Removal Of Uracil Research Articles

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.

Structural basis for the removal and exclusion of uracil from DNA

Structural basis for the removal and exclusion of uracil from DNA

Human immunodeficiency virus type 1 Vpr protein binds to the uracil DNA glycosylase DNA repair enzyme

The role of the accessory gene product Vpr during human immunodeficiency virus type 1 infection remains unclear. We have used the yeast two-hybrid system to identify cellular proteins that interact with Vpr and could be involved in its function. A cDNA clone which encodes the human uracil DNA glycosylase (UNG), a DNA repair enzyme involved in removal of uracil in DNA, has been isolated. Interaction between Vpr and UNG has been demonstrated by in vitro protein-protein binding assays using translated, radiolabeled Vpr and UNG recombinant proteins expressed as a glutathione S-transferase fusion protein. Conversely, purified UNG has been demonstrated to interact with Vpr recombinant protein expressed as a glutathione S-transferase fusion protein. Coimmunoprecipitation experiments confirmed that Vpr and UNG are associated within cells expressing Vpr. By using a panel of C- and N-terminally deleted Vpr mutants, we have determined that the core protein of Vpr, spanning amino acids 15 to 77, is involved in the interaction with UNG. We also demonstrate by in vitro experiments that the enzymatic activity of UNG is retained upon interaction with Vpr.

The phosphodiester bond 3' to a deoxyuridine residue is crucial for substrate binding for uracil DNA N-glycosylase.

Using the method of water-soluble carbodiimide-induced chemical ligation, four 27-member oligodeoxyribonucleotides containing a pyrophosphate internucleotide bond near or adjacent to a deoxyuridine residue were prepared. Escherichia coli uracil DNA N-glycosylase (UDG) activity was found to be sensitive to the presence of an internucleotide pyrophosphate bond in both single- and double-stranded DNA. The rate of uracil excision from single-stranded DNA containing a pyrophosphate bond adjacent to the uracil residue, either 3' or 5', was 0.01% and 0.1% of the rate of uracil removal from control DNA without a pyrophosphate bond, respectively. The rate of uracil excision from duplex DNA containing a pyrophosphate bond 3' or 5' to the uracil residue was also reduced, being 0.1% and 1% the rate of uracil removal from the corresponding duplex DNA control. Placing the pyrophosphate bond one nucleotide 5' or 3' away from the deoxyuridine in both single- and double-stranded oligodeoxyribonucleotides provided much better substrates for UDG. Kinetic measurements showed that the pyrophosphate bond placed adjacent to the deoxyuridine residue drastically reduced the affinity of UDG toward the modified DNA substrate, with the greatest effect occurring when the pyrophosphate bond was 3' adjacent to the deoxyuridine. The enzyme was able to excise a 3'-terminal uracil at the nicked site of a nicked duplex, DNA, provided that the terminal deoxyuridine was 3'-phosphorylated. The effect of the pyrophosphate bond on the substrate susceptibility of oligonucleotides containing deoxyuridine is discussed with respect to the mechanism of action of UDG.

Processivity of Escherichia coli and rat liver mitochondrial uracil-DNA glycosylase is affected by NaCl concentration.

Escherichia coli uracil-DNA glycosylase was shown to catalyze the hydrolysis of a site-specific uracil residue from a defined single-stranded oligonucleotide (25-mer). With duplex 25-mer, the rate of uracil removal from double-stranded DNA containing a U.G mispair was approximately 2-fold greater than a U.A base pair. The mechanism by which E. coli and rat liver mitochondrial uracil-DNA glycosylase located sequential uracil residues within double-stranded DNA was investigated. Two concatemeric polynucleotide substrates were constructed by ligation of homologous 5'-end 32P-labeled 25-mer double-stranded oligonucleotides that contained either a site-specific U.G or U.A target site at intervals of 25 nucleotides along one strand of the DNA. Reaction of uracil-DNA glycosylase with these concatemeric DNAs, followed by alkaline hydrolysis of the resultant AP-sites, would produce predominantly [32P]25-mer products, if a processive mechanism was used to locate successive uracil residues, or oligomeric multiples of [32P]25-mer, if a distributive mode was exhibited. Both the bacterial and the mitochondrial enzymes were found to act processively on U.A- and U.G-containing DNA in the absence of NaCl, based on the initial rate of 25-mer produced relative to the total amount of uracil excised. Approximately 50% of the total uracil excised resulted in the release of 25-mer product. The addition of NaCl (> or = 50 mM) caused reduced processivity on both U.A- and U.G-containing DNA substrates. The mode of action of uracil-DNA glycosylase was very similar to that observed for the EcoRI endonuclease cleavage of restriction sites contained in the same DNA substrate which was used as a positive control.

Modified substrates as probes for studying uracil-DNA glycosylase

In order to study the mechanism of action of uracil-DNA glycosylase (UDG) from human placenta, single-stranded (ss) and double-stranded (ds) oligodeoxyribonucleotides (oligos), containing deoxyuridine (dU) and a wide variety of their analogs were used. It was shown that UDG has a twofold preference for ss oligos over ds oligos and a twofold preference for intermolecular duplexes over similar hairpin-like duplexes. The replacement of dU with 1-(β- d-2′-deoxythreo-pentofuranosil)uracil (xU) or 1-(β- d-3′-deoxy-threo-pentofuranosil)uracil (tU), which results in a change in sugar hydroxyl configuration, has no influence on UDG binding to such substrates, but inhibits uracil removal. A oligo containing 2′-deoxy-2′-fluorouridine (flU), with a 3'-endo conformation of modified sugar is recognized by UDG 100–200-fold less efficiently than the natural ones. For Br atoms or a methyl group were introduced at position 5 of a dU residue in an oligo. It was shown that the nature of a substituent at this position is essential for UDG function.

Sequence specificity for removal of uracil from U·A pairs and U·G mismatches by uracil-DNA glycosylase from Escherichia coli, and correlation with mutational hotspots

The rate of removal of uracil from different positions in double-stranded DNA by uracil-DNA glycosylase from Escherichia coli varied more than 15-fold. Consensus sequences for good and poor removal were 5′-(A/T)UA(A/T)-3′ and 5′-(G/C)U(T/G/C)-3′, respectively. In general, the sequence context surrounding U was more important for the rate of removal than whether U was present in U·A pairs or U·G mispairs. Rates of removal of U from sites of amber mutations in the lacI gene, where mutation frequencies and deamination rates were known, indicated that the observed variation in removal is biologically significant.

Structure and function of the multifunctional DNA-repair enzyme exonuclease III.

The repair of DNA requires the removal of abasic sites, which are constantly generated in vivo both spontaneously and by enzymatic removal of uracil, and of bases damaged by active oxygen species, alkylating agents and ionizing radiation. The major apurinic/apyrimidinic (AP) DNA-repair endonuclease in Escherichia coli is the multifunctional enzyme exonuclease III, which also exhibits 3'-repair diesterase, 3'-->5' exonuclease, 3'-phosphomonoesterase and ribonuclease activities. We report here the 1.7 A resolution crystal structure of exonuclease III which reveals a 2-fold symmetric, four-layered alpha beta fold with similarities to both deoxyribonuclease I and RNase H. In the ternary complex determined at 2.6 A resolution, Mn2+ and dCMP bind to exonuclease III at one end of the alpha beta-sandwich, in a region dominated by positive electrostatic potential. Residues conserved among AP endonucleases from bacteria to man cluster within this active site and appear to participate in phosphate-bond cleavage at AP sites through a nucleophilic attack facilitated by a single bound metal ion.

Uracil DNA N-glycosylase distributively interacts with duplex polynucleotides containing repeating units of either TGGCCAAGCU or TGGCCAAGCTTGGCCAAGCU.

Uracil DNA N-glycosylase (UDG) has been used as a model enzyme to test a novel universal approach to discriminate between two possible enzymatic mechanisms of specific site location in DNA, processive (DNA-scanning mechanism) and distributive (random diffusion-mediated mechanism). Two double-stranded concatemeric polynucleotides of defined length (440-480 nucleotides) containing deoxyuridine at either every 10th or 20th nucleotide in the DNA chain were prepared by the ligation of self-complementary 10- or 20-mer oligodeoxyribonucleotides. Incubation of these polynucleotides with Escherichia coli UDG, followed by thermal breakage of the abasic sites, formed fragments that were multiples of either the 10- or the 20-mer. Since the processive and distributive mechanisms of uracil removal by UDG would be very different, the fragment distribution, generated at each time interval during the UDG reaction, should be unique. To show this, we developed a computer model illustrating both possible mechanisms of UDG functioning. The distribution of DNA fragments experimentally generated during the time course of the UDG reaction was compared with the results of the computer programs that modeled the distributive and processive mechanisms. The data indicated that uracil removal, catalyzed by UDG, is consistent with a distributive model.

Efficient removal of uracil from G.U mispairs by the mismatch-specific thymine DNA glycosylase from HeLa cells.

The uracil DNA glycosylases (EC 3.2.2.3) characterized to date remove uracil from DNA irrespective of whether it is base paired with adenine or mispaired with guanine in double-stranded substrates or whether it is found in single-stranded DNA. We report here the characterization of uracil glycosylase activity that can remove the base solely from a mispair with guanine. It does not recognize uracil either in A.U pairs or in single-stranded substrates. The enzyme, a 55-kDa polypeptide, was previously characterized as a mismatch-specific thymine DNA glycosylase and was thought to be responsible solely for the correction (to G.C) of G.T mispairs that arise as a result of spontaneous hydrolytic deamination of 5-methylcytosine to thymine. Given the broader substrate specificity of the enzyme (in addition to uracil and thymine, the protein can also remove 5-bromouracil from mispairs with guanine), we propose that its biological role in vivo may also include the correction of a subset of G.U mispairs inefficiently removed by the more abundant ubiquitous uracil glycosylases, such as those arising from cytosine deamination in G+C-rich regions of the genome.

Expression and occurrence of uracil-DNA glycosylase in higher plants.

Uracil-DNA glycosylase (UDG) is the first enzyme in the base excision repair pathway for removal of uracil in DNA. DNA repair capacity is likely to be a critical factor in mutagenesis and thereby in the capacity to prevent genetic damage and unwanted variation. We have studied expression of UDG in 9 higher plant species. The highest expression of UDG was measured in Solanum tuberosum. A comparison of 6 Solanum tuberosum cultivars showed that the specific activity ranged from 30 pmol mg1 protein min-1 in the cultivar Laila to 80 pmol mg-1 protein min-1 in the cultivar Ostara. Measurement of UDG in Begonia X cheimantha gave no indications of enzyme activity. The possible effects of no or low UDG activity is discussed. In vitro cultures of Solanum tuberosum and Thymus vulgaris were used to examine the effect of auxin and cytokinin on the UDG activity. Axillary shoots of Solanum tuberosum were cultured on medium including 20 variations in hormone concentration. Auxin (1-naphtaleneacetic acid) increased the expression of UDG. Plants cultured on medium supplemented with 3 mg 1-1 1-naphtaleneacetic acid showed a specific UDG activity which was approximately 3-fold higher than the activity in controls. The cytokinin benzyladenine reduced the specific UDG activity at concentrations in the range 0.25-10 mg 1-1 . In vitro cultured Saintpaulia ionantha was used to examine UDG activity during initiation, conditioning and multiplication cycles. In general, highest expression of UDG was measured in the conditioning cycle on hormone free medium. Measurement of UDG expression during single subculture periods, clearly showed that UDG expression may vary over one culture period. Expression of UDG was in general highest three weeks after transfer to fresh medium. Of different seedling organs from 0- to 15-day-old Brassica napus L., roots and hypocotyls showed the highest UDG activities. In cotyledons a very low and nearly constant specific activity was observed. In 12-day-old seedlings the activity in roots was approximately 20 times higher than the activity in cotyledons.

Consensus sequences for good and poor removal of uracil from double stranded DNA by uracil-DNA glycosylase.

We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,000-fold and studied its biochemical properties, including sequence specificity. The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody directed towards human UDG. SDS-PAGE and western analysis indicate an apparent M(r) = 27,500. Bovine UDG has a 1.7-fold preference for single stranded over double stranded DNA as a substrate. Sequence specificity for uracil removal from dsDNA was examined for bovine and Escherichia coli UDG, using DNA containing less than one dUMP residue per 100 nucleotides and synthetic oligonucleotides containing one dUMP residue. Comparative studies involving about 40 uracil sites indicated similar specificities for both UDGs. We found more than a 10-fold difference in rates of uracil removal between different sequences. 5'-G/CUT-3' and 5'-G/CUG/C-3' were consensus sequences for poor repair whereas 5'-A/TUAA/T-3' was a consensus for good repair. Sequence specificity was verified in double stranded oligonucleotides, but not in single stranded ones, suggesting that the structure of the double stranded DNA helix has influence on sequence specificity. Rate of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches. The results indicate that sequence specific repair may be a determinant to be considered in mutagenesis.

Uracil-DNA glycosylases preferentially excise mispaired uracil.

We have investigated the substrate specificity of human, viral and bacterial uracil-DNA glycosylases employing as substrate double-stranded oligonucleotides containing in the same position of the 5'-32P-labelled strand an uracil residue facing, on the complementary strand, guanine (mimicking cytosine deamination) or adenine (mimicking dUTP misincorporation). The enzyme removal of uracil was monitored and quantified by the generation of alkali-sensitive apyrimidinic sites. All three uracil-DNA glycosylases excise uracil from mispaired oligonucleotides (U/G) more efficiently than from paired oligonucleotides (U/A). The enzymes also remove uracil from single-stranded oligonucleotide with an efficiency similar to that observed with U/A paired oligonucleotide. The efficient recognition of U/G mispair by uracil-DNA glycosylase is important in minimizing miscoding transcripts and C/G-->T/A transitions in proliferating cells.

The spectrum of spontaneous mutations in a Saccharomyces cerevisiae uracil-DNA-glycosylase mutant limits the function of this enzyme to cytosine deamination repair

Uracil-DNA-glycosylase has been proposed to function as the first enzyme in strand-directed mismatch repair in eukaryotic organisms, through removal of uracil from dUMP residues periodically inserted into the DNA during DNA replication (Aprelikova, O. N., V. M. Golubovskaya, T. A. Kusmin, and N. V. Tomilin, Mutat. Res. 213:135-140, 1989). This hypothesis was investigated with Saccharomyces cerevisiae. Mutation frequencies and spectra were determined for an ung1 deletion strain in the target SUP4-o tRNA gene by using a forward selection scheme. Mutation frequencies in the SUP4-o gene increased about 20-fold relative to an isogenic wild-type S. cerevisiae strain, and the mutator effect was completely suppressed in the ung1 deletion strain carrying the wild-type UNG1 gene on a multicopy plasmid. Sixty-nine independently derived mutations in the SUP4-o gene were sequenced. All but five of these were due to GC----AT transitions. From this analysis, we conclude that the mutator phenotype of the ung1 deletion strain is the result of a failure to repair spontaneous cytosine deamination events occurring frequently in S. cerevisiae and that the UNG1 gene is not required for strand-specific mismatch repair in S. cerevisiae.

Effect of uracil situated in the vicinity of a mispair on the directionality of mismatch correction inEscherichia coli

We wanted to establish whether strand breaks and gaps, arising during the removal of uracil from newly-synthesized DNA, can be utilized as strand discrimination signals by the methyl-directed mismatch repair system of Escherichia coli. For this purpose, we constructed a series of M13 heteroduplexes that contained a single uracil residue situated either upstream or downstream from a G/T or an A/C mispair. Transfections of these constructs into E. coli strains, either proficient of deficient in mismatch or uracil repair, allowed us to follow the fate of these mispairs in vivo. Our data show that the intermediates of uracil repair cannot substitute for the strand-discrimination signals generated by the MutH protein, which is thought to initiate the methyl-directed mismatch repair process by nicking the unmethylated strand of a newly-synthesized DNA duplex at d(GATC) sites. However, processing of uracil residues situated upstream from the mispair was shown to reduce the yield of the progeny phage arising from the uracil-containing strand, presumably as a result of co-repair of the base analogue and the mispair.

Uracil-DNA glycosylase affects mismatch repair efficiency in transformation and bisulfite-induced mutagenesis inStreptococus pneumoniae

The generalized mismatch repair system of Streptococcus pneumoniae (the Hex system) can eliminate base pair mismatches arising in heteroduplex DNA during transformation or by DNA polymerase errors during replication. Mismatch repair is most likely initiated at nicks or gaps. The present work was started to examine the hypothesis that strand discontinuities arising after removal of uracil by uracil DNA-glycosylase (Ung) can be utilised as strand discrimination signals. We show that mismatch repair efficiency is enhanced 3- to 6-fold when using uracil-containing DNA as donor in transformation. In order to assess the contribution of Ung to nascent strand discrimination for postreplication mismatch repair, we developed a positive selection procedure to isolate S. pneumoniae Ung- mutants. We succeeded in isolating Ung- mutants using this procedure based on chromosomal integration of uracil-containing hybrid DNA molecules. Cloning and characterization of the ung gene was achieved. Comparison of spontaneous mutation rates in strains either proficient or deficient in mismatch and/or uracil repair gave no support to the hypothesis that Ung plays a major role in targeting the Hex system to neosynthesized DNA strands. However Ung activity is responsible for the increased efficiency of mismatch repair observed in transformation with uracil-containing DNA. In addition Ung is involved in repair of bisulfite-treated transforming DNA.

Human uracil-DNA glycosylase complementsE.coli ungmutants

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.

Cell cycle regulation andin vitrohybrid arrest analysis of the major human uracil-DNA glycosylase

Uracil-DNA glycosylase (UDG) is the first enzyme in the excision repair pathway for removal of uracil in DNA. In vitro transcription/translation of a cloned human cDNA encoding UDG resulted in easily measurable UDG activity. The apparent size of the primary translation product was 34 kD. Two lines of evidence indicated that this cDNA encodes the major nuclear UDG. First, in vitro translation of human fibroblast mRNA isolated from S-phase cells resulted in measurable UDG activity and this UDG translation was specifically inhibited 90% by an anti-sense UDG mRNA transcript. Secondly, cell cycle analysis revealed an 8-12 fold increase in transcript level late in the G1-phase preceding a 2-3 fold increase in total UDG activity in the S-phase. UDG degradation was found to be very slow (T1/2 approximately 30h), therefore, the rate of UDG synthesis could be derived from the rate of UDG accumulation, and was found to correlate temporarily and quantitatively with the transcript level. Inhibitor studies showed that RNA and protein synthesis was required for induction of UDG. However, specific inhibition of DNA replication with aphidicolin indicated that entrance of fibroblasts into the S-phase was not required for UDG accumulation.

Uptake rates of various nitrogen sources by nitrate-grownTetraselmis (Platymonas) striata

Cells of Tetraselmis striata, grown in a medium with nitrate as nitrogen source, were tested for their ability to remove a variety of other nitrogen sources from the growth medium in short term (6 h) experiments. There was no detectable removal of cytosine, thymine, uracil, adenine and allantoin. The purines guanine, xanthine, hypoxanthine and uric acid were all immediately removed at high rates (c. 1 pg N cell-1 h-1). The amino acids alanine, arginine, glutamic acid, glycine and tryptophan were initially either not removed significantly, or were removed at rates which were inadequate to sustain normal growth and reproduction. However, within 1–2 h of presentation of these nitrogen sources the rates of removal from the medium increased from zero to about 0·16 pg N cell-1 h-1 for arginine and glutamate, and from zero to 0·3 pg N cell-1 h-1 for alanine. The amino acid amides asparagine and glutamine were initially removed slowly, but much higher rates (0·5–0·7 pg cell-1 h-1) were found after 2·5 h from pres...

Misincorporation of Uracil Into DNA as Possible Contributor to Neuronal aging and Abiotrophy

Neuronal aging and abiotrophy may be related to the abnormal presence of uracil in DNA. Evidence which could support this hypothesis exists: 1) DNA polymerase beta, the only nuclear DNA polymerase present in adult neurons which is able to repair damaged DNA, incorporates dUTP or dTTP with the same efficiency. This suggests that in adult neurons the incorporation of dUTP into DNA is solely dependent on the relative intracellular concentration of dUTP; 2) uracil into DNA also arises from cytosine deamination; 3) at birth, when neurons stop proliferating, uracil DNA-glycosylase, the enzyme responsible of the removal of uracil from DNA, nearly disappears; 4) a significant replacement of thymine by uracil in DNA could produce genetic instability which in turn could affect the recognition of DNA sequences by enzymes and/or by regulatory DNA binding proteins. Thus enzymatic defects which might alter the intracellular dUTP pool, in different neuronal systems, could account for the multiplicity of the clinical manifestations of aging and neurodegenerative disorders. The increase of the age-specific rate of abiotrophic diseases may be due to accumulation with time of uracil containing DNA.