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.     

Lethality of a dut (deoxyuridine triphosphatase) mutation in Escherichia coli.

A chloramphenicol resistance gene was cloned into a plasmid-borne dut gene, producing an insertion mutation that was then transferred to the chromosome by allelic exchange. The mutation could not be acquired by haploid strains through substitutive recombination, even when two flanking markers were simultaneously transduced. The insertion was easily transferred, via generalized transduction, into the chromosomal dut region of strains harboring a lambda dut + transducing phage; however, the resulting dut mutant/lambda dut + merodiploid could not then be cured of the prophage. This apparent lethality of the mutation could not be explained by effects on adjacent genes; the dfp gene retained complementing activity, and a ttk insertion mutant was viable. The dut gene product, deoxyuridine triphosphatase, is known to reduce incorporation of uracil into DNA and to be required in the de novo synthesis of thymidylate. Therefore, an attempt was made to determine whether the dut insertion would be tolerated in strains carrying the following compensatory mutations: dcd (dCTP deaminase) and cdd (deoxycytidine deaminase), which should reduce dUTP formation; ung (uracil-DNA glycosylase), which should reduce fatally excessive excision repair; deoA (thymidine phosphorylase), which should enhance the utilization of exogenous thymidine; and sulA, which should reduce the lethal side effects of SOS regulon induction. These mutations, either alone or in various combinations, did not permit the survival of a haploid dut insertion mutant, suggesting that the dut gene product might have an essential function apart from its deoxyuridine triphosphatase activity.     

Escherichia coli mutants deficient in deoxyuridine triphosphatase.

Mutants deficient in deoxyuridine triphosphatase (dUTPase) were identified by enzyme assays of randomly chosen heavily mutagenized clones. Five mutants of independent origin were obtained. One mutant produced a thermolabile enzyme, and it was presumed to have a mutation in the structural gene for dUTPase, designated dut. The most deficient mutant had the following associated phenotypes: less than 1% of parental dUTPase activity, prolonged generation time, increased sensitivity to 5'-fluorodeoxyuridine, increased rate of spontaneous mutation, increased rate of recombination (hyper-Rec), an inhibition of growth in the presence of 2 mM uracil, and a decreased ability to support the growth of phage P1 (but not T4 or lambda). This mutation also appeared to be incompatible with pyrE mutations. A revertant selected by its faster growth had regained dUTPase activity and lost its hyper-Rec phenotype. Many of the properties of the dut mutants are compatible with their presumed increased incorporation of uracil into DNA and the subsequent transient breakage of the DNA by excision repair.     

Role of exonuclease III in the base excision repair of uracil-containing DNA.

Mutants of Escherichia coli K-12 deficient in both exonuclease III (the product of the xth gene) and deoxyuridine triphosphatase (the dut gene product) are inviable at high temperatures and undergo filamentation when grown at such temperatures. In dut mutants, the dUTP pool is known to be greatly enhanced, resulting in an increased substitution of uracil for thymine in DNA during replication. The subsequent removal of uracil from the DNA by uracil-DNA glycosylase produces apyrimidinic sites, at which exonuclease III is known to have an endonucleolytic activity. The lethality of dut xth mutants, therefore, indicates that exonuclease III is important for this base-excision pathway and suggests that unrepaired apyrimidinic sites are lethal. Two confirmatory findings were as follows. (i) dut xth mutants were viable if they also had a mutation in the uracil-DNA glycosylase (ung) gene; such mutants should not remove uracil from DNA and should not, therefore, generate apyrimidinic sites. (ii) In the majority of the temperature-resistant revertants isolated, viability had been restored by a mutation in the dCTP deaminase (dcd) gene; such mutations should decrease dUTP production and hence uracil misincorporation. The results indicate that, in dut mutants, exonuclease III is essential for the repair of uracil-containing DNA and of apyrimidinic sites.     

Isolation and partial characterization of a bacteriophage T5 mutant unable to induce thymidylate synthetase and its use in studying the effect of uracil incorporation into DNA on early gene expression.

A mutant of phage T5 which is unable to induce thymidylate synthetase was isolated. T5 thy mutants synthesized less DNA than did wild-type T5, and the burst size of progeny phage was correspondingly reduced two- to threefold in thy+ Escherichia coli. No DNA or progeny phage were made in E. coli thy hosts grown in the absence of exogenous thymine. When the T5 thy mutation was recombined with a T5 dut mutation (unable to induce dUTPase), replication resulted in progeny which contained significant amounts of uracil in their DNA, and these phage failed to produce plaques unless the plating host was deficient in uracil-DNA glycosylase. T5 phage containing various amounts of uracil in their DNA were prepared and used to determine the effect of uracil on the induction of the early enzyme dTMP kinase. The presence of uracil in the parental DNA increased the rate of induction of this enzyme by about 2.5-fold. The T5 thy gene was mapped and is located near the T5 frd gene on the B region of the T5 genome.     

The properties of a bacteriophage T5 mutant unable to induce deoxyuridine 5'-triphosphate nucleotidohydrolase. Synthesis of uracil-containing T5 deoxyribonucleic acid.

Bacteriophage T5 induces a deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) activity during infection of Escherichia coli. A T5 mutant (T5 dut) unable to induce this dUTPase activity has been isolated. Although this mutant is viable, the E. coli dUTPase activity is not sufficiently active to exclude uracil from the progeny DNA and about 3% of the thymine is replaced by uracil. When the mutant is grown in an E. coli dut host about 12% of the thymine in the progeny DNA is replaced by uracil. T5 phage containing 12% uracil can replicate in uracil-DNA glycosylase-deficient (ung) hosts with high efficiency, but fail to replicate in ung+ hosts. The amount of thymine replaced by uracil in the progeny produced in dut hosts is nearly independent of the ung genotype, indicating that the host uracil-DNA glycosylase-dependent repair pathway is not operating efficiently to remove uracil from T5 progeny DNA.     

Toxoplasma gondii: the enzymic defect of a mutant resistant to 5-fluorodeoxyuridine.

We have previously described a mutant of Toxoplasma gondii that was 100-fold more resistant to 5-fluorodeoxyuridine, as measured by growth in human fibroblast cultures. Various pyrimidine salvage enzymes were measured in the wild type and the mutant parasites to determine the biochemical basis for resistance to fluorodeoxyuridine. Both the resistant mutant and the wild type parasite had little or no uridine kinase, an enzyme readily detectable in the human fibroblast host cells. Uridine and deoxyuridine phosphorylases were found in both parasites while human fibroblasts had much less of these enzymes. The critical difference between the mutant and the wild type parasites proved to be a 100-fold lower concentration of uracil phosphoribosyltransferase in the fluorodeoxyuridine-resistant mutant. A back mutant of the resistant strain, selected for its ability to use uracil, simultaneously regained uracil phosphoribosyltransferase and sensitivity to fluorodeoxyuridine. This enzymic evidence together with previously published data show that in wild type T. gondii, deoxyuridine is incorporated into nucleic acids through a phosphorolysis to produce uracil which is then converted to uridylic acid by uracil phosphoribosyltransferase.     

dcd (dCTP deaminase) gene of Escherichia coli: mapping, cloning, sequencing, and identification as a locus of suppressors of lethal dut (dUTPase) mutations.

In Escherichia coli, most of the dUMP that is used as a substrate for thymidylate synthetase is generated from dCTP through the sequential action of dCTP deaminase and dUTPase. Some mutations of the dut (dUTPase) gene are lethal even when the cells are grown in the presence of thymidine, but their lethality can be suppressed by extragenic mutations that can be produced by transposon insertion. Six suppressor mutations were tested, and all were found to belong to the same complementation group. The affected gene was cloned, it was mapped by hybridization with a library of recombinant DNA, and its nucleotide sequence was determined. The gene is at 2,149 kb on the physical map. Its product, a 21.2-kDa polypeptide, was overproduced 1,000-fold via an expression vector and identified as dCTP deaminase, the enzyme affected in previously described dcd mutants. Null mutations in dcd probably suppress the lethality of dut mutations by reducing the accumulation of dUTP, which would otherwise lead to the excessive incorporation of uracil into DNA.     

Effect of an Asp80Ala substitution on the binding of dUTP and dUMP to Trypanosoma cruzi dUTPase

dUTPase (deoxyuridine 5'-triphosphate nucleotide hydrolase) is an enzyme responsible for maintaining low levels of intracellular dUTP and thus prevents uracil incorporation into DNA by DNA polymerases during replication and repair processes. The thermodynamics of binding for both dUTP and dUMP (deoxyuridine 5'-monophosphate) to the D80A mutant form of Trypanosoma cruzi dUTPase have been investigated by fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry. In the presence of magnesium, approximately a 30-fold decrease in the value of the k(cat) and a 15-fold increase in the K(m) for dUTP hydrolysis was calculated while a 5-fold decrease was observed in the affinity for dUMP. In the absence of magnesium, the affinity for dUTP binding was similar for both enzymes while that for dUMP was lowered 3-fold as a consequence of the mutation. Calorimetric titrations in several buffers with different ionization heats rendered similar proton exchanges during the binding of dUMP. Thus, apparently the side chain of Asp 80 does not seem to vary its protonation state during the binding process. The enthalpy change values for the D80A mutant hardly change with temperature and, in addition, were Mg(2+) independent. We conclude that the D80A mutation induces only a slight conformational change in the active site yet results in a significant alteration of nucleotide binding and modifies the ability of the enzyme to discriminate between dUTP and dUMP when magnesium is present.     

Fragmentation of replicating chromosomes triggered by uracil in DNA

The dut mutants of Escherichia coli fail to hydrolyze dUTP and thus incorporate uracil into their DNA, suffering from chromosomal fragmentation. The postulated mechanism for the double-strand DNA breaks is clustered uracil excision, which requires high density of DNA-uracils. However, we did not find enough uracil residues or excision nicks in the DNA of dut mutants to account for clustered uracil excision. Using a dut recBC(Ts) mutant of E.coli to inquire into the mechanism of uracil-triggered chromosomal fragmentation, we show that this fragmentation requires DNA replication and, in turn, inhibits replication of the chromosomal terminus. As a result, origin-containing sub-chromosomal fragments accumulate in dut recBC conditions, indicating preferential demise of replication bubbles. We propose that the basic mechanism of the uracil-triggered chromosomal fragmentation is replication fork collapse at uracil-excision nicks. Possible explanations for the low level terminus fragmentation are also considered.     

dUTPase activity is critical to maintain genetic stability in Saccharomyces cerevisiae

We identified a viable allele (dut1-1) of the DUT1 gene that encodes the dUTPase activity in Saccharomyces cerevisiae. The Dut1-1 protein possesses a single amino acid substitution (Gly82Ser) in a conserved motif nearby the active site and exhibits a greatly reduced dUTPase activity. The dut1-1 single mutant exhibits growth delay and cell cycle abnormalities and shows a strong spontaneous mutator phenotype. All phenotypes of the dut1-1 mutant are suppressed by the simultaneous inactivation of the uracil DNA N-glycosylase, Ung1. However, the ung1 dut1-1 double mutant accumulates uracil in its genomic DNA. The viability of the dut1-1 mutant is greatly impaired by the simultaneous inactivation of AP endonucleases. These data strongly suggest that the phenotypes of the dut1-1 mutant result from the incorporation of dUMPs into DNA subsequently converted into AP sites. The analysis of the dut1-1 strain mutation spectrum showed that cytosines are preferentially incorporated in front of AP sites in a Rev3-dependent manner during translesion synthesis. These results point to a critical role of the Dut1 protein in the maintenance of the genetic stability. Therefore, the normal cellular metabolism, and not only its byproducts, is an important source of endogenous DNA damage and genetic instability in eukaryotic cells.     

Cloning of the dut (deoxyuridine triphosphatase) gene of Escherichia coli

Through the molecular cloning of DNA, cells were obtained that could produce a 300-fold increased level of deoxyuridine triphosphatase (dUTPase). First, lambda pyrE-dut phages were constructed from restriction endonuclease fragments. They contained a segment of Escherichia coli DNA that spanned the structural genes for dUTPase (dut) and orotidylate pyrophosphorylase (pyrE). The initial isolates demonstrated poor enzyme production and impaired growth. Improved enzyme yields were then obtained from large-plaque derivatives and from mutants with partial deletions of the cloned DNA. The deletion mutants were isolated after the induction of a recombinant prophage whose DNA was too large to be packaged. Finally, a 3.3-kb segment of DNA, containing the dut gene, was transferred to plasmid vectors. The recombinants and their levels of dUTPase overproduction (relative to that of wild type cells) were as follows: a thermoinducible lambda pyrE-dut phage, 45-fold (10-fold for orotidylate pyrophosphorylase); a dut-ColE1 type plasmid, 15-fold; and a thermoinducible dut-lambda-ColE1 chimera, 14-fold before induction and 300-fold after induction.     

Pyrimidine excretion by cultured fibroblasts: effect of mutational deficiency in pyrimidine salvage enzymes

In cultured fibroblasts, a mutation resulting in deficiency of a pyrimidine salvage enzyme leads to excretion of related pyrimidines. For example, absence of thymidine kinase led to loss of thymidine and deoxyuridine, and absence of deoxycytidine kinase to loss of deoxyuridine. Both wild type and mutant cells excreted uracil; if established lines are representative in this respect, a fully adequate salvage system for uracil does not seem to be present in the fibroblast.     

5-chloro-2'-deoxyuridine cytotoxicity results from base excision repair of uracil subsequent to thymidylate synthase inhibition

The lack of a phenotypic alteration of 5-hydroxymethyluracil (hmUra) DNA glycosylase (hmUDG) deficient Chinese hamster V79mut1 cells exposed to DNA-damaging agents known to produce hmUra has raised the question whether there might be DNA substrates other than hmUra for hmUDG. Based on the structural similarity between 5-chlorouracil (ClUra) and hmUra and the observations that 5-chloro-2'-deoxyuridine (CldUrd) induces base excision repair (BER) events, we asked whether hmUDG or some other DNA BER enzyme is responsible for the removal of ClUra from DNA. An in vivo flow cytometry assay with FITC-anti-BrdUrd (which cross-reacts with CldUrd) showed that exogenous CldUrd is incorporated into DNA. However, both in vivo and in vitro experiments indicated that ClUra is not excised from DNA by hmUDG or other DNA glycosylase activities. The absence of removal of ClUra by hmUDG raised the question whether DNA strand breaks occurred subsequent to thymidylate synthase inhibition, leading to deoxyuridine incorporation, followed by cleavage of uracil from DNA by uracil DNA glycosylase (UDG). An in vivo thymidylate synthase activity assay in V79 cells demonstrated that CldUrd treatment inhibits thymidylate synthase as effectively as 5-fluoro-2'-deoxyuridine (FdUrd) treatment. Uracil, a known UDG inhibitor, partially reverses the cytotoxic effects of CldUrd on V79 cells, thus confirming that CldUrd induced cytotoxicity is a result of UDG activity. Our results demonstrated that while CldUrd is not directly repaired from DNA, its cytotoxicity is directly due to the UDG removing uracil subsequent to inhibition of thymidylate synthase by CldUMP.     

Nuclear localization of de novo thymidylate biosynthesis pathway is required to prevent uracil accumulation in DNA

Uracil accumulates in DNA as a result of impaired folate-dependent de novo thymidylate biosynthesis, a pathway composed of the enzymes serine hydroxymethyltransferase (SHMT), thymidylate synthase (TYMS), and dihydrofolate reductase. In G1, this pathway is present in the cytoplasm and at S phase undergoes small ubiquitin-like modifier-dependent translocation to the nucleus. It is not known whether this pathway functions in the cytoplasm, nucleus, or both in vivo. SHMT1 generates 5,10-methylenetetrahydrofolate for de novo thymidylate biosynthesis, a limiting step in the pathway, but also tightly binds 5-methyltetrahydrofolate in the cytoplasm, a required cofactor for homocysteine remethylation. Overexpression of SHMT1 in cell cultures inhibits folate-dependent homocysteine remethylation and enhances thymidylate biosynthesis. In this study, the impact of increased Shmt1 expression on folate-mediated one-carbon metabolism was determined in mice that overexpress the Shmt1 cDNA (Shmt1tg+ mice). Compared with wild type mice, Shmt1tg+ mice exhibited elevated SHMT1 and TYMS protein levels in tissues and evidence for impaired homocysteine remethylation but surprisingly exhibited depressed levels of nuclear SHMT1 and TYMS, lower rates of nuclear de novo thymidylate biosynthesis, and a nearly 10-fold increase in uracil content in hepatic nuclear DNA when fed a folate- and choline-deficient diet. These results demonstrate that SHMT1 and TYMS localization to the nucleus is essential to prevent uracil accumulation in nuclear DNA and indicate that SHMT1-mediated nuclear de novo thymidylate synthesis is critical for maintaining DNA integrity.     

Specific mutator effects of ung (uracil-DNA glycosylase) mutations in Escherichia coli.

Studies of trpA reversions revealed that G:C leads to A:T transitions were stimulated about 30-fold in E. coli ung mutants, whereas other base substitutions were not affected. A dUTPase (dut) mutation, which increases the incorporation of uracil into DNA in place of thymine, had no significant effect on the rate of G:C leads to A:T transitions. The results support the proposal that the glycosylase functions to reduce the mutation rate in wild-type cells by acting in the repair of DNA cytosine residues that have undergone spontaneous deamination to uracil. Further support was provided by the finding that when lambda bacteriophages were treated with bisulfite, an agent known to produce cytosine deamination, the frequency of clear-plaque mutants was increased an additional 20-fold by growth on an ung host. Bisulfite-induced mutations of the cellular chromosome, however, were about equal in ung+ and ung strains; it was found that during the treatment of ung+ cells with bisulfite, the glycosylase was inactivated.     

In situ autoradiographic detection of folylpolyglutamate synthetase activity

The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of [6-3H]deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of [6-3H]deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which function in mammalian cells.     

Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast.

(Deoxy)thymidylate (dTMP) kinase is an enzyme which phosphorylates dTMP to dTDP in the presence of ATP and magnesium. This enzyme is important in cellular DNA synthesis because the synthesis of dTTP, either via the de novo pathway or through the exogenous supply of thymidine, requires the activity of this enzyme. It has been suggested that the activities of the enzymes involved in DNA precursor biosynthesis, such as thymidine kinase, thymidylate synthase, thymidylate kinase, and dihydrofolate reductase, are subjected to cell cycle regulation. Here we describe the cloning of a human dTMP kinase cDNA by functional complementation of a yeast dTMP kinase temperature-sensitive mutant at the non-permissive temperature. The nucleotide sequence of the cloned human cDNA is predicted to encode a 24 KD protein that shows considerable homology with the yeast and vaccinia virus dTMP kinase enzymes. The human enzyme activity has been investigated by expressing it in yeast. In this work, we demonstrate that the cloned human cDNA, when expressed in yeast, produces dTMP kinase activity.     

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.     

Mycoplasma Pneumoniae Thymidine Phosphorylase

Mycoplasma pneumoniae (Mpn) is a human pathogen causing acute respiratory diseases and accounts for approximately 30% cases of community-acquired pneumonia. Co-infection with Mycoplasmas compromises the efficacy of anticancer and antiviral nucleoside analog-based drugs due to the presence of Mycoplasma thymidine phosphorylase (TP). In this study, a TP-deficient strain of Mpn was generated in order to study the effect of Mpn TP in the metabolism of nucleoside analogs. Deficiency in TP activity led to increased uptake and incorporation of radiolabeled deoxyuridine and uracil but thymidine uptake was not affected. The activities of enzymes in the salvage of thymidine and deoxyuridine, e.g., thymidine kinase and uracil phosphoribosyltransferase were upregulated in the TP-deficient mutant, which may explain the increased uptake of deoxyuridine and uracil. Thirty FDA-approved anticancer and antiviral nucleoside and nucleobase analogs were used to screen their inhibitory activity toward the TP mutant and the wild type strain. Seven analogs were found to inhibit strongly the growth of both wild type and TP mutant. Differences in the inhibitory effect of several purine analogs between the two strains were observed. Further study is needed in order to understand the mechanism of inhibition caused by these analogs. Our results indicated that TP is not an essential gene for Mpn survival and TP deficiency affects other enzymes in Mpn nucleotide metabolism, and suggested that Mycoplasma nucleotide biosynthesis pathway enzymes are potential targets for future development of antibiotics.