The DNA-repair enzyme uracil-DNA glycosylase in the human hematopoietic system

The expression of the DNA base-excision-repair enzyme uracil-DNA glycosylase in the human hematopoietic system followed a tightly regulated pattern: high enzyme activities were recorded in proliferating bone marrow progenitor cells and in peripheral blood T- and B-cells, both groups of cells requiring the integrity of their genetic information for their proper function. The blood quiescent immunocompetent cells retained their DNA-uracil exclusion capacity, even in the oldest age groups. Peripheral blood mature end cells, granulocytes, platelets and red cells had little activity, consistent with the fact that these cells are anuclear or short-lived, so that no template-primer functions of their DNA are required. Uracil-DNA glycosylase expression is high in all types of human leukemia, providing a selective advantage for survival of leukemic cells. Overall results show that a deficiency of this DNA base-excision-repair pathway is not likely to be an etiopathogenetic factor in the formation of non-random or other chromosomal abnormalities or in the leukemogenesis itself.     

Fidelity of uracil-initiated base excision DNA repair in Escherichia coli cell extracts

The error frequency and mutational specificity associated with Escherichia coli uracil-initiated base excision repair were measured using an M13mp2 lacZalpha DNA-based reversion assay. Repair was detected in cell-free extracts utilizing a form I DNA substrate containing a site-specific uracil residue. The rate and extent of complete uracil-DNA repair were measured using uracil-DNA glycosylase (Ung)- or double-strand uracil-DNA glycosylase (Dug)-proficient and -deficient isogenic E. coli cells. In reactions utilizing E. coli NR8051 (ung(+) dug(+)), approximately 80% of the uracil-DNA was repaired, whereas about 20% repair was observed using NR8052 (ung(-) dug(+)) cells. The Ung-deficient reaction was insensitive to inhibition by the PBS2 uracil-DNA glycosylase inhibitor protein, implying the involvement of Dug activity. Under both conditions, repaired form I DNA accumulated in conjunction with limited DNA synthesis associated with a repair patch size of 1-20 nucleotides. Reactions conducted with E. coli BH156 (ung(-) dug(+)), BH157 (ung(+) dug(-)), and BH158 (ung(-) dug(-)) cells provided direct evidence for the involvement of Dug in uracil-DNA repair. The rate of repair was 5-fold greater in the Ung-proficient than in the Ung-deficient reactions, while repair was not detected in reactions deficient in both Ung and Dug. The base substitution reversion frequency associated with uracil-DNA repair was determined to be approximately 5.5 x 10(-)(4) with transversion mutations dominating the mutational spectrum. In the presence of Dug, inactivation of Ung resulted in up to a 7.3-fold increase in mutation frequency without a dramatic change in mutational specificity.     

Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.     

Normal uracil-DNA glycosylase activity in Bloom's syndrome cells

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

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.     

A mollicute (mycoplasma) DNA repair enzyme: purification and characterization of uracil-DNA glycosylase.

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

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.     

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.     

Highly efficient base excision repair (BER) in human and rat male germ cells

The quality of germ cell DNA is critical for the fate of the offspring, yet there is limited knowledge of the DNA repair capabilities of such cells. One of the main DNA repair pathways is base excision repair (BER) which is initiated by DNA glycosylases that excise damaged bases, followed by incision of the generated abasic (AP) sites. We have studied human and rat methylpurine-DNA glycosylase (MPG), uracil-DNA glycosylase (UNG), and the major AP endonuclease (HAP1/APEX) in male germ cells. Enzymatic activities and western analyses indicate that these enzymes are present in human and rat male germ cells in amounts that are at least as high as in somatic cells. Minor differences were observed between different cellular stages of rat spermatogenesis and spermiogenesis. Repair of methylated DNA was also studied at the cellular level using the Comet assay. The repair was highly efficient in both human and rat male germ cells, in primary spermatocytes as well as round spermatids, compared to rat mononuclear blood cells or hepatocytes. This efficient BER removes frequently occurring DNA lesions that arise spontaneously or via environmental agents, thereby minimising the number of potential mutations transferred to the next generation.     

Uracil-DNA glycosylase in benign and malignant maturing human hematopoietic cells

The expression of uracil-DNA glycosylase was studied in human normal hematopoietic bone marrow cells and in malignant counterparts obtained from patients with chronic granulocytic leukemia. We observed that the expression of the enzyme was highest in the proliferating granulocytic compartment (myeloblasts through myelocytes) and that it was diminished in more mature cells. Furthermore, we demonstrated that uracil-DNA glycosylase activity was higher in immature red blood cells or reticulocytes than in more mature red cells. The same tendency was also demonstrated in human malignant monoblasts, which were induced to terminal maturation by phorbol ester. It can be concluded from these results that uracil-DNA glycosylase expression is equal in benign and malignant hematopoietic progenitor cells; no selectivity towards malignant vs. benign progenitors can be expected in possible chemotherapeutic approaches relying on uracil-DNA glycosylase.     

Glycosylases that excise modified DNA pyrimidines in young and senescent human WI-38 fibroblasts

Cellular DNA is continuously subject to damages by both endogenous and exogenous oxidizing agents. Excision repair in human cells is initiated by DNA glycosylases which remove oxidized bases from DNA. 5-Hydroxymethyluracil-DNA glycosylase excises 5-hydroxymethyluracil from DNA. A different enzyme has glycosylic activity against many ring-saturated DNA pyrimidines. Levels of these enzymes were examined in WI-38 fibroblasts of different culture ages. All glycosylases were assayed by measurements of direct release of modified free bases from their respective DNA substrates. Levels of 5-hydroxymethyluracil-DNA glycosylase were reduced in aging cells. Specific activities of the glycosylase that releases ring-saturated pyrimidines and of uracil-DNA glycosylase were not substantially altered in senescent cells. Therefore, although aging cells might have reduced excision of DNA 5-hydroxymethyluracil, there is no overall age-dependent decrease of DNA glycosylase activities.     

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.     

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.     

Molecular cloning of human uracil-DNA glycosylase, a highly conserved DNA repair enzyme.

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

Mutational analysis of arginine 276 in the leucine-loop of human uracil-DNA glycosylase

Uracil residues are eliminated from cellular DNA by uracil-DNA glycosylase, which cleaves the N-glycosylic bond between the uracil base and deoxyribose to initiate the uracil-DNA base excision repair pathway. Co-crystal structures of the core catalytic domain of human uracil-DNA glycosylase in complex with uracil-containing DNA suggested that arginine 276 in the highly conserved leucine intercalation loop may be important to enzyme interactions with DNA. To investigate further the role of Arg(276) in enzyme-DNA interactions, PCR-based codon-specific random mutagenesis, and site-specific mutagenesis were performed to construct a library of 18 amino acid changes at Arg(276). All of the R276X mutant proteins formed a stable complex with the uracil-DNA glycosylase inhibitor protein in vitro, indicating that the active site structure of the mutant enzymes was not perturbed. The catalytic activity of the R276X preparations was reduced; the least active mutant, R276E, exhibited 0.6% of wildtype activity, whereas the most active mutant, R276H, exhibited 43%. Equilibrium binding studies utilizing a 2-aminopurine deoxypseudouridine DNA substrate showed that all R276X mutants displayed greatly reduced base flipping/DNA binding. However, the efficiency of UV-catalyzed cross-linking of the R276X mutants to single-stranded DNA was much less compromised. Using a concatemeric [(32)P]U.A DNA polynucleotide substrate to assess enzyme processivity, human uracil-DNA glycosylase was shown to use a processive search mechanism to locate successive uracil residues, and Arg(276) mutations did not alter this attribute.     

Affinity Purification and Comparative Analysis of Two Distinct Human Uracil-DNA Glycosylases

Evidence is presented on two forms of uracil-DNA glycosylase (UDG1 and UDG2) that exist in human cells. We have developed an affinity technique to isolate uracil-DNA glycosylases from HeLa cells. This technique relies on the use of a uracil-DNA glycosylase inhibitor (Ugi) produced by theBacillus subtilisbacteriophage, PBS2. Affinity-purified preparations of uracil-DNA glycosylase, derived from total HeLa cell extracts, reveal a group of bands in the 36,000 molecular weight range and a single 30,000 molecular weight band when analyzed by SDS–PAGE and silver staining. In contrast, only the 30,000 molecular weight band is seen in HeLa mitochondrial preparations. Separation of HeLa cell nuclei from the postnuclear supernatant reveals that uracil-DNA glycosylase activity is evenly distributed between the nuclear compartment and the postnuclear components of the cell. Immunostaining of a nuclear extract with antisera to UDG1 indicates that the nuclear associated uracil-DNA glycosylase activity is not associated with the highly conserved uracil-DNA glycosylase, UDG1. With the use of Ugi-Sepharose affinity chromatography, we show that a second and distinct uracil-DNA glycosylase is associated with the nuclear compartment. Immunoblot analysis, utilizing antisera generated against UDG1, reveals that the 30,000 molecular weight protein and a protein in the 36,000 range share common epitopes. Cycloheximide treatment of HeLa cells indicates that upon inhibition of protein synthesis, the higher molecular weight species disappears and is apparently posttranslationally processed into a lower molecular weight form. This is substantiated by mitochondrial import studies which reveal thatin vitroexpressed UDG1 becomes resistant to trypsin treatment within 15 min of incubation with mitochondria. Within this time frame, a lower molecular weight form of uracil-DNA glycosylase appears and is associated with the mitochondria. Antibodies generated against peptides from specific regions of the cyclin-like uracil-DNA glycosylase (UDG2), demonstrate that this nuclear glycosylase is a phosphoprotein with a molecular weight in the range of 36,000. SDS–PAGE analysis of Ugi affinity-purified and immunoprecipitated UDG2 reveals two closely migrating phosphate-containing species, indicating that UDG2 either contains multiple phosphorylation sites (resulting in heterogeneous migration) or that two distinct forms of UDG2 exist in the cell. Cell staining of various cultured human cell lines corroborates the finding that UDG1 is largely excluded from the nucleus and that UDG2 resides mainly in the nucleus. Our results indicate that UDG1 is targeted to the mitochondria and undergoes proteolytic processing typical of resident mitochondrial proteins that are encoded by nuclear DNA. These results also indicate that the cyclin-like uracil-DNA glycosylase (UDG2) may be a likely candidate for the nuclear located base-excision repair enzyme.     

DNA glycosylases

Summary Various DNA glycosylases exist, which initiate the first step in base-excision repair. A summary of the kinetic and physical characteristics of three classes of DNA glycosylase are presented here. The first class discussed, include glycosylases which recognize alkylated DNA. Various data from enzymes derived from both prokaryotic and eukaryatic sources is discussed. The second class deals with a glycosylase that recognizes and initiates the excision of pyrimidine dimers in DNA. To date, this enzyme has only been uncovered from two sources, Micrococcus luteus and the T4 bacteriophage of E. coli. The third class consists of the most studied of the glycosylases, the uracil-DNA glycosylase enzymes. Various characteristics are presented for the uracil-DNA glycosylases derived from various sources. Recent information from our laboratory is presented implicating that herpes simplex virus may mediate a uracil-DNA glycosylase activity in productively infected cells.     

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.