Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells.

The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior to their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. These results suggest that there may be a relationship between the sensitivity of xeroderma pigmentosum cells from each complementation group to specific DNA damaging agents and their inability to regulate nucleotide excision repair during cell stimulation.

Infidelity of DNA synthesis in vitro: screening for potential metal mutagens or carcinogens.

Thirty-one metal salts have been tested for their ability to affect the accuracy of DNA synthesis in vitro. All ten salts of metal carcinogens decreased the fidelity of DNA synthesis. Of the three metals which beforehand were considered to be possible mutagens or carcinogens, only one decreased fidelity. In contrast, 17 noncarcinogenic metal salts did not affect fidelity even when present at concentrations that were clearly inhibitory.

Induction of the DNA repair enzyme uracil-DNA glycosylase in stimulated human lymphocytes.

The capacity of human cells to modulate the synthesis of DNA repair enzymes has been investigated by measuring the induction of the uracil-DNA glycosylase during lymphocyte stimulation. Treatment of peripheral lymphocytes with phytohemagglutinin increased glycosylase activity 10-fold. Glycosylase stimulation was coordinate with the activation of DNA synthesis and DNA polymerase activity. Two chromatographically distinct species of the glycosylase have been resolved; only one species is induced during phytohemagglutinin stimulation. The effect of actinomycin D and cycloheximide on glycosylase induction was determined. Treatment with either inhibitor at 96 hr after phytohemagglutinin addition (maximal induction) decreased glycosylase activity after an appreciable lag period. This suggested that induction of the uracil-DNA glycosylase requires transcription and translation although the enzyme may be quite stable once induced.

Physical association of base excision repair enzymes with parental and replicating DNA in BHK-21 cells.

The physical association of mammalian excision repair enzymes with DNA was examined as a function of cell proliferation. The molecular weight distribution of two nuclear base excision repair enzymes, the uracil DNA glycosylase and the hypoxanthine DNA glycosylase, were examined by sucrose step gradient analysis. The sedimentation of DNA polymerase activity as well as the distribution of parental and replicating DNA were determined simultaneously. In confluent BHK-21 fibroblasts, basal levels of both DNA glycosylases, DNA polymerase beta, and parental DNA sedimented to the 20%/40% sucrose border. In proliferating BHK-21 cells, induced levels of both DNA glycosylases, DNA polymerase alpha, and replicating DNA sedimented to the 40%/50% sucrose border. The physical association of the repair enzymes with DNA was demonstrated by detergent treatment and by DNase digestion. As defined by [35S] methionine pulse labeling analysis, newly synthesized DNA repair enzymes were localized with either parental or replicating DNA. These results suggested that the physical association of mammalian DNA repair enzymes with nuclear DNA was dependent on the proliferative state of the cell.

Restriction of carcinogen-induced error incorporation during in vitro DNA synthesis.

The in vitro accuracy of DNA replication has been investigated through the measurement of the frequency with which noncomplementary nucleotides were incorporated during polynucleotide replication. The effect of beta-propiolactone treatment of deoxynucleotide templates, ribopolynucleotide templates, and the DNA polymerase from avian myeloblastosis virus was determined. Treatment of the deoxynucleotide template, poly(dA) (see article) oligo(dT) 12-18, by beta-propiolactone resulted in an increased frequency of noncomplementary nucleotide incorporation during DNA polymerization. Carcinogen treatment of the ribonucleotide templates, poly(rA) (see article) oligo(dT) 12-18, and poly(rC) (see article) oligo(dG) 12-18, and carcinogen treatment of avian myeloblastosis virus DNA polymerase did not alter the frequency of noncomplementary nucleotide incorporation. This suggested that carcinogen-induced error incorporation during DNA synthesis was restricted solely to the treatment of a deoxynucleotide template.

Stimulation of the nuclear uracil DNA glycosylase in proliferating human fibroblasts.

The cell cycle stimulation of individual species of the uracil DNA glycosylase was examined in WI-38 normal diploid fibroblasts. The nuclear uracil DNA glycosylase was induced as WI-38 cells traversed the cell cycle. In contrast, the specific activity of the mitochondrial glycosylase remained constant during cell proliferation. The two enzyme activities can be further distinguished by their elution patterns on DNA-cellulose, by differential cation sensitivity, and by kinetic differences. The singular stimulation of the nuclear glycosylase in the cell cycle is a further suggestion that normal human cells actively regulate excision repair pathways.

Immunocytochemical localization of the base excision repair enzyme uracil DNA glycosylase in quiescent and proliferating normal human cells.

The immunocytochemical localization of the base excision repair enzyme uracil DNA glycosylase was examined as a function of cell proliferation. Two nontransformed normal human fibroblast cell strains were analyzed using an anti-human uracil DNA glycosylase monoclonal antibody. In quiescent cells, basal levels of a nonnuclear immunocytochemically reactive glycosylase protein were detected. No nuclear immunofluorescence was observed. In contrast, in proliferating cells, intense immunofluorescence could be detected exclusively in the nuclear or perinuclear regions. As proliferation diminished, basal levels of the nonnuclear immunocytochemically reactive glycosylase were again observed. The subcellular distribution of the glycosylase was examined in parallel by in vitro biochemical assay. In quiescent cells, glycosylase activity was observed in both the nuclear and membrane fractions. A small amount of enzyme activity could be detected in the soluble cytoplasmic fraction. Immunoblot analysis demonstrated a Mr 37,000 glycosylase protein in each subcellular fraction. During cell proliferation, there was an increase in glycosylase activity in each of the subcellular fractions. These results suggest a correlation between the proliferative state of normal human cells and the preferential nuclear or perinuclear localization of an immunocytochemically reactive glycosylase protein. Further, immunofluorescence of the nuclear enzyme may be dependent on defined conformational states of that nuclear glycosylase in the cell cycle.

Regulation of hypoxanthine DNA glycosylase in normal human and Bloom's syndrome fibroblasts.

The regulation of the base excision repair enzyme hypoxanthine DNA glycosylase was examined in normal human skin fibroblasts (NHS) and fibroblasts from a patient with Bloom's syndrome. Using randomly proliferating cells and those synchronized at specific intervals in the cell cycle, enzyme levels were shown to become elevated severalfold in a proliferation-associated manner. In NHS synchronized in G0 by serum deprivation or in G1 by isoleucine deprivation, maximal enzyme levels were reached prior to maximal rates of DNA synthesis. In Bloom's syndrome cells synchronized in this manner, these two activities were coincident. Cells synchronized at the G1-S border by hydroxyurea exhibit an initial wave of DNA synthesis upon removal of the drug. The cells then undergo another DNA synthetic cycle climaxing 18-21 h after release. Maximal hypoxanthine glycosylase activity of hydroxyurea-synchronized Bloom's cells was observed during the second round of DNA synthesis. However, in NHS the peak of enzyme activity was observed as early as 9 h prior to the second round of DNA synthesis. To determine if hypoxanthine glycosylase could be induced in the absence of DNA synthesis, serum-synchronized NHS were released in the presence of hydroxyurea. The results showed that inhibition of DNA synthesis did not diminish glycosylase induction which demonstrated that DNA replication was not required for glycosylase induction.

Positive correlation between the extent of cell proliferation and the regulation of base excision repair.

The regulation of base excision repair during cell proliferation was examined as a function of the rate of cell growth. Normal human skin fibroblasts (mean generation time, 27.64 hr) and hamster fibroblasts (mean generation time, 13.79 hr) were utilized to examine this relationship. The regulation of base excision repair in each cell type was examined by quantitating (a) the activity of the base excision repair enzyme, uracil DNA glycosylase, during asynchronous cell proliferation; and (b) glycosylase activity during cell proliferation after exposure to dimethyl sulfate. In both cell types, uracil DNA glycosylase was increased as a function of cell proliferation. The extent of enhancement was greater in the baby hamster kidney cells than in the normal human skin cells (9.5-fold versus 4-fold, respectively). In baby hamster kidney cells, cell proliferation as well as the enhancement of uracil DNA glycosylase was unaffected by exposure to 20 microM dimethyl sulfate. In contrast, the exposure of normal human skin cells to 20 microM dimethyl sulfate reduced the extent of cell growth and, consequently, the proliferative-dependent stimulation of uracil DNA glycosylase. These results suggest that the regulation of base excision repair is directly proportional to the proliferative rate characteristic of a given cell population.

Biosynthesis of the human base excision repair enzyme uracil-DNA glycosylase.

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.

New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered a classical glycolytic protein examined for its pivotal role in energy production. It was also used as a model protein for analysis of protein structure and enzyme mechanisms. The GAPDH gene was utilized as a prototype for studies of genetic organization, expression and regulation. However, recent evidence demonstrates that mammalian GAPDH displays a number of diverse activities unrelated to its glycolytic function. These include its role in membrane fusion, microtubule bundling, phosphotransferase activity, nuclear RNA export, DNA replication and DNA repair. These new activities may be related to the subcellular localization and oligomeric structure of GAPDH in vivo. Furthermore, other investigations suggest that GAPDH is involved in apoptosis, age-related neurodegenerative disease, prostate cancer and viral pathogenesis. Intriguingly, GAPDH is also a unique target of nitric oxide. This review discusses the functional diversity of GAPDH in relation to its protein structure. The mechanisms through which mammalian cells may utilize GAPDH amino acid sequences to provide these new functions and to determine its intracellular localization are considered. The interrelationship between new GAPDH activities and its role in cell pathologies is addressed.

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.

Purification and properties of the human placental uracil DNA glycosylase.

Human placental uracil DNA glycosylase was purified 3700-fold to apparent homogeneity as defined by SDS gel analysis. Its immunological characteristics were examined using three monoclonal antibodies prepared against partially purified human placental uracil DNA glycosylase. Immunoblot analysis demonstrated that, even in crude isolates, only one glycosylase species of molecular weight 37,000 could be detected. Each of the three monoclonal antibodies quantitatively recognized the highly purified enzyme by ELISA. The glycosylase is a single polypeptide with a molecular weight of 37,000 as defined by both Sephadex gel filtration and by SDS-polyacrylamide gel electrophoresis analysis. The enzyme is heat-stable, with a t 1/2 of greater than 30 min at 42 degrees C or at 45 degrees C. Surprisingly, inhibitor analysis demonstrated that the glycosylase was inhibited by preincubation with either 5-fluorouracil or 5-bromouracil. However, no significant inhibition was observed when either compound was added directly to the enzyme assay.

Effects of metals in in vitro bioassays.

The capacity of in vitro bioassays to detect the potential carcinogenicity of metal compounds is reviewed. The in vitro bioassays discussed include: bacterial reversion analysis to determine the capacity of metal salts to revert Salmonella typhimurium histidine auxotrophs or to revert Escherichia coli WP 2 tryp- to tryptophan prototrophy; examination of the ability of metal salts to preferentially inhibit cell growth in Bacillus subtilis cells deficient in DNA repair pathways; determination of the ability of metal salts to induce resistance to base analogs in mammalian cells; the capacity of metal salts to enhance viral transformation of mammalian cells or to transform cells in the absence of virus; and the ability of metal salts to induce chromosomal aberrations in mammalian cells. Using each of these in vitro bioassays, diverse metal compounds have been identified as potential carcinogens. Furthermore, the use of different compounds of a specific metal may allow a determination of the valence which may be required for carcinogenesis.

Minireview. Emerging new functions of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in mammalian cells.

Recent evidence indicates new, intriguing roles for the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in fundamental mammalian cell processes. These include its role in DNA repair, in the translational control of gene expression, in DNA replication and in endocytosis. These findings have the potential to alter our basic understanding of the molecular mechanisms through which human or mammalian cells utilize individual proteins in vital, yet unrelated, cell processes.

Cell cycle regulation of DNA repair in normal and repair deficient human cells.

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14--15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2--4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.

On the fidelity of DNA synthesis:effects of steroids and intercalating agents.

Studies on the fidelity of DNA synthesis in vitro were extended to pharmacological agents that interact non-covalently to DNA. Several steroids, intercalators and cancer chemotherapeutic agents increased the relative frequency of incorrect deoxynucleotide incorporation in the fidelity assay. The relationship between non-covalent interactions with DNA and the decreased fidelity of DNA synthesis in vitro is considered.

Developmental regulation of the base excision repair enzyme uracil DNA glycosylase in the rat.

The developmental regulation of the mammalian DNA-repair enzyme uracil DNA glycosylase was examined in the rat at specific intervals ranging from -4 days before to 106 days after birth. Enzyme activity was quantitated by in vitro biochemical assay. In the adult animal, as measured in crude cell extracts, three organs (liver, kidney and spleen) had significant levels of activity. In contrast, three organs (brain, heart and lung) had low activity. Partial purification of this enzyme identified one major species of molecular weight 32,700 Da, demonstrating the quantitation of the nuclear glycosylase. During development, with the exception of the liver, the specific activity of the glycosylase paralleled the regulation of DNA synthesis. In these organs the highest levels of the glycosylase and the rate of DNA replication were observed around the time of birth. In the liver, DNA replication was similarly regulated. However, glycosylase activity was minimal at early stages of life. Instead, maximal levels were observed at 14-21 days after birth. At that time DNA replication was severely reduced. These results demonstrate that individual organs express this DNA-repair enzyme in a distinct and specific pattern during development. Accordingly, the regulation of the uracil DNA glycosylase during development may provide a model system to examine the differential regulation of DNA-repair genes.

Proliferation-dependent regulation of DNA glycosylases in progeroid cells.

The regulation of the base excision repair enzymes uracil DNA glycosylase and hypoxanthine DNA glycosylase was examined in 2 different progeroid cell strains. The immunoreactivity of the uracil DNA glycosylase in progeroid cells was examined by enzyme linked immunosorbent assay (ELISA) and by immunoblot analysis. The enzyme was recognized in a quantitative manner by 2 different anti-human uracil DNA glycosylase monoclonal antibodies in the ELISA. Western blot analysis identified a glycosylase protein of Mr = 37,000. In randomly proliferating progeroid cells, the uracil DNA glycosylase was enhanced 3-fold during cell growth. In synchronous cells, uracil DNA glycosylase and hypoxanthine DNA glycosylase were induced with an extent of induction (5-6-fold) comparable to that observed for normal human cells. Further, the activity of each base excision repair enzyme was enhanced with a comparable temporal sequence prior to the induction of DNA synthesis and DNA polymerase activity. These results indicate a normal cell cycle regulation of base excision repair in progeroid cells.

DNA nucleotide excision-repair synthesis is independent of perturbations of deoxynucleoside triphosphate pool size.

We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [3H]TTP from [3H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [3H]TTP pool was formed within 30 min of the addition of [3H]thymidine and remained relatively constant for the next 6 h. Addition of 2-10 mM hydroxyurea (HU) caused a gradual 2-4-fold increase in the [3H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [3H]TTP pool size in cells exposed to 20 J/m2 and unirradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8-10-fold reduction in the size of the [3H]TTP pool relative to the initial studies. In the UV excision-repair studies the presence of hydroxyurea did not alter the specific activity of [3H] thymidine 5'-monophosphate incorporated into parental DNA due to repair replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair synthesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [3H]TTP pool.