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.     

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.     

Vaccinia virus uracil DNA glycosylase has an essential role in DNA synthesis that is independent of its glycosylase activity: catalytic site mutations reduce virulence but not virus replication in cultured cells

Previous findings that the vaccinia virus uracil DNA glycosylase is required for virus DNA replication, coupled with an inability to isolate a mutant with an active site substitution in the glycosylase gene, were surprising, as such enzymes function in DNA repair and bacterial, yeast, and mammalian null mutants are viable. To further study the role of the viral protein, we constructed recombinant vaccinia viruses with single or double mutations (D68N and H181L) in the uracil DNA glycosylase conserved catalytic site by using a complementing cell line that constitutively expresses the viral enzyme. Although these mutations abolished uracil DNA glycosylase activity, they did not prevent viral DNA replication or propagation on a variety of noncomplementing cell lines or human primary skin fibroblasts. In contrast, replication of a uracil DNA glycosylase deletion mutant occurred only in the complementing cell line. Therefore, the uracil DNA glycosylase has an essential role in DNA replication that is independent of its glycosylase activity. Nevertheless, the conservation of the catalytic site in all poxvirus orthologs suggested an important role in vivo. This idea was confirmed by the decreased virulence of catalytic-site mutants when administered by the intranasal route to mice.     

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.     

Cloning and expression in Escherichia coli of a gene for an alkylbase DNA glycosylase from Saccharomyces cerevisiae; a homologue to the bacterial alkA gene.

An alkylation repair deficient mutant of Escherichia coli (tag ada), lacking DNA glycosylase activity for removal of alkylated bases, was transformed by a genomic yeast DNA library and clones selected which survived plating on medium containing the alkylating agent methylmethane sulphonate. Three distinct yeast clones were identified which were able to suppress the alkylation sensitive phenotype of the bacterial mutant. Restriction enzyme analysis revealed common DNA fragments present in all three clones spanning 2 kb of yeast DNA. DNA from this region was sequenced and analysed for possible translation of polypeptides with any homology to either the Tag or the AlkA DNA glycosylases of E. coli. One open reading frame of 296 amino acids was identified encoding a putative protein with significant homology to AlkA. DNA containing the open reading frame was subcloned in E. coli expression vectors and cell extracts assayed for alkylbase DNA glycosylase activity. It appeared that such activity was expressed at levels sufficiently high for enzyme purification. The molecular weight of the purified protein was determined by SDS-PAGE to be 35,000 daltons, in good agreement with the 34,340 value calculated from the sequence. The yeast enzyme was able to excise 7-methylguanine as well as 3-methyladenine from dimethyl sulphate treated DNA, confirming the related nature of this enzyme to the AlkA DNA glycosylase from E. coli.     

Effect of the thymidylate synthase inhibitors on dUTP and TTP pool levels and the activities of DNA repair glycosylases on uracil and 5-fluorouracil in DNA

5-Fluorouracil (5-FU), 5-fluorodeoxyuridine (5-dUrd), and raltitrixed (RTX) are anticancer agents that target thymidylate synthase (TS), thereby blocking the conversion of dUMP into dTMP. In budding yeast, 5-FU promotes a large increase in the dUMP/dTMP ratio leading to massive polymerase-catalyzed incorporation of uracil (U) into genomic DNA, and to a lesser extent 5-FU, which are both excised by yeast uracil DNA glycosylase (UNG), leading to DNA fragmentation and cell death. In contrast, the toxicity of 5-FU and RTX in human and mouse cell lines does not involve UNG, but, instead, other DNA glycosylases that can excise uracil derivatives. To elucidate the basis for these divergent findings in yeast and human cells, we have investigated how these drugs perturb cellular dUTP and TTP pool levels and the relative abilities of three human DNA glycosylases (hUNG2, hSMUG1, and hTDG) to excise various TS drug-induced lesions in DNA. We found that 5-dUrd only modestly increases the dUTP and dTTP pool levels in asynchronous MEF, HeLa, and HT-29 human cell lines when growth occurs in standard culture media. In contrast, treatment of chicken DT40 B cells with 5-dUrd or RTX resulted in large increases in the dUTP/TTP ratio. Surprisingly, even though UNG is the only DNA glycosylase in DT40 cells that can act on U·A base pairs derived from dUTP incorporation, an isogenic ung(-/-) DT40 cell line showed little change in its sensitivity to RTX as compared to control cells. In vitro kinetic analyses of the purified human enzymes show that hUNG2 is the most powerful catalyst for excision of 5-FU and U regardless of whether it is found in base pairs with A or G or present in single-stranded DNA. Fully consistent with the in vitro activity assays, nuclear extracts isolated from human and chicken cell cultures show that hUNG2 is the overwhelming activity for removal of both U and 5-FU, despite its bystander status with respect to drug toxicity in these cell lines. The diverse outcomes of TS inhibition with respect to nucleotide pool levels, the nature of the resulting DNA lesion, and the DNA repair response are discussed.     

Increased spontaneous mutation frequency in human cells expressing the phage PBS2-encoded inhibitor of uracil-DNA glycosylase

The Ugi protein inhibitor of uracil-DNA glycosylase encoded by bacteriophage PBS2 inactivates human uracil-DNA glycosylases (UDG) by forming a tight enzyme:inhibitor complex. To create human cells that are impaired for UDG activity, the human glioma U251 cell line was engineered to produce active Ugi protein. In vitro assays of crude cell extracts from several Ugi-expressing clonal lines showed UDG inactivation under standard assay conditions as compared to control cells, and four of these UDG defective cell lines were characterized for their ability to conduct in vivo uracil-DNA repair. Whereas transfected plasmid DNA containing either a U:G mispair or U:A base pairs was efficiently repaired in the control lines, uracil-DNA repair was not evident in the lines producing Ugi. Experiments using a shuttle vector to detect mutations in a target gene showed that Ugi-expressing cells exhibited a 3-fold higher overall spontaneous mutation frequency compared to control cells, due to increased C:G to T:A base pair substitutions. The growth rate and cell cycle distribution of Ugi-expressing cells did not differ appreciably from their parental cell counterpart. Further in vitro examination revealed that a thymine DNA glycosylase (TDG) previously shown to mediate Ugi-insensitive excision of uracil bases from DNA was not detected in the parental U251 cells. However, a Ugi-insensitive UDG activity of unknown origin that recognizes U:G mispairs and to a lesser extent U:A base pairs in duplex DNA, but which was inactive toward uracil residues in single-stranded DNA, was detected under assay conditions previously shown to be efficient for detecting TDG.     

Characterization of an 8-oxoguanine DNA glycosylase from Methanococcus jannaschii.

A thermostable 8-oxoguanine (oxoG) DNA glycosylase from Methanococcus jannaschii has been expressed in Escherichia coli, purified, and characterized. The enzyme, which has been named mjOgg, belongs to the same diverse DNA glycosylase superfamily as the 8-oxoguanine DNA glycosylases from yeast (yOgg1) and human (hOgg1) but is substantially different in sequence. In addition, unlike its eukaryotic counterparts, which have a strong preference for oxoG.C base pairs, mjOgg has little specificity for the base opposite oxoG. mjOgg has both DNA glycosylase and DNA lyase (beta-elimination) activity, and the combined glycosylase/lyase activity occurs at a rate comparable with the glycosylase activity alone. Mutation of Lys-129, analogous to Lys-241 of yOgg1, abolishes glycosylase activity.     

Immunofluorescent localization of the murine 8-oxoguanine DNA glycosylase (mOGG1) in cells growing under normal and nutrient deprivation conditions

OGG1 is a major DNA glycosylase in mammalian cells, participating in the repair of 7,8-dihydro-8-oxoguanine (8-oxoguanine, 8-oxoG), the most abundant known DNA lesion induced by endogenous reactive oxygen species in aerobic organisms. 8-oxoG is therefore often used as a marker for oxidative DNA damage. In this study, polyclonal and monoclonal antibodies were raised against the purified wild-type recombinant murine 8-oxoG DNA glycosylase (mOGG1) protein and their specificity against the native enzyme and the SDS-denatured mOGG1 polypeptide were characterized. Specific antibodies directed against the purified wild-type recombinant mOGG1 were used to localize in situ this DNA repair enzyme in established cell lines (HeLa cells, NIH3T3 fibroblasts) as well as in primary culture mouse embryo fibroblasts growing under either normal or oxidative stress conditions. Results from these studies showed that mOGG1 is localized to the nucleus and the cytoplasm of mammalian cells in culture. However, mOGG1 levels increase and primarily redistribute to the nucleus and its peripheral cytoplasm in cells exposed to oxidative stress conditions. Immunofluorescent localization results reported in this study suggest that susceptibility to oxidative DNA damage varies among mammalian tissue culture cells and that mOGG1 appears to redistribute once mOGG1 cell copy number increases in response to oxidative DNA damage.     

Murine Wnt-1 with an internal c-myc tag recombinantly produced in Escherichia coli can induce intracellular signaling of the canonical Wnt pathway in eukaryotic cells

Wnt-1 belongs to the Wnt family of secreted glycoproteins inducing an intracellular signaling pathway involved in cell proliferation, differentiation, and pattern formation. The canonical branch is one of three known branches. This is also valid in vitro, and Wnts can be considered beneficial for culturing primary cells from organs, provided Wnts are available and applicable even with cells of different species. It was shown here that internally c-myc-tagged murine Wnt-1 produced in the heterologous host Escherichia coli was appropriate for inducing intracellular signaling of the canonical Wnt pathway in eukaryotic cells via stabilization of cytosolic beta-catenin. The pioneering injection of the protein into the blastocoels of Xenopus laevis embryos led to axis duplication and suppression of head formation. Applying the recombinant murine Wnt-1 to metanephric mesenchyme activated the tubulogenic program. The signal-inducing activity of the recombinant protein was also positively demonstrated in the TOP-flash reporter assay. Although Wnts were purified recently from the growth media of stably transfected eukaryotic cell lines, the production of active Wnt proteins in pro- or eukaryotic microorganisms reportedly has never been successful. Here soluble production in E. coli and translocation into the oxidizing environment of the periplasm were achieved. The protein was purified using the internal c-myc tag. The effect on the eukaryotic cells implies that activity was retained. Thus, this approach could make recombinant murine Wnt-1 available as a good starting point for other Wnts needed, for example, for maintaining and differentiating stem cells, organ restoration therapy, and tissue engineering.     

Induction of uracil-DNA glycosylase and dUTP nucleotidohydrolase activity in herpes simplex virus-infected human cells

HeLa BU cells infected with either the type 1 or the type 2 forms of herpes simplex virus show an increase in the activities of uracil-DNA glycosylase and dUTP nucleotidohydrolase. Under optimal conditions, uracil-DNA glycosylase activity increases approximately 40-fold in HSV type 2-infected cells. In herpes simplex virus (HSV) type 1-infected cells, uracil-DNA glycosylase activity increases only 6-fold. At a KCl concentration of 100 mM, uracil-DNA glycosylase derived from HSV type 2-infected cells is activated 2-fold, while the glycosylase extracted from mock infected HeLa BU cells is inhibited almost 90% at 100 mM KCl. dUTP nucleotidohydrolase activity increases 4-fold and 3-fold, respectively, in HSV type 1- and HSV type 2-infected HeLa BU cells. Nondenaturing polyacrylamide gel electrophoresis of extracts derived from the type 1- and type 2-infected cells indicates distinct electrophoretic mobilities from the host cell enzyme. dUTP nucleotidohydrolase RF values for the mock infected cells, HSV type 1, and HSV type 2 are 0.5, 0.25, and 0.33, respectively. Serum from rabbits immunized against cells infected with herpes simplex virus type 1 or type 2 specifically neutralizes the dUTPase and uracil-DNA glycosylase activities extracted from herpes simplex virus-infected cells. This serum does not neutralize dUTPase or uracil-DNA glycosylase activity derived from mock infected cells.     

Adherence of Candida albicans to buccal and vaginal epithelial cells: ultrastructural observations

The adherence of Candida albicans to human buccal and vaginal epithelial cells was studied by transmission electron microscopy. Adherence to epithelial cells was confirmed by both a radiometric assay as well as direct microscopic examination of stained cell preparations. Ultramicroscopic preparations revealed that yeast cells were closely appressed to epithelial cell surfaces and were often partially enclosed within phagocyticlike invaginations of the epithelial cells. A murine model of vaginitis caused by C. albicans was also used to study adherence to epithelial cells and to follow the course of colonization. Ultramicroscopic preparations of murine vaginal tissue revealed that within 2 h postinfection, yeast cells could be seen adhering to epithelial cells. At 6 h postinfection, hyphae and yeast cells were not only found on the epithelial cell surface but also within the submucosal tissue. When observed on the epithelial cell surface, Candida cells were either attached to host cells, or when infected tissue was stained with ruthenium red, Candida cells were observed on the epithelial surface embedded within an electron-dense matrix. Fungal elements were abundant in the submucosa at 24 h postinfection and were still observed on the epithelial cell surface; all of this was accompanied by an inflammatory response.     

Two sensitive, convenient, and widely applicable assays for marker enzyme activities specific to endoplasmic reticulum

Two assay protocols are described for enzyme activities known to reside in the endoplasmic reticulum of a wide variety of species and tissue types, with the intent that they be used as marker enzyme assays in subcellular fractionations. The enzyme activities assayed are choline phosphotransferase and dolichol-P-mannosyl synthase, both of which result in synthesis of lipid products. The assays are constructed to make them easy to perform and sensitive enough to detect enzyme activity even using microgram quantities of cell protein. The assay methodologies are effective not only in vertebrate cells, but in insect cells and yeast cells as well. This implies that these assays should be useful as marker enzyme assays for a wide variety of eukaryotic cells.     

The polyanion-binding domain of cytoplasmic Lys-tRNA synthetase from Saccharomyces cerevisiae is not essential for cell viability.

Cytoplasmic Lys-tRNA synthetase (LysRS) from Saccharomyces cerevisiae is a dimeric enzyme made up of identical subunits of 68 kDa. By limited proteolysis, this enzyme can be converted to a truncated dimer without loss of activity. Whereas the native enzyme strongly interacts with polyanionic carriers, the modified form displays reduced binding properties. KRS1 is the structural gene for yeast cytoplasmic LysRS. It encodes a polypeptide with an amino-terminal extension composed of about 60-70 amino acid residues, compared to its prokaryotic counterpart. This segment, containing 13 lysine residues, is removed upon proteolytic treatment of the native enzyme. The aim of the present study was to probe in vivo the significance of this amino-terminal extension. We have constructed derivatives of the KRS1 gene, encoding enzymes lacking 58 or 69 amino-terminal residues and, by site-directed mutagenesis, we have changed four or eight lysine residues from the amino-terminal segment of LysRS into glutamic acids. Engineered proteins were expressed in vivo after replacement of the wild-type KRS1 allele. The mutant enzymes displayed reduced specific activities (2-100-fold). A series of carboxy-terminal deletions, encompassing 3, 10 or 15 amino acids, were introduced into the LysRS mutants with modified amino-terminal extensions. The removal of three residues led to a 2-7-fold increase in the specific activity of the mutant enzymes. This partial compensatory effect suggests that interactions between the two extreme regions of yeast LysRS are required for a proper conformation of the native enzyme. All KRS1 derivatives were able to sustain growth of yeast cells, although the mutant cell lines displaying a low LysRS activity grew more slowly. The expression, as single-copy genes, of mutant enzymes with a complete deletion of the amino-terminal extension or with four Lys----Glu mutations, that displayed specific activities close to that of the wild-type LysRS, had no discernable effect on cell growth. We conclude that the polycationic extensions of eukaryotic aminoacyl-tRNA synthetases are dispensable, in vivo, for aminoacylation activities. The results are discussed in relation to the triggering role in in situ compartmentalization of protein synthesis that has been ascribed to the polypeptide-chain extensions that characterize most, if not all, eukaryotic aminoacyl-tRNA synthetases.     

Eukaryotic DNA ligases

Recent studies on eukaryotic DNA ligases are briefly reviewed. The two distinguishable enzymes from mammalian cells, DNA ligase I and DNA ligase II, have been purified to homogeneity and characterized biochemically. Two distinct DNA ligases have also been identified in Drosophila melanogaster embryos. The genes encoding DNA ligases from Schizosaccharomyces pombe, Saccharomyces cerevisiae and vaccinia virus have been cloned and sequenced. These 3 proteins exhibit about 30% amino acid sequence identity; the 2 yeast enzymes share 53% amino acid sequence identity or conserved changes. Altered DNA ligase I activity has been found in cell lines from patients with Bloom's syndrome, although a causal link between the enzyme deficiency and the disease has not yet been proven.     

Cloning and expression in Escherichia coli of a human cDNA encoding the DNA repair protein N-methylpurine-DNA glycosylase

A 871-base pair cDNA encoding the human N-methylpurine-DNA glycosylase (MPG) was cloned from a HeLa S3 cDNA expression library in a pUC vector by phenotypic screening of MPG-negative (tag- alkA-) Escherichia coli cells exposed to methylmethane sulfonate. The active MPG is expressed as a 31-kDa fusion protein. The human cDNA-encoded MPG releases 3-methyladenine, 7-methylguanine, and 3-methylguanine from DNA and thus has a substrate range similar to that of the indigenous enzyme and the E. coli AlkA protein. The cDNA hybridizes with distinct restriction fragments of mammalian DNAs but not with E. coli or yeast DNA. A search in the GenBank data bank failed to show any other cloned DNA with a similar sequence. Although the human protein has 62% sequence homology with the corresponding rat enzyme, only a few amino acid residues are conserved between the human protein and the E. coli and yeast MPGs. However, a conserved glutamine residue in all MPGs that release 3-alkyladenine and an arginine residue in eukaryotic MPGs and E. coli AlkA that act additionally on N-alkylguanines suggest that these residues are involved in recognition of adenine and guanine adducts in DNA, respectively. Although the 1.1-kilobase mRNAs of MPG from human and rodents are similar in size, liver and cultured cells of rat have much lower levels of MPG mRNA than do human and mouse cells. A hamster cell line variant isolated as being resistant to methylmethane sulfonate does not have a higher level of MPG mRNA than the parent cell line.     

Sensitization of rad mutants of Dictyostelium discoideum to ultraviolet light by postirradiation treatment with caffeine

The capacity of normal human cells to regulate DNA-repair pathways was examined. Synchronous populations of WI-38 human diploid fibroblasts were used to determine whether base-excision repair was increased as a function of the cell cycle. 2 parameters of the base-excision repair pathway were examined: (1) The induction of the DNA-repair enzyme uracil DNA glycosylase which functions in an initial step in base excision repair: (2) cell-mediated base-excision repair as measured by unscheduled DNA synthesis after exposure to sodium bisulfite or to methyl methanesulfonate. The glycosylase activity was increased 5-fold during cell proliferation; unscheduled DNA synthesis was enhanced 4- to 30-fold in a similar fashion. Equivalent results were observed where repair replication was quantitated using density-gradient analysis in the absence of hydroxyurea. The increase of the activity of the uracil DNA glycosylase and the enhancement of DNA repair occurred prior to the induction of DNA replication. Furthermore, at the maximal stimulation of DNA replication both glycosylase activity and DNA repair had substantially diminished. As the cells entered the second cell cycle, the glycosylase activity was again increased and then was again diminished. These results suggest that human cells actively modulate this DNA-repair pathway. The temporal stimulation of base-excision repair suggests the possibility that a DNA-repair complex may be formed prior to DNA replication to prescreen DNA and thus ensure the transfer of the correct genetic information to daughter cells.     

Formamidopyrimidine DNA glycosylase in the yeast Saccharomyces cerevisiae.

A DNA glycosylase that excises, 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (Fapy) from double stranded DNA has been purified 28,570-fold from the yeast Saccharomyces cerevisiae. Gel filtration chromatography shows that yeast Fapy DNA glycosylase has a molecular weight of about 40 kDa. The Fapy DNA glycosylase is active in the presence of EDTA, but is completely inhibited by 0.2 M KCl. Yeast Fapy DNA glycosylase does not excise N7-methylguanine, N3-methyladenine or uracil. A repair enzyme for 7,8-dihydro-8-oxoguanine (8-OxoG) co-purifies with the Fapy DNA glycosylase. This repair activity causes strand cleavage at the site of 8-OxoG in DNA duplexes. The highest rate of incision of the 8-OxoG-containing strand was observed for duplexes where 8-OxoG was opposite guanine. The mode of incision at 8-OxoG was not established yet. The results however suggest that the Fapy- and 8-OxoG-repair activities are associated with a single protein.