Structure of Escherichia coli uracil DNA glycosylase and its complexes with nonhydrolyzable substrate analogues in solution observed by synchrotron small-angle X-ray scattering

The structure of native and modified uracil DNA glycosylase from E. coli in solution was studied by synchrotron small-angle X-ray scattering. The modified enzyme (6His-uracyl DNA glycosylase) differs from the native one by the presence of an additional N-terminal 11-meric sequence amino acid residues including a block of six His residues. It was found that the conformations of these enzymes in solution at moderate ionic strength (60 mM NaCI) substantially differ in spite of minimal differences in the amino acid sequences and functional activity. The structure of native uracil DNA glycosylase in solution is close to that in crystal, showing a tendency for association. The interaction of this enzyme with nonhydrolyzable analogues of DNA ligands causes a partial dissociation of associates and a compactization of protein structure. At the same time, 6His-uracyl DNA glycosylase has a compact structure essentially different from the crystal one. A decrease in the ionic strength of solution results in a partial disruption of compact structure of the modified protein, without changes in its functional activity.     

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.     

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.     

An effective scheme to produce recombinant uracil-DNA glycosylase of Escherichia coli for PCR diagnostics

An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori vector within the same reading frame with six residues of histidine in the C-terminal sequence. Using this vector and the E. coli DH5α, a host-vector expression system has been developed and conditions for protein synthesis have been optimized. To purify the protein, metal affinity chromatography with further dialysis was used to remove imidazole. The enzyme yield was no less than 60 mg of the end protein per 1 L of the culture medium. The concordance between amino acid sequences of the recombinant and native enzymes was proved by peptide mass fingerprinting and mass spectrometry. A rapid test to determine the activity of the enzyme preparation was suggested. It was found that the activity of 1.0 mg of the recombinant protein is no less than 3 × 10³ units. The recombinant enzyme was most stable at pH 8.0 and an ionic strength of the solution equal to 200 mM; it lost its activity completely for 10 min at 60°C. Storage during 1 year at ?20°C resulted in the loss of no more than 30% of activity. In the enzyme preparation, the activity of DNase was absent. The free energy of the unfolding of the protein globule of the recombinant uracil-DNA glycosylase is 23.1 ± 0.2 kJ/mol. The data obtained indicate that the recombinant enzyme may be recommended for use in PCR diagnostics to prevent the appearance of false positive results caused by pollution of the reaction mixture by products of the preceding reactions.     

Staphylococcus aureus protein SAUGI acts as a uracil-DNA glycosylase inhibitor

DNA mimic proteins are unique factors that control the DNA binding activity of target proteins by directly occupying their DNA binding sites. The extremely divergent amino acid sequences of the DNA mimics make these proteins hard to predict, and although they are likely to be ubiquitous, to date, only a few have been reported and functionally analyzed. Here we used a bioinformatic approach to look for potential DNA mimic proteins among previously reported protein structures. From ∼14 candidates, we selected the Staphylococcus conserved hypothetical protein SSP0047, and used proteomic and structural approaches to show that it is a novel DNA mimic protein. In Staphylococcus aureus, we found that this protein acts as a uracil-DNA glycosylase inhibitor, and therefore named it S. aureus uracil-DNA glycosylase inhibitor (SAUGI). We also determined and analyzed the complex structure of SAUGI and S. aureus uracil-DNA glycosylase (SAUDG). Subsequent BIAcore studies further showed that SAUGI has a high binding affinity to both S. aureus and human UDG. The two uracil-DNA glycosylase inhibitors (UGI and p56) previously known to science were both found in Bacillus phages, and this is the first report of a bacterial DNA mimic that may regulate SAUDG’s functional roles in DNA repair and host defense.     

Purification and properties of mitochondrial uracil-DNA glycosylase from rat liver

Uracil-DNA glycosylase from rat liver mitochondria, an inner membrane protein, has been purified approximately 575,000-fold to apparent homogeneity. During purification two distinct activity peaks, designated form I and form II, were resolved by phosphocellulose chromatography. Form I constituted approximately 85% while form II was approximately 15% of the total activity; no interconversion between the forms was observed. The major form was purified as a basic protein with an isoelectric point of 10.3. This enzyme consists of a single polypeptide with an apparent Mr of 24,000 as determined by recovering glycosylase activity from a sodium dodecyl sulfate-polyacrylamide gel. A native Mr of 29,000 was determined by glycerol gradient sedimentation. The purified enzyme had no detectable exonuclease, apurinic/apyrimidinic endonuclease, DNA polymerase, or hydroxymethyluracil-DNA glycosylase activity. A 2-fold preference for single-stranded uracil-DNA over a duplex substrate was observed. The apparent Km for uracil residues in DNA was 1.1 microM, and the turnover number is about 1000 uracil residues released per minute. Both free uracil and apyrimidinic sites inhibited glycosylase activity with Ki values of approximately 600 microM and 1.2 microM, respectively. Other uracil analogues including 5-(hydroxymethyl)uracil, 5-fluorouracil, 5-aminouracil, 6-azauracil, and 2-thiouracil or analogues of apyrimidinic sites such as deoxyribose and deoxyribose 5'-phosphate did not inhibit activity. Both form I and form II had virtually identical kinetic properties, and the catalytic fingerprints (specificity for uracil residues located in a defined nucleotide sequence) obtained on a 152-nucleotide restriction fragment of M13mp2 uracil-DNA were almost identical. These properties differentiated the mitochondrial enzyme from that of the uracil-DNA glycosylase purified from nuclei of the same source.     

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.     

The mouse uracil-DNA glycosylase gene: isolation of cDNA and genomic clones and mapping ung to mouse chromosome 5

Uracil-DNA glycosylase (UDG) is the enzyme responsible for the first step in the base-excision repair pathway that specifically removes uracil from DNA. Here we report the isolation of the cDNA and genomic clones for the mouse uracil-DNA glycosylase gene (ung) homologous to the major placental uracil-DNA glycosylase gene (UNG) of humans. The complete characterization of the genomic organization of the mouse uracil-DNA glycosylase gene shows that the entire mRNA coding region for the 1.83-kb cDNA of the mouse ung gene is contained in an 8.2-kb SstI genomic fragment which includes six exons and five introns. The cDNA encodes a predicted uracil-DNA glycosylase (UDG) protein of 295 amino acids (33 kDa) that is highly similar to a group of UDGs that have been isolated from a wide variety of organisms. The mouse ung gene has been mapped to mouse chromosome 5 using fluorescence in situ hybridization (FISH).     

Characterization of the full length uracil-DNA glycosylase in the extreme thermophile Thermotoga maritima

A full length (192 amino acids) uracil-DNA glycosylase (TMUDG) has been expressed and purified from the extreme thermophile Thermotoga maritima. This protein is active up to 85 degrees C. The enzyme is product inhibited by abasic sites in DNA and weakly inhibited by uracil. TMUDG was originally cloned from an ORF which encoded a protein of 185 amino acids. This shorter protein was stable up to 70-75 degrees C and it seemed unusual that this enzyme had an optimal activity temperature below the growth temperature of the organism (80-90 degrees C). Following the publication of the complete genomic sequence of T. maritima, it was shown that the gene contains an additional seven amino acids (LYTREEL) at the N-terminal end of the protein. It is suggested that these seven residues are important in maintaining proper protein folding that results in increased temperature stability. We have also demonstrated that TMUDG can substitute for the Escherichia coli uracil-DNA glycosylase and initiate base excision repair using a closed circular DNA substrate containing a unique U:G base pair.     

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 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.     

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.     

Intracellular localization of rat-liver uracil-DNA glycosylase. Purification and properties of the chromatin enzyme

Most of the uracil-DNA glycosylase of the rat liver cell is located in chromatin; there is, however, some activity in the nuclear sap and in the cytoplasm. The chromatin uracil-DNA glycosylase has been purified; the preparation is devoid of endonuclease and exonuclease activities; the enzyme does not need divalent cations, has a broad optimum pH around 8, is strongly inhibited by increasing ionic strength and free uracil. The apparent Km is independent of the strandedness of the DNA substrate containing uracil, but V is slightly higher with the single-stranded substrate. The frequency of uracil substitution in the double-stranded DNA influences the kinetic parameters: a higher frequency increases both Km and V. The inhibitory effects of NaCl and free uracil are greater when the substrate is double-stranded rather than single-stranded. It is speculated that, acting either on the DNA or on the enzyme, both oppose the opening of the double helix necessary for the formation of the enzyme-substrate complex. The increased reaction rate with a higher frequency of uracil residues in double-stranded DNA is interpreted as a tendency for the repair enzyme to work in a processive way. It is supposed that processivity also occurs with single-stranded DNA and that it is opposed by both NaCl and free uracil, explaining a greater inhibition when the single-stranded substrate has a higher uracil content.     

Characterization of the Escherichia coli uracil-DNA glycosylase.inhibitor protein complex.

The Bacillus subtilis bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein was characterized and shown to form a stable complex with Escherichia coli uracil-DNA glycosylase (Ung). As determined by mass spectrometry, the Ugi protein had a molecular weight of 9,474. We confirmed this value by sedimentation equilibrium centrifugation and determined that Ugi exists as a monomeric protein in solution. Amino acid analysis performed on both Ugi and Ung proteins was in excellent agreement with the amino acid composition predicted from the respective nucleotide sequence of each gene. The Ung.Ugi complex was resolved from its constitutive components by nondenaturing polyacrylamide gel electrophoresis and shown to possess a 1:1 stoichiometry. Analytical ultracentrifugation studies revealed that the Ung.Ugi complex had a molecular weight of 35,400, consistent with the complex containing one molecule each of Ung and Ugi. The acidic isoelectric points of the protein species were 6.6 (Ung) and 4.2 (Ugi), whereas the Ung.Ugi complex had an isoelectric point of 4.9. Dissociation of the Ung.Ugi complex by SDS-polyacrylamide gel electrophoresis revealed no apparent alteration in the molecular weight of either polypeptide subsequent to binding. Furthermore, when the Ung.Ugi complex was treated with urea and resolved by urea-polyacrylamide gel electrophoresis, both uracil-DNA glycosylase and inhibitor activities were recovered from the dissociated complex. Thus, the complex seems to be reversible. In addition, we demonstrated that the Ugi interaction with Ung prevents enzyme binding to DNA and dissociates uracil-DNA glycosylase from a preformed DNA complex.     

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.     

Purification of the human O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase, the latter to apparent homogeneity

Uracil-DNA glycosylase, the enzyme that catalyzes the release of free uracil from single-stranded and double-stranded DNA, has been purified 26,600-fold from HeLa S3 cell extracts. The enzyme preparation was essentially homogeneous as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme is a small monomeric protein of molecular mass 29 kDa. A minor uracil-DNA glycosylase preparation was also obtained in the final chromatographic step. This preparation is homogeneous with a molecular mass of 29 kDa and may represent the mitochondrial enzyme. This report also presents a 700-fold purification of HeLa S3 cell O6-methylguanine-DNA methyltransferase. The glycosylase and methyltransferase showed very similar chromatographic properties. The report indicates that the lability of the methyltransferase upon purification may be a consequence of the total separation of the two DNA repair enzymes or of the possibility that some other stabilizing factor is involved.     

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.     

Mutations in active-site residues of the uracil-DNA glycosylase encoded by vaccinia virus are incompatible with virus viability.

The D4R gene of vaccinia virus encodes a functional uracil-DNA glycosylase that is essential for viral viability (D. T. Stuart, C. Upton, M. A. Higman, E. G. Niles, and G. McFadden, J. Virol. 67:2503-2513, 1993), and a D4R mutant, ts4149, confers a conditional lethal defect in viral DNA replication (A. K. Millns, M. S. Carpenter, and A. M. DeLange, Virology 198:504-513, 1994). The mutant ts4149 protein was expressed in vitro and assayed for uracil-DNA glycosylase activity. Less than 6% of wild-type activity was observed at permissive temperatures, but the ts4149 protein was completely inactive at the nonpermissive temperature. Mutagenesis of the ts4149 gene back to wild type (Arg-179-->Gly) restored full activity. The ts4149 protein was considerably reduced in lysates of cells infected at the permissive temperature, and its activity was undetectable, even in the presence of the uracil glycosylase inhibitor protein, which inhibits the host uracil-DNA glycosylases but not that of vaccinia virus. Thus the ts4149 protein is thermolabile, correlating uracil removal with vaccinia virus DNA replication. Three active-site amino acids of the vaccinia virus uracil-DNA glycosylase were mutated (Asp-68-->Asn, Asn-120-->Val, and His-181-->Leu), producing proteins that were completely defective in uracil excision but still retained the ability to bind DNA. Each mutated D4R gene was transfected into vaccinia virus ts4149-infected cells in order to assess the recombination events that allowed virus survival at 40 degrees C. Genetic analysis and sequencing studies revealed that the only viruses to survive were those in which recombination eliminated the mutant locus. We conclude that the uracil cleavage activity of the D4R protein is essential for its function in vaccinia virus DNA replication, suggesting that the removal of uracil residues plays an obligatory role.