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.     

Identification and partial characterization of rabbit brain deoxyuridine 5′-triphosphatase

Adult rabbit brain contains the enzymatic machinery to convert deoxyuridine to deoxyuridine triphosphate (dUTP). Although dUTP as dUMP can be readily incorporated into DNA in place of thymidine monophosphate, we detected no (3H)dUMP in newly synthesized (3H)DNA in adult rabbit brain after the intraventricular injection of (3H)deoxyuridine. Only (3H)thymidine was detected. The probable explanation for the lack of incorporation of uracil into adult rabbit brain DNA is the presence of a specific, high affinity dUTPase which converts dUTP to dUMP and PP. After homogenization and ammonium sulfate fractionation of adult rabbit brain (35 to 75% saturation), a high affinity, specific dUTPase was detected in the dialyzed enzyme preparation. The Km and Vmax of the dUTPase were 0.2 microM and 36 pmol/mg protein/min, respectively. No high affinity dUTPase activity was detectable in liver. In brain, another enzyme hydrolyzed dUTP and dTTP (NTPase( to their respective diphosphates. NTPase, unlike dUTPase, was not sensitive to heating at 65 degrees C for five minutes. Thus, brain, like other tissues, contains a high affinity, specific dUTPase presumably to "sanitize" the cells of dUTP and, thus, protect the integrity of newly synthesized 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.     

dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae.

dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) catalyses the hydrolysis of dUTP to dUMP and PPi and thereby prevents the incorporation of uracil into DNA during replication. Although it is widely believed that dUTPase is essential for cell viability because of this role, direct evidence supporting this assumption has not been presented for any eukaryotic system. We have analysed the role of dUTPase (DUT1) in the life cycle of yeast. Using gene disruption and tetrad analysis, we find that DUT1 is necessary for the viability of S. cerevisiae; however, under certain conditions dut1 null mutants survive if supplied with exogenous thymidylate (dTMP). Analyses with isogenic uracil-DNA-glycosylase (UNG1) deficient or proficient strains indicate that in the absence of dUTPase, cell death results from the incorporation of uracil into DNA and the attempted repair of this damage by UNG1-mediated excision repair. However, in dut1 ung1 double mutants, starvation for dTMP causes dividing cells to arrest and die in all phases of the cell cycle. This latter effect suggests that the extensive stable substitution of uracil for thymine in DNA leads to a general failure in macromolecular synthesis. These results are in general agreement with previous models in thymine-less death that implicate dUTP metabolism. They also suggest an alternative approach for chemotherapeutic drug design.     

Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from Campylobacter jejuni

The enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and PPi thus controlling the incorporation of uracil into DNA genomes. In Campylobacter jejuni dUTPase exhibits structural properties of dimeric proteins characteristic of protozoa of the Kinetoplastidae family. In the present study we perform a kinetic analysis of Campylobacter dUTPase using the continuous spectrophotometric method and show that the enzyme is highly specific for deoxyuridine nucleotides. The Michaelis-Menten constant for dUTP was 0.66 microM while the k(cat) was 12.3 s(- 1). dUDP was also efficiently hydrolysed although the specificity constant, k(cat)/K(m), was five fold lower than for dUTP. The reaction product and the non hydrolysable analogue alpha,beta imido dUDP are potent inhibitors of the enzyme while several analogues of dUMP with substituents at the 3'- and 5'-positions active against trimeric dUTPases, show poor inhibitory activity. Apparent structural and kinetic differences with other eukaryotic dUTPases suggest that the present enzyme might be exploited as a target for new drugs against campylobacteriosis.     

Purification and characterization of porcine liver DNA polymerase gamma: utilization of dUTP and dTTP during in vitro DNA synthesis.

Porcine liver DNA polymerase gamma has been demonstrated to preferentially incorporate dTMP over dUMP during in vitro DNA synthesis. When polymerase activity was measured in standard reactions containing saturating levels of either dTTP or dUTP, the polymerization rate was slightly faster in the reaction containing dTTP. However, under conditions where both dTTP and dUTP competed, at an equal molar concentration, approximately 3-times more thymine residues were incorporated than uracil residues into DNA. Similarly, preferential incorporation of dTMP was observed on several substrates including poly (dA).oligo p(dT), poly (rA).oligo p(dT) and poly (dA-dT). The discrimination against dUMP incorporation was even more apparent with reduced levels of dUTP. These observations were consistent with the finding that the Km for DNA polymerase gamma was about 3-fold lower for dTTP (0.4 microM) than for dUTP (1.1 microM). On the other hand, the Vmax for these two reactions was very similar. Discrimination against dUMP incorporation was also observed during inhibition of polymerase gamma by dideoxyribonucleoside triphosphates. Dideoxythymidine triphosphate preferentially inhibited dUMP incorporation compared to that of dTMP, whereas ddATP, ddCTP and ddGTP inhibited both reactions equally.     

A bifunctional dCTP deaminase-dUTP nucleotidohydrolase from the hyperthermophilic archaeon Methanocaldococcus jannaschii

By the sequential action of dCTP deaminase and dUTPase, dCTP is converted to dUMP, the precursor of thymidine nucleotides. In addition, dUTPase has an essential role as a safeguard against uracil incorporation in DNA. The putative dCTP deaminase (MJ0430) and dUTPase (MJ1102) from the hyperthermophilic archaeon Methanocaldococcus jannaschii were overproduced in Escherichia coli. Unexpectedly, we found the MJ0430 protein capable of both reactions, i.e. hydrolytic deamination of the cytosine ring and hydrolytic cleavage of the phosphoanhydride bond between the alpha- and beta-phosphates. When the reaction was followed by thin layer chromatography using [3H]dCTP as substrate, dUMP and not dUTP was identified as a reaction product. In the presence of unlabeled dUTP, which acted as an inhibitor, no label was transferred from [3H]dCTP to the pool of dUTP. This finding strongly suggests that the two consecutive steps of the reaction are tightly coupled within the enzyme. The hitherto unknown bifunctionality of the MJ0430 protein appears beneficial for the cells because the toxic intermediate dUTP is never released. The MJ0430 protein also catalyzed the hydrolysis of dUTP to dUMP but with a low affinity for the substrate (Km >100 micro m). According to limited proteolysis, the C-terminal residues constitute a flexible region. The other protein investigated, MJ1102, is a specific dUTPase with a Km for dUTP (0.4 micro m) comparable in magnitude with that found for previously characterized dUTPases. Its physiological function is probably to degrade dUTP derived from other reactions in nucleotide metabolism.     

Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme

Genomes of all free-living organisms encode the enzyme dUTPase (dUTP pyrophosphatase), which plays a key role in preventing uracil incorporation into DNA. In the present paper, we describe the biochemical and structural characterization of DUT1 (Saccharomyces cerevisiae dUTPase). The hydrolysis of dUTP by DUT1 was strictly dependent on a bivalent metal cation with significant activity observed in the presence of Mg2+, Co2+, Mn2+, Ni2+ or Zn2+. In addition, DUT1 showed a significant activity against another potentially mutagenic nucleotide: dITP. With both substrates, DUT1 demonstrated a sigmoidal saturation curve, suggesting a positive co-operativity between the subunits. The crystal structure of DUT1 was solved at 2 ? resolution (1 ?=0.1 nm) in an apo state and in complex with the non-hydrolysable substrate α,β-imido dUTP or dUMP product. Alanine-replacement mutagenesis of the active-site residues revealed seven residues important for activity including the conserved triad Asp87/Arg137/Asp85. The Y88A mutant protein was equally active against both dUTP and UTP, indicating that this conserved tyrosine residue is responsible for discrimination against ribonucleotides. The structure of DUT1 and site-directed mutagenesis support a role of the conserved Phe142 in the interaction with the uracil base. Our work provides further insight into the molecular mechanisms of substrate selectivity and catalysis of dUTPases.     

Utilization in vitro of deoxyuridine triphosphate in DNA synthesis by DNA polymerases alpha and beta from calf thymus.

DNA polymerases alpha and beta (EC 2.7.7.7.) from calf thymus could utilize dUTP as a substrate for DNA synthesis as well as DNA polymerase I of Escherichia coli. Deoxyuridylate was incorporated into DNA by replacing deoxythymidylate and supported the further elongation of DNA chains on activated DNA or on the intiated homopolymers, poly(dA) . (dT)10 and poly(rA) . (dT)10. The rate of the incorporation of deoxyuridylate into DNA varied from 50 to 160% of that of deoxythymidylate, depending on the nature of the template primers and the species of DNA polymerase used. The apparent Km values for dUTP were very similar to those for dTTP. Uracil DNA-glycosylase excised efficiently the uracil residues in products of DNA polymerase reactions with either activated calf thymus DNA or initiated homopolymers.     

DNA repair synthesis-related enzymes during spermatogenesis in the mouse

To assess whether uracil DNA glycosylase and dUTP nucleotidohydrolase (dUTPase) can be involved in repair-type DNA synthesis associated to crossing-over or induced by UV and X-ray treatments, we have studied these enzyme activities in male mouse germ cells at specific stages of differentiation.Although the highest uracil DNA glycosylase activity was observed in dividing germ cells (spermatogonia and preleptotene spermatocytes), some activity was also detected in meiotic (3.5%) and post-meiotic (1.0%) cells with a relative maximum of activity at pachytene stage (4.7%) when meiotic crossing-over takes place. These findings suggest that uracil DNA glycosylase is involved, in this biological system, in DNA replication and in repair-type DNA synthesis.dUTPase is present at all the stages of spermatogenesis studied but, unlike thymidylate synthetase which is mainly associated with replicating germ cells, dUTPase activity is maximal in spermatocytes at pachytene stages. The data reported suggest that, in this biological system, the main role of dUTPase is to degrade dUTP to prevent misincorporation of uracil into DNA during crossing-over, rather than to participate in the biosynthetic pathway of dTTP.     

Structure/function analysis of a dUTPase: catalytic mechanism of a potential chemotherapeutic target.

dUTP pyrophosphatase catalyses hydrolysis of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP) and inorganic pyrophosphate (PPi). Elimination of dUTP is vital since its misincorporation into DNA by DNA polymerases can initiate a damaging iterative repair and misincorporation cycle, resulting in DNA fragmentation and cell death. The anti-tumour activity of folate agonists and thymidylate synthase inhibitors is thought to rely on dUTP misincorporation. Furthermore, retroviral cDNA production may be particularly susceptible to the effects of dUTP misincorporation by virtue of the error-prone nature of reverse trans criptase. Consequently, dUTPase activity is an ideal point of intervention in both chemotherapy and anti-retroviral therapy. In particular, the dUTPase encoded by a human endogenous retrovirus (HERV-K) has been suggested to complement HIV infection and so is an attractive target for specific inhibition. Hence, we used site photoaffinity labelling, site-directed mutagenesis and molecular modelling to assign catalytic roles to the conserved amino acid residues in the active site of the HERV-K dUTPase and to identify structural differences with other dUTPase enzymes. We found that dUTP photoaffinity labelling was specific for a beta-hairpin motif in HERV-K dUTPase. Mutagenesis of aspartate residues Asp84 and 86 to asparagine within this beta-hairpin showed the carboxylate moiety of both residues was required for catalysis but not for dUTP binding. An increase in the pKa of both aspartate residues brought about by substitution of a serine residue with a glutamate residue adjacent to the aspartate residues increased activity by a factor of 1.67 at pH 8.0, implicating general base catalysis as the enzyme's catalytic mechanism. Conservative mutagenesis of Tyr87 to Phe resulted in a sevenfold reduction of dUTPase activity and a 3.3-fold reduction in binding activity, whilst substitution with an isoleucine residue totally abolished both catalytic activity and dUTP binding, suggesting that binding/activity is dependent on an aromatic side-chain at the base of the hairpin. Comparison of a homology-based three-dimensional model structure of HERV-K dUTPase with a crystallographic structure of the human dUTPase revealed displacement of a conserved alpha-helix in the HERV-K enzyme causing expansion of the HERV-K active site. This expansion may be responsible for the ability of the HERV-K enzyme to hydrolyse dTTP and bind the bulkier dNTPs in contrast to the majority of dUTPases which are highly specific for dUTP. Knowledge of the dUTPase catalytic mechanism and the distinctive topography of the HERV-K active site provides a molecular basis for the design of HERV-K dUTPase-specific inhibitors.     

A novel process for mutation detection using uracil DNA-glycosylase.

A novel process is presented for the detection of known mutations and polymorphisms in DNA. This process, termed glycosylase mediated polymorphism detection (GMPD) involves amplification of the target DNA using three normal dNTPs and a fourth modified dNTP, whose base is a substrate for a specific DNA-glycosylase once incorporated into the DNA. The work described here utilises uracil DNA-glycosylase as the specific glycosylase and dUTP as the modified dNTP. Primers are designed so that during extension, the position of the first uracil incorporated into the extended primers differs depending on whether a mutation is present or absent. Subsequent glycosylase excision of the uracil residues followed by cleavage of the apyrimidinic sites allows detection of the mutation in the amplified fragment as a fragment length polymorphism. Variation in the sizes of the fragment length polymorphisms generated, can be readily achieved through the use of inosine bases in place of adenine bases in the upper and/or lower primers. The GMPD process is also adaptable to solid phase analysis. The use of the process for detection of mutations in the RYR1 and CFTR genes is demonstrated. Overall, the simplicity, specificity, versatility and flexibility of the GMPD process make it an attractive candidate for both small and large scale application in mutation detection and genome analysis.     

Uracil in DNA

The data confirming the formation of dUMP residues in DNA to be a continuous process taking place in the living cells are reviewed. All living organisms produce specific enzymes repairing the lesion of this type. The possible ways for uracil incorporation into DNA are described. The main of them are as follows: cytosine deamination in DNA molecules and utilization of dUTP by DNA polymerases during replication. The spontaneous mutability, the decrease in chain length of the newly synthesized DNA and the increase in recombination frequencies are discussed as possible consequences of this phenomenon.     

Increased uracil insertion in DNA is cytotoxic and increases the frequency of mutation,double strand break formation and VSG switching in Trypanosoma brucei

Deoxyuridine 5′-triphosphate pyrophosphatase (dUTPase) and uracil-DNA glycosylase (UNG) are key enzymes involved in the control of the presence of uracil in DNA. While dUTPase prevents uracil misincorporation by removing dUTP from the deoxynucleotide pool, UNG excises uracil from DNA as a first step of the base excision repair pathway (BER). Here, we report that strong down-regulation of dUTPase in UNG-deficient Trypanosoma brucei cells greatly impairs cell viability in both bloodstream and procyclic forms, underscoring the extreme sensitivity of trypanosomes to uracil in DNA. Depletion of dUTPase activity in the absence of UNG provoked cell cycle alterations, massive dUTP misincorporation into DNA and chromosomal fragmentation. Overall, trypanosomatid cells that lack dUTPase and UNG activities exhibited greater proliferation defects and DNA damage than cells deficient in only one of these activities. To determine the mutagenic consequences of uracil in DNA, mutation rates and spectra were analyzed in dUTPase-depleted cells in the presence of UNG activity. These cells displayed a spontaneous mutation rate 9-fold higher than the parental cell line. Base substitutions at A:T base pairs and deletion frequencies were both significantly enhanced which is consistent with the generation of mutagenic AP sites and DNA strand breaks. The increase in strand breaks conveyed a concomitant increase in VSG switching in vitro. The low tolerance of T. brucei to uracil in DNA emphasizes the importance of uracil removal and regulation of intracellular dUTP pool levels in cell viability and genetic stability and suggests potential strategies to compromise parasite survival.     

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.     

Purification and characterization of a dUTPase from Acholeplasma laidlawii B-PG9

dUTP was purified 120-fold from extracts of Acholeplasma laidlawii B-PG9 by Blue-Sepharose, Phenyl-Sepharose, hydroxyapatite, and DEAE-Sephacel chromatography techniques. The only substrate for the enzyme was dUTP with an apparent Km of 4.5 microM. The only reaction products were dUMP and PPi. The dUTPase did not exhibit any specific divalent cation requirement, but it was inhibited by EDTA. The enzyme was not inhibited by Pi or p-hydroxymercuribenzoate. The molecular weight of the enzyme was estimated by gel filtration chromatography to be 48,000, and its isoelectric point was 5.3. The enzyme was thermostable at 55 degrees C for 1 h. A. laidlawii dUTPase was distinguishable from KB (human epidermoid carcinoma) dUTPase by differences in electrophoretic migration, isoelectric point, and thermostability. The enzyme is important in preventing dUTP from being incorporated into DNA and may have a significant role in both the synthesis of thymidine- and PPi-dependent phosphorylations.     

The incorporation of uracil into animal cell DNA in vitro

In the presence of dUTP, net DNA synthesis in vitro is substantially reduced. Small DNA fragments that arise during in vitro DNA synthesis in the presence of dUTP are produced as a result of dUMP incorporation and subsequent post-replication excision repair process initiated by uracil-DNA-glycosylase. The size of the fragments is dependent upon the amount of dUMP incorporated, but unlike the normal 4S intermediates of DNA synthesis, these repair products are not precursors to high molecular weight DNA but are further degraded. The high levels of dUTPase as well as the presence of RNA primers on most nascent DNA pieces (Tseng and Goulian, 1977) suggest that repair of uracil-containing DNA does not contribute to the generation of the small, nascent DNA pieces found during DNA synthesis in this in vitro system.     

Excision repair of uracil incorporated in DNA as a result of a defect in dUTPase.

Mutants of Escherichia coli that are severely defective in the enzyme dUTPase (dut) accumulate short (4 to 5 S) Okazaki fragments following brief pulses with [³H]thymidine. The transient appearance of DNA fragments in these mutants is plausibly explained by the misincorporation of uracil in DNA as a result of an increase in available dUTP, followed by its rapid excision and repair. The evidence in support of this interpretation is the following: (1) accumulation of short DNA fragments can be partially suppressed by a mutation in dCTP deaminase, presumably by decreasing the intracellular level of dUTP relative to dTTP; (2) accumulation of the short DNA fragments can be almost completely suppressed by a mutation in uracil N-glycosidase, probably by preventing the introduction of nicks at the sites of uracil incorporation; (3) introduction of DNA polymerase I or DNA ligase mutations into dUTPase-defective strains results in the persistence of the 4 to 5 S fragments and rapid cessation of DNA synthesis. Uracil N-glycosidase, DNA polymerase I and DNA ligase must therefore be involved in the excision repair of uracil-containing DNA.     

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.