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.     

W-mutagenesis in the bisulfite-treated lambda phage

Survival of phage lambda cI857 inactivated by bisulfite (pH 5.6, 37 degrees C) is higher (the dose modification factor approx. 1.2) and frequency of bisulfite-induced c-mutations 2-4-fold lower on the lawn of the wild-type strain ung+, as compared to ung-1 mutant deficient in uracil-DNA glycosylase. Irradiation of host cells by a moderate UV dose inducing SOS repair system enhances the frequency of bisulfite-induced c-mutations 2-3-fold in the wild-type (ung+) host, but not in the ung-1 mutant. It is suggested that W-mutagenesis in bisulfite-treated lambda phage in the ung+ cells is due to SOS repair of apyrimidinic sites which are produced during excision of uracil residues, the products of cytosine deamination.     

Uracil-DNA glycosylase (UNG)-deficient mice reveal a primary role of the enzyme during DNA replication

Gene-targeted knockout mice have been generated lacking the major uracil-DNA glycosylase, UNG. In contrast to ung- mutants of bacteria and yeast, such mice do not exhibit a greatly increased spontaneous mutation frequency. However, there is only slow removal of uracil from misincorporated dUMP in isolated ung-/- nuclei and an elevated steady-state level of uracil in DNA in dividing ung-/- cells. A backup uracil-excising activity in tissue extracts from ung null mice, with properties indistinguishable from the mammalian SMUG1 DNA glycosylase, may account for the repair of premutagenic U:G mispairs resulting from cytosine deamination in vivo. The nuclear UNG protein has apparently evolved a specialized role in mammalian cells counteracting U:A base pairs formed by use of dUTP during DNA synthesis.     

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

The steady-state levels of uracil residues in DNA extracted from strains of Escherichia coli were measured and the influence of defects in the genes for uracil-DNA glycosylase (ung), double-strand uracil-DNA glycosylase (dug), and dUTP pyrophosphatase (dut) on uracil accumulation was determined. A sensitive method, called the Ung-ARP assay, was developed that utilized E. coli Ung, T4pdg, and the Aldehyde Reactive Probe reagent to label abasic sites resulting from uracil excision with biotin. The limit of detection was one uracil residue per million DNA nucleotides (U/10(6)nt). Uracil levels in the genomic DNA of E. coli JM105 (ung+ dug+) were at the limit of detection, as were those of an isogenic dug mutant, regardless of growth phase. Inactivation of ung in JM105 resulted in 31+/-2.6 U/10(6)nt during early log growth and 19+/-1.7 U/10(6)nt in saturated phase. An ung dug double mutant (CY11) accumulated 33+/-2.9 U/10(6)nt and 23+/-1.8U/10(6)nt during early log and saturated phase growth, respectively. When cultures of CY11 were supplemented with 20 ng/ml of 5-fluoro-2'-deoxyuridine, uracil levels in early log phase growth DNA rose to 125+/-1.7 U/10(6)nt. Deoxyuridine supplementation reduced the amount of uracil in CY11 DNA, but uridine did not. Levels of uracil in DNA extracted from CJ236 (dut-1 ung-1) were determined to be 3000-8000 U/10(6)nt as measured by the Ung-ARP assay, two-dimensional thin-layer chromatography of metabolically-labeled 32P DNA, and LC/MS of uracil and thymine deoxynucleosides. DNA sequencing revealed that the sole molecular defect in the CJ236 dUTP pyrophosphatase gene was a C-->T transition mutation that resulted in a Thr24Ile amino acid change.     

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.     

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.     

Altered CTP synthetase activity confers resistance to 5-bromodeoxyuridine toxicity and mutagenesis

A V79 Chinese hamster fibroblast cell line selected for resistance to the toxic effects of 5-fluorouracil (Kaufman, 1984b) was found to be cross-resistant to the toxic effects of the thymidine analog 5-bromodeoxyuridine (BrdUrd). When tested for sensitivity to BrdUrd mutagenesis, the fluorouracil-resistant cells were found to be resistant to mutagenesis induced by high concentrations of BrdUrd in the medium (INC mutagenesis) but not to mutagenesis induced by the replication of DNA containing 5-bromouracil (REP mutagenesis). Analyses of deoxyribonucleoside triphosphate pools indicated that high endogenous dCTP levels in the mutant prevented the high BrdUTP/dCTP ratio associated with INC mutagenesis. However, the mutant phenotype had no effect on the nucleotide pool imbalance associated with REP mutagenesis. This mutant provides further genetic evidence for the existence of two independent mechanisms for BrdUrd mutagenesis in mammalian cells.     

Escherichia coli K-12 mutants deficient in uracil-DNA glycosylase.

A new assay specific for uracil-DNA glycosylase is described, Escherichia coli mutants partially and totally deficient in uracil-DNA glycosylase activity have been isolated by using this assay in mass-screening procedures. These have been designated ung mutants. The ung gene maps between tyrA and nadB on the E. coli chromosome. T4 phage containing uracil in their DNA grow on the most glycosylase-deficient hosts but are unable to grow on wild-type bacteria. This provides a simple spot test for the ung genotype. The ung mutants show slightly higher rates of spontaneous mutation to antibiotic resistance. Taken together, these results suggest a central role for uracil-DNA glycosylase in the initiation of an excision repair pathway for the exclusion of uracil from DNA.     

Synthesis and metabolism of uracil-containing deoxyribonucleic acid in Escherichia coli

Significant amounts of uracil were found in the deoxyribonucleic acids (DNAs) of Escherichia coli mutants deficient in both uracil-DNA glycosylase (ung) and deoxyuridine 5'-triphosphate nucleotidohydrolase (dut) activities, whereas little uracil was found in the DNAs of wild-type cells and cells deficient in only one of these two activities. The amounts of uracil found in the DNAs of dut ung mutants were directly related to the growth temperature of the cultures, apparently because the deoxyuridine 5'-triphosphate nucleotidohydrolase synthesized by dut mutants was temperature sensitive. The dut mutant used failed to grow exponentially, became filamentous at temperatures above 25 degrees C, and exhibited a hyperrec phenotype; however, the ung mutation suppressed all of these effects. Although the dut ung mutants grew exponentially at all temperatures, their growth rates were always slower than the growth rate of the wild type. Since pool size measurements indicated that both deoxyuridine triphosphate and deoxythymidine triphosphate pools were markedly elevated in dut mutants, the reduced growth rate of dut ung cells apparently was due to the actual presence of uracil in the DNA, rather than to a deficiency of deoxyuridine triphosphate and deoxyribosylthymine triphosphate for DNA synthesis. The presence of uracil in E. coli donor DNA also markedly reduced the recombination frequency when the recipient cells were ung+, indicating that DNA repair commenced before the entering DNA could be replicated.     

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.     

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.     

UNG-dependent correction of molecular heteroduplexes of M13 phage DNA in Escherichia coli cells

Correction of heteroduplex DNA obtained by hybridization of uracil-containing single-stranded M13mp18 phage DNA and "mutant" synthetic oligonucleotide with deletion of cytosine in SalGI site was studied in ung+ and ung- E. coli strains. Uracil-containing DNA was prepared after growth of phage in an E. coli strain dut- ung-. The DNA was hybridized with "mutant" oligonucleotide then complementary DNA chain was synthesized by T4 DNA polymerase. Ung+ and ung- E. coli cells were transformed by DNA. In all experiments mutation frequency in ung+ was higher than in ung- cells (approximately 6-fold) and reached 11-50%. Absolute number of mutants was higher in ung+ cells. The results indicate that high level of mutagenesis depends on uracil repair system polarizing the correction of heteroduplex DNA.     

Mutation by [5-3H]cytosine decay in DNA of Escherichia coli lacking uracil-DNA glycosylase activity

The mutagenic local effect of tritium decay at the 5 position of cytosine in DNA of Escherichia coli was determined in wild-type and in ung strains defective in uracil-DNA glycosylase. In the absence of this in vivo activity any genetic consequences of uracil residues formed in DNA should be enhanced. However, the mutation frequency response was no greater in the mutant strain than in the wild type. This finding is inconsistent with the earlier suggestion that efficient production of C to T transitions by the local effect of [5-3H]cytosine decay results from the formation of uracil in cellular DNA. Some other intermediate should be considered, one that is not a substrate for uracil-DNA glycosylase.     

Repair of thymine.guanine and uracil.guanine mismatched base-pairs in bacteriophage M13mp18 DNA heteroduplexes

Repair of thymine.guanine (T.G) and uracil.guanine (U.G) mismatched base-pairs in bacteriophage M13mp18 replicative form (RF) DNA was compared upon transfection into repair-proficient or repair-deficient Escherichia coli strains. Oligonucleotide-directed mutagenesis was used to prepare covalently closed circular heteroduplexes that contained the mismatched base-pair at a restriction recognition site. The heteroduplexes were unmethylated at dam (5'-GATC-3') sites to avoid methylation-directed biasing of repair. In an E. coli host containing uracil-DNA glycosylase (ung+), about 97% of the transfecting U.G-containing heteroduplexes had the U residue excised by the uracil-excision repair system. With the analogous T.G mispair, mismatch repair operated on almost all of the transfecting heteroduplexes and removed the T residue in about 75% of them when the mismatched T was on the minus strand of the RF DNA. Similar preferential excision of the minus-strand's mismatched base was observed whether the heteroduplex RF DNA molecules had only one or both strands unmethylated at dcm (5'-CC(A/T)GG-3') sites and whether the RF DNA was prepared by primer extension in vitro or by reannealing mutant and non-mutant DNA strands. Also, the extent and directionality of repair was the same at a U.G mispair in ung- host cells as at the analogous T.G mispair in ung- or ung+ cells. Only in a mismatch repair-deficient (mutH-) host was the plus strand of the transfecting M13mp18 heteroduplex DNA preferentially repaired. It is suggested that the plus strand nick made by the M13-encoded gene II protein might be employed by a mutH- host to initiate repair on that strand.     

Role of mismatch-specific uracil-DNA glycosylase in repair of 3,N4-ethenocytosine in vivo

The 3,N(4)-ethenocytosine (epsilon C) residue might have biological role in vivo since it is recognized and efficiently excised in vitro by the E. coli mismatch-specific uracil-DNA glycosylase (MUG) and the human thymine-DNA glycosylase (hTDG). In the present work we have generated mug defective mutant of E. coli by insertion of a kanamycin cassette to assess the role of MUG in vivo. We show that human TDG complements the enzymatic activity of MUG when expressed in a mug mutant. The epsilon C-DNA glycosylase defective strain did not exhibit spontaneous mutator phenotype and did not show unusual sensitivity to any of the following DNA damaging treatments: methylmethanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet light, H(2)O(2), paraquat. However, plasmid DNA damaged by 2-chloroacetaldehyde treatment in vitro was inactivated at a greater rate in a mug mutant than in wild-type host, suggesting that MUG is required for the in vivo processing of the ethenobases. In addition, 2-chloroacetaldehyde treatment induces preferentially G.C --> C.G and A.T --> T.A transversions in mug mutant. Comparison of the mutation frequencies induced by the site-specifically incorporated epsilon C residue in E. coli wild-type versus mug indicates that MUG repairs more than 80% of epsilon C residues in vivo. Furthermore, the results show that nucleotide excision repair and recombination are not involved in the processing of epsilon C in E. coli. Based on the mutagenesis data we suggest that epsilon C may be less toxic and less mutagenic than expected. The increased spontaneous mutation rate for G.C --> A.T transition in the ung mug double mutant as compared to the single ung mutant suggest that MUG may be a back-up repair enzyme to the classic uracil-DNA glycosylase.     

DNA intermediates at the Escherichia coli replication fork. II. Studies using dut and ung mutants in vitro.

The incorporation of uracil into and excision from DNA were studied in vitro using lysates on cellophane discs made from Escherichia coli strains with defects in the enzymes dUTPase (dut) and uracil-DNA glycosylase (ung).Results with dut ung lysates indicate that dUTP is competitively incorporated with dTTP at the replication fork. Such incorporation is not due to DNA polymerase I. There is a mild discrimination (2.5-fold) against incorporation of dUTP versus dTTP. These data, together with in vivo uracil incorporation data (Tye et al., 1978) permit a rough estimate of the pool of dUTP in vivo (~0.5% of the dTTP pool).These in vitro data indicate that uracil-DNA glycosylase is the initial step in at least 90% of uracil excision events. However, in a strain defective in uracil-DNA glycosylase (ung-1), uracil-containing DNA is still more subject to single-strand scission than non-uracil-containing DNA, albeit at a rate at least tenfold less than in an ung⁺ strain.A number of qualitative statements may also be made about different steps in uracil incorporation and subsequent excision and repair events. When high levels of dUTP are added in vitro, a dut ung⁺ strain has a higher steady-state level of uracil in newly synthesized DNA than does an isogenic dut⁺ ung strain. Thus the dUTPase in these lysates has a higher capacity to be overloaded than does the excision system (i.e. uracil DNA glycosylase). However, the DNA sealing system (presumably DNA polymerase I and DNA ligase) apparently can handle all single-strand interruptions being introduced by uracil excision at the maximal rate, at least so that DNA synthesis can continue.     

Identification of uracil as a major lesion in E. coli DNA following the incorporation of 5-bromouracil, and some of the accompanying effects

Cultivation of E. coli cells in the presence of 5-bromodeoxyuridine (BUdR) leads to formation of lesions in the cellular DNA which affect its secondary structure, as reflected by changes in temperature profiles. Such DNA contains single-stranded regions susceptible to endonuclease S1. One of the major sources of the BU-induced lesions appears to be dehalogenation of incorporated 5-bromouracil (BU) residues, with accompanying formation of uracil. The presence of uracil residues in such DNA was demonstrated directly by chromatography of hydrolyzates, and by the susceptibility of such residues to uracil-DNA glycosylase. The number of uracil residues was dependent on the extent of damage in the DNA, and decreased during the DNA repair that accompanied reactivation of bromouracil-inactivated cells. Dehalogenation of incorporated BU presumably results in formation of apyrimidinic sites by uracil-DNA glycosylase, and then single-strand nicks either by AP-endonuclease and/or dehalogenation. The findings are relevant to the mechanism of BU-induced mutagenesis.     

Multiple uracil-DNA glycosylase activities in Deinococcus radiodurans

The extremely radiation resistant bacterium, Deinococcus radiodurans, contains a spectrum of genes that encode for multiple activities that repair DNA damage. We have cloned and expressed the product of three predicted uracil-DNA glycosylases to determine their biochemical function. DR0689 is a homologue of the Escherichia coli uracil-DNA glycosylase, the product of the ung gene; this activity is able to remove uracil from a U : G and U : A base pair in double-stranded DNA and uracil from single-stranded DNA and is inhibited by the Ugi peptide. DR1751 is a member of the class 4 family of uracil-DNA glycosylases such as those found in the thermophiles Thermotoga maritima and Archaeoglobus fulgidus. DR1751 is also able to remove uracil from a U : G and U : A base pair; however, it is considerably more active on single-stranded DNA. Unlike its thermophilic relatives, the enzyme is not heat stable. Another putative enzyme, DR0022, did not demonstrate any appreciable uracil-DNA glycosylase activity. DR0689 appears to be the major activity in the organism based on inhibition studies with D. radiodurans crude cell extracts utilizing the Ugi peptide. The implications for D. radiodurans having multiple uracil-DNA glycosylase activities and other possible roles for these enzymes are discussed.     

Role of uracil-DNA glycosylase in the repair of deaminated cytosine residues of DNA in Escherichia coli.

Uracil-DNA glycosylase, which acts specifically on uracil-containing DNA, was purified 250-fold from an extract of Escherichia coli 1100. The enzyme releases free uracil from DNA, producing alkali-labile apyrimidinic sites in the DNA. The enzyme is active on both native and heat-denatured DNA of phage PBS1, which contains uracil in place of thymine. piX174 DNA which had been treated with bisulfite and then at alkaline pH was susceptible to the action of uracil-DNA glycosylase. Since DNA treated with bisulfite alone was less susceptible to the enzyme, it is likely that the enzyme recognizes deaminated cytosine, namely uracil, but not bisulfite adducts of uracil and cytosine in the treated DNA. DNA treated with nitrite or hydroxylamine was not attacked by the enzyme. Enzyme activity acting on bisulfite-treated DNA was absent from an extract of E. coli mutant BD10 (ung). The mutant exhibited higher sensitivity to bisulfite than did the wild-type strain and was unable to reactivate phage T1 pre-exposed to bisulfite and weak alkali.