Coding properties of poly(deoxycytidylic acid) templates containing uracil or apyrimidinic sites: in vitro modulation of mutagenesis by deoxyribonucleic acid repair enzymes

Heat treatment of poly(deoxycytidylic acid)-[poly(dC)] induces the formation of dUMP residues, which code for dAMP when replicated by Escherichia coli DNA polymerases I and III. The specificity of dUMP coding properties is indicated by the quantitative relation between the dAMP incorporated and the frequency of dUMP residues in the heat-treated poly(dC). The dAMP incorporation is prevented by preincubation of uracil containing poly(dC) with uracil-DNA glycosylase. The excision of uracil by uracil-DNA glycosylase leads to the formation of apyrimidinic sites (AP sites), which are barely replicated in vitro under physiological conditions. However, the alteration of E. coli DNA polymerase I fidelity of replication by Mn2+ greatly stimulates the replication of AP sites. There is a preferential incorporation of dAMP, as compared to dTMP, opposite the AP sites. The dAMP incorporation is prevented by preincubation of poly(dC) containing AP sites with Micrococcus luteus AP endonuclease B. The results show a close association between DNA repair by base excision and the prevention of mutagenic processes in vitro. Furthermore, since the alteration of DNA polymerase fidelity allows some replication of the noncoding DNA lesion (AP site), this could imply a role in SOS-induced mutagenesis in vivo.     

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.     

Uracil in Ori<Subscript>s</Subscript> of herpes simplex 1 alters its specific recognition by origin binding protein (OBP): does virus induced uracil-DNA glycosylase play a key role in viral reactivation and replication?

We have recently demonstrated that mammalian uracil-DNA glycosylase activity is undetectable in adult neurons. On the basis of this finding we hypothesized that uracil, derived either from oxidative deamination of cytosine or misincorporation of dUMP in place of dTMP during DNA repair by the unique nuclear DNA polymerase present in adult neurons, DNA polymerase β, might accumulate in neuronal DNA. Uracil residues could also arise in the herpes simplex 1 (HSV1) genome during latency in nerve cells. We therefore suggest a role for the virus encoded uracil-DNA glycosylase in HSV1 reactivation and in the first steps of DNA replication. We show here 1) that the viral DNA polymerase incorporates dUTP in place of dTTP with a comparable efficiencyin vitro; 2) that virus specific DNA/protein interactions between the virus encoded origin binding protein and its target DNA sequence is altered by the presence of uracil residues in its central region TCGCA. Thus uracil, present in viral Ori_S or other key sequences could hamper the process leading to viral reactivation. Hence, HSV1 uracil-DNA glycosylase, dispensable in viral proliferation in tissue culture, could be essential in neurons for the “cleansing” of the viral genome of uracil residues before the start of replication.     

Uracil DNa-glycosylase from HeLa cells: general properties, substrate specificity and effect of uracil analogs.

Uracil-DNA glycosylase was partially purified from HeLa cells. Various substrates containing [3H]dUMP residues were prepared by nick-translation of calf thymus DNA. The standard substrate was double-stranded DNA with [3H]dUMP located internally in the chain. Compared to the release of uracil from this substrate, a 3-fold increase in the rate was seen with single-stranded DNA, and a 20-fold reduction in the rate was observed when the [3H]dUMP-residue was located at the 3'end. The rate of [3H]uracil release decreased progressively when one, two or three of the dNMP residues were replaced by the corresponding rNMP; in the extreme case when the substrate contained [3H]dUMP in addition to rCMP, rGMP, and rAMP, the rate of [3H]uracil release was less than 3% of that of the control. The enzyme was inhibited to the same extent by uracil and the uracil analogs 6-aminouracil and 5-azauracil, but very weakly, or not at all, by 5 other analogs. Our results suggest strongly that uracil-DNA glycosylase has a high degree of selectivity for uracil in dUMP residues located internally in DNA chains and that the recognition of the correct substrate also depends on the residues flanking dUMP being deoxyribonucleotides.     

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.     

Post-replicative base excision repair in replication foci.

Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication. We report that the major nuclear uracil-DNA glycosylase (UNG2) increases in S phase, during which it co-localizes with incorporated BrdUrd in replication foci. Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei. Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uracil-DNA glycosylase responsible. PCNA and replication protein A (RPA) co-localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two-hybrid assays, a peptide SPOT assay and enzyme-linked immunosorbent assays. These results demonstrate rapid post-replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork.     

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.     

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.     

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl(2). The functional significance of the UNG2.PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30-810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-(3)H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl(2). The stimulatory effect of PCNA was increased by the addition of MgCl(2); however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl(2) and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA.     

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.     

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.     

Genomic uracil and human disease

Uracil is present in small amounts in DNA due to spontaneous deamination of cytosine and incorporation of dUMP during replication. While deamination generates mutagenic U:G mismatches, incorporated dUMP results in U:A pairs that are not directly mutagenic, but may be cytotoxic. In most cells, mutations resulting from uracil in DNA are prevented by error-free base excision repair. However, in B-cells uracil in DNA is also a physiological intermediate in acquired immunity. Here, activation-induced cytosine deaminase (AID) introduces template uracils that give GC to AT transition mutations in the Ig locus after replication. When uracil-DNA glycosylase (UNG2) removes uracil, error-prone translesion synthesis over the abasic site causes other mutations in the Ig locus. Together, these processes are central to somatic hypermutation (SHM) that increases immunoglobulin diversity. AID and UNG2 are also essential for generation of strand breaks that initiate class switch recombination (CSR). Patients lacking UNG2 display a hyper-IgM syndrome with recurrent infections, increased IgM, strongly decreased IgG, IgA and IgE and skewed SHM. UNG2 is also involved in innate immune response against retroviral infections. Ung(-/-) mice have a similar phenotype and develop B-cell lymphomas late in life. However, there is no evidence indicating that UNG deficiency causes lymphomas in humans.     

Generation of single-nucleotide repair patches following excision of uracil residues from DNA.

The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase beta.     

hSMUG1 can functionally compensate for Ung1 in the yeast Saccharomyces cerevisiae

There are at least four distinct families of enzymes that recognize and remove uracil from DNA. Family-3 (SMUG1) enzymes have recently been identified and have a preference for uracil in single-stranded DNA when assayed in vitro. Here we investigate the in vivo function of SMUG1 using the yeast Saccharomyces cerevisiae as a model system. These organisms lack a SMUG1 homologue and use a single enzyme, Ung1 to carry out uracil-repair. When a wild-type strain is treated with antifolate agents to induce uracil misincorporation into DNA, S-phase arrest and cellular toxicity occurs. The arrest is characteristic of checkpoint activation due to single-strand breaks caused by continuous uracil removal and self-defeating DNA repair. When uracil-DNA glycosylase is deleted (deltaung1), cells continue through S-phase and arrest at G(2)/M, presumably due to the effects of stable uracil misincorporation in DNA. Pulsed field gel electrophoresis (PFGE) demonstrates that cells are able to complete DNA replication with uracil-substituted DNA and do not experience the extensive strand breakage attributed to uracil-DNA glycosylase-mediated repair. As a result, these cells experience early protection from antifolate-induced cytotoxicity. When either UNG1 or SMUG1 functions are reintroduced back into the null strain and then subjected to antifolate treatment, the cells revert back to the wild-type phenotype as shown by a restored sensitivity to drug and S-phase arrest. The arrest is accompanied by the accumulation of replication intermediates as determined by PFGE. Collectively, these data indicate that SMUG1 can act as a functional homolog of the family-1 uracil-DNA glycosylase enzymes.     

Arabidopsis Uracil DNA Glycosylase (UNG) Is Required for Base Excision Repair of Uracil and Increases Plant Sensitivity to 5-Fluorouracil

Uracil in DNA arises by misincorporation of dUMP during replication and by hydrolytic deamination of cytosine. This common lesion is actively removed through a base excision repair (BER) pathway initiated by a uracil DNA glycosylase (UDG) activity that excises the damage as a free base. UDGs are classified into different families differentially distributed across eubacteria, archaea, yeast, and animals, but remain to be unambiguously identified in plants. We report here the molecular characterization of AtUNG (Arabidopsis thaliana uracil DNA glycosylase), a plant member of the Family-1 of UDGs typified by Escherichia coli Ung. AtUNG exhibits the narrow substrate specificity and single-stranded DNA preference that are characteristic of Ung homologues. Cell extracts from atung^−/− mutants are devoid of UDG activity, and lack the capacity to initiate BER on uracil residues. AtUNG-deficient plants do not display any apparent phenotype, but show increased resistance to 5-fluorouracil (5-FU), a cytostatic drug that favors dUMP misincorporation into DNA. The resistance of atung^−/− mutants to 5-FU is accompanied by the accumulation of uracil residues in DNA. These results suggest that AtUNG excises uracil in vivo but generates toxic AP sites when processing abundant U:A pairs in dTTP-depleted cells. Altogether, our findings point to AtUNG as the major UDG activity in Arabidopsis.     

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.     

Human cytomegalovirus uracil DNA glycosylase is required for the normal temporal regulation of both DNA synthesis and viral replication.

Human cytomegalovirus (CMV) encodes a gene, UL114, whose product is homologous to the uracil DNA glycosylase and is highly conserved in all herpesviruses. This DNA repair enzyme excises uracil residues in DNA that result from the misincorporation of dUTP or spontaneous deamination of cytosine. We constructed a recombinant virus, RC2620, that contains a large deletion in the UL114 open reading frame and carries a 1.2-kb insert containing the Escherichia coli gpt gene. RC2620 retains the capacity to replicate in primary human fibroblasts and reaches titers that are similar to those produced by the parent virus but exhibits a significantly longer replication cycle. Although the rate of expression of alpha and beta gene products appears to be unaffected by the mutation, DNA synthesis fails to proceed normally. Once initiated, DNA synthesis in mutant virus-infected cells proceeds at the same rate as with wild-type virus, but initiation is delayed by 48 h. The mutant virus also exhibits two predicted phenotypes: (i) hypersensitivity to the nucleoside analog 5-bromodeoxyuridine and (ii) retention of more uracil residues in genomic DNA than the parental virus. Together, these data suggest UL114 is required for the proper excision of uracil residues from viral DNA but in addition plays some role in establishing the correct temporal progression of DNA synthesis and viral replication. Although such involvement has not been previously observed in herpesviruses, a requirement for uracil DNA glycosylase in DNA replication has been observed in poxviruses.     

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.     

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.     

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.