DNA glycosylase activity. Finally, this excision repair enzyme seems to be turned off as cells enter their differentiated state.     

Excision of deaminated cytosine from the vertebrate genome: role of the SMUG1 uracil-DNA glycosylase

Gene-targeted mice deficient in the evolutionarily conserved uracil-DNA glycosylase encoded by the UNG gene surprisingly lack the mutator phenotype characteristic of bacterial and yeast ung(-) mutants. A complementary uracil-DNA glycosylase activity detected in ung(-/-) murine cells and tissues may be responsible for the repair of deaminated cytosine residues in vivo. Here, specific neutralizing antibodies were used to identify the SMUG1 enzyme as the major uracil-DNA glycosylase in UNG-deficient mice. SMUG1 is present at similar levels in cell nuclei of non-proliferating and proliferating tissues, indicating a replication- independent role in DNA repair. The SMUG1 enzyme is found in vertebrates and insects, whereas it is absent in nematodes, plants and fungi. We propose a model in which SMUG1 has evolved in higher eukaryotes as an anti-mutator distinct from the UNG enzyme, the latter being largely localized to replication foci in mammalian cells to counteract de novo dUMP incorporation into DNA.     

Purification and properties of the uracil DNA glycosylase from Bloom's syndrome.

Bloom's syndrome uracil DNA glycosylase was highly purified from two non-transformed cell strains derived from individuals from different ethnic groups. Their properties were then compared to two different highly purified normal human uracil DNA glycosylases. A molecular mass of 37 kDa was observed for each of the four human enzymes as defined by gel-filtration column chromatography and by SDS-PAGE. Each of the 37 kDa proteins was identified as a uracil DNA glycosylase by electroelution from the SDS polyacrylamide gel, determination of glycosylase activity by in vitro biochemical assay and identification of the reaction product as free uracil by co-chromatography with authentic uracil. Bloom's syndrome enzymes differed substantially in their isoelectric point and were thermolabile as compared to the normal human enzymes. Bloom's syndrome enzymes displayed a different Km, Vmax and were strikingly insensitive to 5-fluorouracil and 5-bromouracil, pyrimidine analogues which drastically decreased the activity of the normal human enzymes. In particular, each Bloom's syndrome enzyme required 10-100-fold higher concentrations of each analogue to achieve comparable inhibition of enzyme activity. Potential mechanisms are considered through which an altered uracil DNA glycosylase characterizing this cancer-prone human genetic disorder may arise.     

Trypanosomes lacking uracil-DNA glycosylase are hypersensitive to antifolates and present a mutator phenotype

Cells contain low amounts of uracil in DNA which can be the result of dUTP misincorporation during replication or cytosine deamination. Elimination of uracil in the base excision repair pathway yields an abasic site, which is potentially mutagenic unless repaired. The Trypanosoma brucei genome presents a single uracil-DNA glycosylase responsible for removal of uracil from DNA. Here we establish that no excision activity is detected on U:G, U:A pairs or single-strand uracil-containing DNA in uracil-DNA glycosylase null mutant cell extracts, indicating the absence of back-up uracil excision activities. While procyclic forms can survive with moderate amounts of uracil in DNA, an analysis of the mutation rate and spectra in mutant cells revealed a hypermutator phenotype where the predominant events were GC to AT transitions and insertions. Defective elimination of uracil via the base excision repair pathway gives rise to hypersensitivity to antifolates and oxidative stress and an increased number of DNA strand breaks, suggesting the activation of alternative DNA repair pathways. Finally, we show that uracil-DNA glycosylase defective cells exhibit reduced infectivity in vivo demonstrating that efficient uracil elimination is important for survival within the mammalian host.     

Uracil in Ori<Subscript>s</Subscript> of herpes simplex 1 alters its specific recognition by origin binding protein (OBP): does virus induced uracil-DNA glycosylase play a key role in viral reactivation and replication?

We have recently demonstrated that mammalian uracil-DNA glycosylase activity is undetectable in adult neurons. On the basis of this finding we hypothesized that uracil, derived either from oxidative deamination of cytosine or misincorporation of dUMP in place of dTMP during DNA repair by the unique nuclear DNA polymerase present in adult neurons, DNA polymerase β, might accumulate in neuronal DNA. Uracil residues could also arise in the herpes simplex 1 (HSV1) genome during latency in nerve cells. We therefore suggest a role for the virus encoded uracil-DNA glycosylase in HSV1 reactivation and in the first steps of DNA replication. We show here 1) that the viral DNA polymerase incorporates dUTP in place of dTTP with a comparable efficiencyin vitro; 2) that virus specific DNA/protein interactions between the virus encoded origin binding protein and its target DNA sequence is altered by the presence of uracil residues in its central region TCGCA. Thus uracil, present in viral Ori_S or other key sequences could hamper the process leading to viral reactivation. Hence, HSV1 uracil-DNA glycosylase, dispensable in viral proliferation in tissue culture, could be essential in neurons for the “cleansing” of the viral genome of uracil residues before the start of replication.     

Bloom's syndrome: DNA replication in cultured fibroblasts and lymphocytes

Summary Analysis of DNA fiber autoradiograms from Bloom's syndrome skin fibroblasts and blood lymphocytes shows a retarded rate of replication fork movement compared to normal adult controls. Other measurements from the autoradiograms—replication unit length, incidence of bidirectional replication, and degree of initiation synchrony—are normal in Bloom's syndrome cells. These results suggest that a slow rate of fork movement is a specific manifestation of defective DNA synthesis in all Bloom's syndrome cells.     

Human uracil-DNA glycosylase complements E. coli ung mutants.

We have previously isolated a cDNA encoding a human uracil-DNA glycosylase which is closely related to the bacterial and yeast enzymes. In vitro expression of this cDNA produced a protein with an apparent molecular weight of 34 K in agreement with the size predicted from the sequence data. The in vitro expressed protein exhibited uracil-DNA glycosylase activity. The close resemblance between the human and the bacterial enzyme raised the possibility that the human enzyme may be able to complement E. coli ung mutants. In order to test this hypothesis, the human uracil-DNA glycosylase cDNA was established in a bacterial expression vector. Expression of the human enzyme as a LacZ alpha-humUNG fusion protein was then studied in E. coli ung mutants. E. coli cells lacking uracil-DNA glycosylase activity exhibit a weak mutator phenotype and they are permissive for growth of phages with uracil-containing DNA. Here we show that the expression of human uracil-DNA glycosylase in E. coli can restore the wild type phenotype of ung mutants. These results demonstrate that the evolutionary conservation of the uracil-DNA glycosylase structure is also reflected in the conservation of the mechanism for removal of uracil from DNA.     

Mutations in active-site residues of the uracil-DNA glycosylase encoded by vaccinia virus are incompatible with virus viability.

The D4R gene of vaccinia virus encodes a functional uracil-DNA glycosylase that is essential for viral viability (D. T. Stuart, C. Upton, M. A. Higman, E. G. Niles, and G. McFadden, J. Virol. 67:2503-2513, 1993), and a D4R mutant, ts4149, confers a conditional lethal defect in viral DNA replication (A. K. Millns, M. S. Carpenter, and A. M. DeLange, Virology 198:504-513, 1994). The mutant ts4149 protein was expressed in vitro and assayed for uracil-DNA glycosylase activity. Less than 6% of wild-type activity was observed at permissive temperatures, but the ts4149 protein was completely inactive at the nonpermissive temperature. Mutagenesis of the ts4149 gene back to wild type (Arg-179-->Gly) restored full activity. The ts4149 protein was considerably reduced in lysates of cells infected at the permissive temperature, and its activity was undetectable, even in the presence of the uracil glycosylase inhibitor protein, which inhibits the host uracil-DNA glycosylases but not that of vaccinia virus. Thus the ts4149 protein is thermolabile, correlating uracil removal with vaccinia virus DNA replication. Three active-site amino acids of the vaccinia virus uracil-DNA glycosylase were mutated (Asp-68-->Asn, Asn-120-->Val, and His-181-->Leu), producing proteins that were completely defective in uracil excision but still retained the ability to bind DNA. Each mutated D4R gene was transfected into vaccinia virus ts4149-infected cells in order to assess the recombination events that allowed virus survival at 40 degrees C. Genetic analysis and sequencing studies revealed that the only viruses to survive were those in which recombination eliminated the mutant locus. We conclude that the uracil cleavage activity of the D4R protein is essential for its function in vaccinia virus DNA replication, suggesting that the removal of uracil residues plays an obligatory role.     

Post-replicative base excision repair in replication foci.

Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication. We report that the major nuclear uracil-DNA glycosylase (UNG2) increases in S phase, during which it co-localizes with incorporated BrdUrd in replication foci. Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei. Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uracil-DNA glycosylase responsible. PCNA and replication protein A (RPA) co-localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two-hybrid assays, a peptide SPOT assay and enzyme-linked immunosorbent assays. These results demonstrate rapid post-replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork.     

hSMUG1 can functionally compensate for Ung1 in the yeast Saccharomyces cerevisiae

There are at least four distinct families of enzymes that recognize and remove uracil from DNA. Family-3 (SMUG1) enzymes have recently been identified and have a preference for uracil in single-stranded DNA when assayed in vitro. Here we investigate the in vivo function of SMUG1 using the yeast Saccharomyces cerevisiae as a model system. These organisms lack a SMUG1 homologue and use a single enzyme, Ung1 to carry out uracil-repair. When a wild-type strain is treated with antifolate agents to induce uracil misincorporation into DNA, S-phase arrest and cellular toxicity occurs. The arrest is characteristic of checkpoint activation due to single-strand breaks caused by continuous uracil removal and self-defeating DNA repair. When uracil-DNA glycosylase is deleted (deltaung1), cells continue through S-phase and arrest at G(2)/M, presumably due to the effects of stable uracil misincorporation in DNA. Pulsed field gel electrophoresis (PFGE) demonstrates that cells are able to complete DNA replication with uracil-substituted DNA and do not experience the extensive strand breakage attributed to uracil-DNA glycosylase-mediated repair. As a result, these cells experience early protection from antifolate-induced cytotoxicity. When either UNG1 or SMUG1 functions are reintroduced back into the null strain and then subjected to antifolate treatment, the cells revert back to the wild-type phenotype as shown by a restored sensitivity to drug and S-phase arrest. The arrest is accompanied by the accumulation of replication intermediates as determined by PFGE. Collectively, these data indicate that SMUG1 can act as a functional homolog of the family-1 uracil-DNA glycosylase enzymes.     

Uracil-DNA glycosylase inhibitor of bacteriophage PBS2: cloning and effects of expression of the inhibitor gene in Escherichia coli.

The uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 was cloned, and the effects of this inhibitor on Escherichia coli cells that contain uracil-DNA glycosylase activity were determined. A PBS2 genomic library was constructed by inserting EcoRI restriction fragments of PBS2 DNA into a plasmid pUC19 vector. The library was used to transform wild-type (ung+) E. coli, and the presence of the functional inhibitor gene was determined by screening for colonies that supported growth of M13mp19 phage containing uracil-DNA. A clone was identified that carried a 4.1-kilobase EcoRI DNA insert in the vector plasmid. Extracts of cells transformed with this recombinant plasmid lacked detectable uracil-DNA glycosylase activity and contained a protein that inhibited the activity of purified E. coli uracil-DNA glycosylase in vitro. The uracil-DNA glycosylase inhibitor expressed in these E. coli was partially purified and characterized as a heat-stable protein with a native molecular weight of about 18,000. Hence, we conclude that the PBS2 uracil-DNA glycosylase inhibitor gene was cloned and that the gene product has properties similar to those from PBS2-infected Bacillus subtilis cells. Inhibitor gene expression in E. coli resulted in (i) a weak mutator phenotype, (ii) a growth rate similar to that of E. coli containing pUC19 alone, (iii) a sensitivity to the antifolate drug aminopterin similar to that of cells lacking the inhibitor gene, and (iv) an increased resistance to the lethal effects of 5-fluoro-2'-deoxyuridine. These physiological properties are consistent with the phenotypes of other ung mutants.     

Uracil-DNA glycosidase studied with an immune serum

Immune antiserum to uracil-DNA glycosylase was obtained by immunizing rabbits with an enzyme isolated from the rat liver. Antiserum was found to suppress the activity of uracil-DNA glycosylase not only in the extracts of rat liver, but also in the extracts of brain, cardiac muscle, kidney, spleen, thymus of rats, and in those of human placenta too. This enables us to make a conclusion about the similarity in antigenic properties of the enzyme in cells of various types of differentiation. Indirect immunofluorescent test shows a slight staining of the periphery of the nucleus in normal liver hepatocytes and the intensive staining of the inner part of the nucleus in hepatocytes of regenerating liver. Therefore it is concluded that the enzymatic activity increases as cells proliferate. This may be the result of the appearance of uracil in DNA during replication.     

Excision of uracil from bromodeoxyuridine-substituted and U.V.-irradiated DNA in cultured mouse lymphoma cells.

A uracil-DNA glycosylase activity was detected in cell-free extracts from cultured mouse lymphoma L5178 cells. We investigated whether or not this enzyme plays a role in the removal of uracil from chromosomal DNA. U.V. light (254nm) irradiation of the cells with BUdR-substituted DNA produced not only single-strand breaks but also 'internal' uracil residues that were recognized as substrate sites by uracil-DNA glycosylase. These 'internal' uracil residues were lost from the DNA upon reincubation of the irradiated cells. The product released from the DNA was identified as uracil. Thus, the intracellular action of the uracil-DNA glycosylase was demonstrated and the subsequent reconstitution of the DNA strand was inferred in cultured mammalian cells.     

Uracil-DNA glycosylase in insects. Drosophila and the locust

It has been reported that Drosophila lacks a uracil-DNA glycosylase but that a direct incising activity on uracil-containing DNA appeared developmentally only in third instar larvae. In contrast we have found by two independent assays, that uracil-DNA glycosylase exists in both Drosophila eggs as well as in third instar larvae. The first assay shows the liberation of [3H] uracil from a d(AT)n polymer randomly substituted with [3H]uracil by its synthesis in the presence of [3H] dUTP. The second fluorometric assay for uracil-DNA glycosylase depends on the unique topological properties of circular DNAs and has the advantage of detecting apyrimidinic/apurinic (AP) endonuclease activity as well. To test one other insect, locust eggs were also assayed for uracil-DNA glycosylase. The amount of uracil-DNA glycosylase correlated well with the amount of DNA in actively replicating cells.     

Isolation of insertion, deletion, and nonsense mutations of the uracil-DNA glycosylase (ung) gene of Escherichia coli K-12.

Two uracil-DNA glycosylase (ung) mutation selection procedures based upon the ability of uracil glycosylase to degrade the chromosomes of organisms containing uracil-DNA were devised to obtain a collection of well-defined ung alleles. In an enrichment procedure, lysogens were selected from Escherichia coli cultures infected with lambda pKanr phage containing uracil in their DNA. (These uracil-DNA phage were prepared by growth on host cells deficient in both dUTPase and uracil-DNA glycosylase.) The lysogenic Kanr population was enriched for uracil glycosylase-deficient mutants by a factor of 10(4). In a phage suicide selection procedure, lambda pung+ phage were unable to form plaques on dut ung cells containing uracil-DNA in their chromosomes, and all of the progeny were lambda pung-. Deletion, insertion (ung::Mu and ung::Tn10), nonsense, and missense mutants were isolated by using these procedures. Extracts of three insertion mutants contained no detectable enzyme activity. All of the other mutant isolates had less than 1% of the normal uracil glycosylase specific activity. The previously studied ung-1 allele, which was derived by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, produced about 0.02% of the normal amount of uracil glycosylase activity. No significant phenotypic differences between ung-1 and ung::Tn10 alleles were observed. Variations of the lysogen selection procedure may be helpful for isolating other DNA glycosylase mutations in E. coli and other organisms.     

Reduced 5-hydroxymethyluracil-DNA glycosylase activity in Werner's syndrome cells.

Werner's syndrome (WS) is an autosomal recessive disease marked by early symptoms of accelerated aging. There is evidence indicating accumulation of oxidized DNA bases to be a major factor in cellular aging. The first step of excision repair of such bases in human cells is their removal from DNA by glycosylases. 5-Hydroxymethyluracil (HMU)-DNA glycosylase excises HMU from DNA; another glycosylase removes many non-aromatic pyrimidine derivatives. Levels of glycosylases that excise oxidized pyrimidines from DNA were compared between confluent and proliferating populations of WS cells, age-matched controls, and young control cells. They were assayed by measurements of direct release of free bases from their respective DNA substrates. Specific activities of the glycosylase that releases various modified pyrimidines and of uracil-DNA glycosylase (which removes uracil from DNA) were essentially the same in all cell lines. Cell cycle variations of these enzymes also did not differ between WS and control cells. HMU-DNA glycosylase specific activity was reduced in WS cells. Reduction of HMU-DNA glycosylase has been described in senescent human WI-38 cells. Therefore, while neither WS nor senescent cells have overall deficiencies of DNA glycosylase activities, they both might have reduced excision of HMU from DNA. This indicates a possible role of HMU accumulation in the aging process.