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PubMed1976

Pyrimidine nucleoside analogues.X. 5-Substituted 1-(1,3-dihydroxypropyl-2) uracils.

A convenient method is suggested for synthesis of uracil-1-malonic acid diethyl ester by alkylating 2,4-bis(trimethylsilyl) uracil with bromo-malonic acid diethyl ester. This compound has been shown to hydrolyze with NaOH yielding either uracil or uracil-1-acetic acid, depending on reaction conditions. Similarly, thymine-1-malinic acid diethyl ester and 5-fluorouracil-1,3-dimalonic acid tetraethyl ester were obtained. 1-(1,3-Dihydroxypropyl)uracil has been obtained by reducing uracil-1-malonic acid diethyl ester with NaBH4.

Hydrogen-Ion Concentration↗
PubMed1987

The Partial Purification and Characterization of Nuclear and Mitochondrial Uracil-DNA Glycosylase Activities from Zea mays Seedlings.

Uracil-DNA glycosylase activities from etiolated Zea mays seedling nuclei and mitochondria were partially purified and characterized. Nuclei and mitochondria were separated using sucrose differential and step gradient centrifugation. Experiments with osmotically shocked organelles indicated that enzyme activity from mitochondria was soluble, whereas nuclear enzyme activity was only partially soluble under the conditions tested. Purification using DEAE-cellulose and Affigel Blue column chromatography yielded distinct elution profiles from both columns for each of the organellar enzyme activities. Final purification was 490- and 850- fold for the nuclear and mitochondrial uracil-DNA glycosylase, respectively. Characterization studies demonstrated significant differences between the nuclear and mitochondrial uracil-DNA glycosylase with respect to K(m), temperature, and pH activity optimum, the effect of salts, and substrate preference. Molecular weight as determined by gel filtration was 18,000 for enzymes from both sources. Both were also sensitive to the sulfhydryl group-blocking agent N-ethylmaleimide. A number of uracil analogs were tested for their ability to inhibit nuclear and mitochondrial uracil-DNA glycosylase activities. 5-Azauracil, uracil, 6-aminouracil, 6-azauracil, 5-aminouracil, and 5-fluorouracil all inhibited both activities to variable degrees.

Journal Article↗
PubMed2002

Structure of product-bound Bacillus caldolyticus uracil phosphoribosyltransferase confirms ordered sequential substrate binding.

Uracil phosphoribosyltransferase (UPRTase) is part of the salvage pathway that leads to the biosynthesis of UMP. It catalyzes the formation of UMP and pyrophosphate from uracil and alpha-D-5-phosphoribosyl-1-pyrophosphate. Unlike enzymes in the de novo synthesis of UMP, UPRTases have only been found in lower organisms and are therefore potential targets for the development of new antibiotics. UPRTase from Bacillus caldolyticus has been crystallized and the structure has been determined by isomorphous replacement and refined to 3.0 A resolution. UPRTase from B. caldolyticus forms a dimer with the active sites pointing away from each other. A long arm from each subunit wraps around the other subunit, contributing half of the dimer interface. The monomer adopts the phosphoribosyltransferase type I fold, with a small C-terminal hood defining the uracil-binding site. The structure contains a well defined UMP molecule in the active site. The binding of UMP involves two sequence segments that are highly conserved among UPRTases. The first segment, Asp131-Ser139, contains the PRPP-binding consensus sequence motif known from other type I phosphoribosyltransferases and binds the ribose-5'-phosphate part of UMP. The second segment, Tyr193-Ala201, which is specific for uracil phosphoribosyltransferases, binds the uracil part of UMP through backbone contacts, partly mediated by a water molecule. Modelling of a PRPP-enzyme complex reveals that uracil can be activated to its tautomeric enol form by the complex. This is consistent with kinetic data, which display ordered sequential binding of substrates, with PRPP binding first. Based on this observation, a reaction mechanism is proposed.

Bacillus↗
PubMed1999

Pea chloroplast glyceraldehyde-3-phosphate dehydrogenase has uracil glycosylase activity.

Pea (Pisum sativum) chloroplastic glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) was tested for uracil DNA glycosylase activity. It was found that both the chloroplast and the recombinant subunit B dehydrogenases remove uracil from poly(dA[3H]dU). The glycosylase activity of the recombinant subunit B enzyme and that of a truncated form corresponding in length to subunit A were associated with the dehydrogenase activity in gel-filtration experiments. Both activities of the chloroplast enzyme were inhibited by antisera raised against recombinant subunit B, and both activities of the recombinant subunit B enzyme were inhibited by antisera raised against pea chloroplast glyceraldehyde-3-P dehydrogenase. Antisera raised against Escherichia coli uracil glycosylase did not affect the glycosylase activity of the recombinant subunit B enzyme. The glycosylase pH activity profile of the chloroplast dehydrogenase was unique. It is distinct from the dehydrogenase pH activity profile and from the pH activity profiles of other plant glycosylases. The glycosylase activity, but not the dehydrogenase activity, of the recombinant subunit B enzyme was inhibited by uracil. Pyridine nucleotides stimulated the glycosylase activity. To our knowledge this is the first example of a nonhuman glyceraldehyde-3-P dehydrogenase, and of an NADP-dependent glyceraldehyde-3-P dehydrogenase, that exhibits uracil glycosylase activity.

Amino Acid Sequence↗
PubMed1995

Incorporation of uracil into viral DNA correlates with reduced replication of EIAV in macrophages.

The retrovirus equine infectious anemia virus (EIAV) encodes a dUTPase situated between reverse transcriptase and integrase. We have described the inability of EIAV with a 270-bp dUTPase deletion, delta DU EIAV, to replicate to wild-type (WT) levels in equine macrophages (D. S. Threadgill, W. K. Steagall, M. T. Flaherty, F. J. Fuller, S. T. Perry, K. E. Rushlow, S. F. J. LeGrice, and S. L. Payne, J. Virol. 67, 2592-2600, 1993). Here we describe the construction of a second dUTPase-deficient virus (DUD71E) containing a single amino acid substitution in dUTPase. delta DU and DUD71E replicate to 2% of WT levels in macrophages by 7 days postinfection, when WT EIAV is highly cytopathic. To identify the replication block(s), we analyzed DNA synthesis, integration, and transcription. DNA synthesis was normal in macrophages, with evidence of full-length viral DNA by 24 hr postinfection. The level of integrated delta DU and DUD71E DNA appeared to be decreased 2- to 3-fold compared to WT. Steady-state levels of full-length viral transcripts were decreased over 100-fold, indicating that replication of dUTPase-deficient EIAV is blocked between viral DNA synthesis and transcription. As dUTP hydrolysis normally plays a role in preventing incorporation of uracil into newly synthesized DNA, we investigated the possibility that dUTPase-deficient EIAV DNA contains uracil. In vitro assays showed that while WT virions do not utilize dUTP, dUTPase-deficient virus and recombinant RT synthesize uracil-containing DNA. The presence of uracil in viral DNA recovered from delta DU- and DUD71E-infected macrophages was also demonstrated. In macrophages, a virally encoded dUTPase may be necessary to prevent the incorporation of uracil into viral DNA.

Animals↗
PubMed1995

Cyclopentenyl uracil: an effective inhibitor of uridine salvage in vivo.

Cyclopentenyl uracil, a non-cytotoxic inhibitor of uridine kinase, was found to effectively block the salvage of circulating uridine by host and tumor tissues in the intact mouse. Dose-response characteristics of the inhibition were determined. Large doses (1 g/kg) of cyclopentenyl uracil were required, and the effect of a single dose fell rapidly over a 24-hr period. A sustained inhibition of uridine salvage of > 64-79% could be maintained by multiple doses of 1 g/kg given on an every 8-hr schedule. Mice given cyclopentenyl uracil (1 g/kg) every 8 hr for 5 days continued to gain weight and showed no signs of toxicity; however, the combination of cyclopentenyl uracil with a non-toxic dose of N-(phosphonacetyl)-L-aspartic acid (PALA; 200 mg/kg daily for 5 days) was lethal to mice, indicating that circulating uridine modifies the toxicity of agents that act on enzymes of the de novo pyrimidine pathway. Although the duration of action and potency of cyclopentenyl uracil are not ideal, this is the first demonstration of an effective inhibition of uridine salvage in the intact mouse with a non-cytotoxic agent. This makes possible the evaluation of concurrent inhibition of de novo and salvage routes to pyrimidine nucleotides as an approach to chemotherapy.

Animals↗
PubMed1983

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.

Bromodeoxyuridine↗
PubMed1993

Implication of uracil in spontaneous mutagenesis on a single-stranded shuttle vector replicated in mammalian cells.

Almost all spontaneous point mutations found on a single-stranded shuttle vector after its transfection and replication in monkey cells were located at cytosine residues. In order to understand this very specific type of targeting we have studied the possible implication of uracil residues in the induction of these spontaneous mutations. The single-stranded shuttle vector pCF3A carrying the supF tRNA gene as a mutagenesis target has been allowed to replicate in mammalian COS7 cells, mutations being screened in bacteria using the beta-galactosidase assay. Progenies from untreated DNA and DNA treated with the uracil-DNA glycosylase prior to transfection were analyzed to determine the amount and classes of mutations. While spontaneous mutation frequency was 9.7 x 10(-4) for control DNA, single-stranded vector treated with the E. coli uracil-DNA glycosylase exhibited a reduced mutation frequency of about 30%. The abolished mutations were mainly confined to the cytosine to thymine transitions for which a decrease by a factor of 5 was indeed observed. This finding fits well with the fact that it is usually admitted that uracil pairs with adenine, indicating therefore that approximately 30% of spontaneous mutations observed in our experimental conditions and 80% of C to T transitions may be due to the presence of uracil instead of cytosine.

Animals↗
PubMed1995

Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA.

Uracil-DNA glycosylase inhibitor (Ugi) is a B. subtilis bacteriophage protein that protects the uracil-containing phage DNA by irreversibly inhibiting the key DNA repair enzyme uracil-DNA glycosylase (UDG). The 1.9 A crystal structure of Ugi complexed to human UDG reveals that the Ugi structure, consisting of a twisted five-stranded antiparallel beta sheet and two alpha helices, binds by inserting a beta strand into the conserved DNA-binding groove of the enzyme without contacting the uracil specificity pocket. The resulting interface, which buries over 1200 A2 on Ugi and involves the entire beta sheet and an alpha helix, is polar and contains 22 water molecules. Ugi binds the sequence-conserved DNA-binding groove of UDG via shape and electrostatic complementarity, specific charged hydrogen bonds, and hydrophobic packing enveloping Leu-272 from a protruding UDG loop. The apparent mimicry by Ugi of DNA interactions with UDG provides both a structural mechanism for UDG binding to DNA, including the enzyme-assisted expulsion of uracil from the DNA helix, and a crystallographic basis for the design of inhibitors with scientific and therapeutic applications.

Binding Sites↗
PubMedDate not supplied

Effects of 5-azacytosine in DNA on enzymic uracil excision.

PBS-2 phage DNA, which contains uracil in place of thymine, was used as substrate for both purified B. subtilis uracil-DNA glycosylase and a crude extract from M. luteus. Addition of [3H]5-azacytidine to the medium after phage infection resulted in substitution of 1.2% azacytosine for cytosine in DNA. Substrate DNA was also labeled with [14C]uracil. Neither enzyme preparation released tritiated bases from DNA. Analysis by S1 nuclease digestion show no increase in single-strandedness of the modified DNA. Enzymic release of uracil by the M. luteus extract was reduced by about 50% from the substituted substrate. By contrast, the rate of uracil excision by the purified enzyme was unaffected by the presence of DNA 5-azacytosine.

Azacitidine↗
PubMed1990

Activity profiles of enzymes that control the uracil incorporation into DNA during neuronal development.

We have shown that DNA polymerase beta, the only nuclear DNA polymerase present in adult neurons, cannot discriminate between dTTP and dUTP, having the same Km for both substrates. This fact suggests that during reparative DNA synthesis, in adult neurons, dUMP residues can be incorporated into DNA. Since uracil DNA-glycosylase functions to prevent the mutagenic effects of uracil in DNA coming as a product of deamination of cytosine residues or as a result of dUMP incorporation by DNA polymerase, we have studied the perinatal activity of uracil DNA-glycosylase and of 2 enzymes (nucleoside diphosphokinase and dUTPase) involved in dUTP metabolism. Our data indicate that during neuronal development there is a rapid decrease in uracil DNA-glycosylase which could impair the removal of uracil present in DNA in adult neurons. However, misincorporation of dUMP into DNA might be kept to a low frequency by the action of dUTPase present at all developmental stages.

Animals↗
PubMed2005

Determination of apoptosis, uracil incorporation, DNA strand breaks, and sister chromatid exchanges under conditions of thymidylate deprivation in a model of BER deficiency.

Thymidylate synthase (TS) is an important target of several chemotherapeutic agents. During TS inhibition, dTTP levels decrease with a subsequent increase in dUTP. Uracil incorporated into the genome is removed by base excision repair (BER). BER has been hypothesized to play a role in the response to thymidylate deprivation, despite a lack of direct evidence. We previously found that beta-pol null murine fibroblasts were approximately six-fold more resistant than wild-type cells to raltitrexed, a folate-based inhibitor specific for TS. In this study, a number of endpoints were determined to understand the influence of BER and beta-pol during raltitrexed treatment. Raltitrexed induced apoptosis in wild-type cells to a greater extent than in beta-pol null cells. A PARP inhibitor decreased the sensitivity to raltitrexed, although the extent was not different between wild-type and beta-pol null cells. No evidence was seen for extensive strand break formation that preceded apoptosis, although raltitrexed induced more sister chromatid exchanges in wild-type cells. Increased levels of uracil in DNA were detected following treatment in wild-type and beta-pol null cells. However, uracil levels were only approximately two-fold higher in DNA from treated cells compared to untreated. Uracil DNA glycosylase activity was slightly higher in beta-pol null cells, although not sufficiently different to explain the difference in sensitivity to raltitrexed. Taken together, the data suggest that the sensitivity of the wild-type cells to raltitrexed is not associated with activation of PARP-1 dependent BER, extensive uracil incorporation into DNA and persistent strand breaks, but rather with changes suggestive of DNA recombination.

Animals↗
PubMed2004

Mycobacterium tuberculosis and Escherichia coli nucleoside diphosphate kinases lack multifunctional activities to process uracil containing DNA.

E. coli nucleoside diphosphate kinase (EcoNDK) is an important cellular enzyme required to maintain balanced nucleotide pools in the cells. Recently, it was reported that EcoNDK is also a multifunctional base excision repair enzyme, possessing uracil-DNA glycosylase (UDG) and AP-DNA processing activities. We investigated for the presence of such activities in M. tuberculosis NDK (MtuNDK), which shares 45.2% identity, and 52.6% similarity with EcoNDK. In contrast to the robust uracil excision activity reported for EcoNDK, MtuNDK preparation exhibited very poor excision of uracil from DNA. However, this activity was undetectable when MtuNDK was purified from an ung(-) strain of E. coli, or when the assays were performed in the presence of extremely low amounts of a highly specific proteinaceous inhibitor, Ugi which forms an extremely tight complex with the host Ung (UDG), showing that MtuNDK preparation was contaminated with UDG. Reinvestigation of uracil processing activity of EcoNDK, showed that even this protein lacked UDG activity. All preparations of NDK were shown to be active by their autophosphorylation activity. Ugi neither displayed a physical interaction with EcoNDK nor did it affect autophosphorylation of NDKs. Further, neither of the NDK preparations processed the AP-DNA generated by UDG treatment of the uracil containing DNA duplexes. However, partially purified preparations of NDK did process such DNA substrates.

Base Sequence↗
PubMed2006

Uracil in DNA--general mutagen, but normal intermediate in acquired immunity.

Deamination of cytosine in DNA results in mutagenic U:G mispairs, whereas incorporation of dUMP leads to U:A pairs that may be genotoxic directly or indirectly. In both cases, uracil is mainly removed by a uracil-DNA glycosylase (UDG) that initiates the base excision repair pathway. The major UDGs are mitochondrial UNG1 and nuclear UNG2 encoded by the UNG-gene, and nuclear SMUG1. TDG and MBD4 remove uracil from special sequence contexts, but their roles remain poorly understood. UNG2 is cell cycle regulated and has a major role in post-replicative removal of incorporated uracils. UNG2 and SMUG1 are both important for prevention of mutations caused by cytosine deamination, and their functions are non-redundant. In addition, SMUG1 has a major role in removal of hydroxymethyl uracil from oxidized thymines. Furthermore, UNG-proteins and SMUG1 may have important functions in removal of oxidized cytosines, e.g. isodialuric acid, alloxan and 5-hydroxyuracil after exposure to ionizing radiation. UNG2 is also essential in the acquired immune response, including somatic hypermutation (SHM) required for antibody affinity maturation and class switch recombination (CSR) mediating new effector functions, e.g. from IgM to IgG. Upon antigen exposure B-lymphocytes express activation induced cytosine deaminase that generates U:G mispairs at the Ig locus. These result in GC to AT transition mutations upon DNA replication and apparently other mutations as well. Some of these may result from the generation of abasic sites and translesion bypass synthesis across such sites. SMUG1 can not complement UNG2 deficiency, probably because it works very inefficiently on single-stranded DNA and is down-regulated in B cells. In humans, UNG-deficiency results in the hyper IgM syndrome characterized by recurrent infections, lymphoid hyperplasia, extremely low IgG, IgA and IgE and elevated IgM. Ung(-/-) mice have a similar phenotype, but in addition display dysregulated cytokine production and develop B cell lymphomas late in life.

Amino Acid Sequence↗
PubMed2006

Base excision repair of both uracil and oxidatively damaged bases contribute to thymidine deprivation-induced radiosensitization.

PURPOSE: Increased cellular sensitivity to ionizing radiation due to thymidine depletion is the basis of radiosensitization with fluoropyrimidine and methotrexate. The mechanism responsible for cytotoxicity has not been fully elucidated but appears to involve both the introduction of uracil into, and its removal from, DNA. The role of base excision repair of uracil and oxidatively damaged bases in creating the increased radiosensitization during thymidine depletion is examined. METHODS AND MATERIALS: Isogenic strains of S. cerevisiae differing only at loci involved in DNA repair functions were exposed to aminopterin and sulfanilamide to induce thymidine deprivation. Cultures were irradiated and survival determined by clonogenic survival assay. RESULTS: Strains lacking uracil base excision repair (BER) activities demonstrated less radiosensitization than the parental strain. Mutant strains continued to show partial radiosensitization with aminopterin treatment. Mutants deficient in BER of both uracil and oxidatively damaged bases did not demonstrate radiosensitization. A recombination deficient rad52 mutant strain was markedly sensitive to radiation; addition of aminopterin increased radiosensitivity only slightly. Radiosensitization observed in rad52 mutants was also abolished by deletion of the APN1, NTG1, and NTG2 genes. CONCLUSION: These data suggest radiosensitization during thymidine depletion is the result of BER activities directed at both uracil and oxidatively damaged bases.

Aminopterin↗
PubMed2002

Role of DNA flexibility in sequence-dependent activity of uracil DNA glycosylase.

Uracil DNA glycosylase (UDG) is a base excision repair enzyme that specifically recognizes and removes uracil from double- or single-stranded DNA. The efficiency of the enzyme depends on the DNA sequence surrounding the uracil. Crystal structures of UDG in complex with DNA reveal that the DNA is severely bent and distorted in the region of the uracil. This suggests that the sequence-dependent efficiency of the enzyme may be related to the energetic cost of DNA distortion in the process of specific damage recognition. To test this hypothesis, molecular dynamics simulations were performed on two sequences representing extreme cases of UDG efficiency, AUA/TAT (high efficiency) and GUG/CAC (low efficiency). Analysis of the simulations shows that the effective bending force constants are lower for the AUA/TAT sequence, indicating that this sequence is more flexible than the GUG/CAC sequence. Fluorescence lifetimes of the adenine analogue 2-aminopurine (2AP), replacing adenine opposite the uracil, are shorter in the context of the AUA/TAT sequence, indicating more dynamic base-base interaction and greater local flexibility than in the GUG/CAC sequence. Furthermore, the K(M) of Escherichia coli UDG for the AUA/TAT sequence is 10-fold smaller than that for the GUG/CAC sequence, while the k(cat) is only 2-fold smaller. This indicates that differences in UDG efficiency largely arise from differences in binding and not catalysis. These results link directly flexibility near the damaged DNA site with the efficiency of DNA repair.

2-Aminopurine↗
PubMed2002

Base-flipping mutations of uracil DNA glycosylase: substrate rescue using a pyrene nucleotide wedge.

We recently introduced a new substrate rescue tool for investigating enzymatic base flipping by uracil DNA glycosylase (UDG) in which a bulky pyrene nucleotide wedge (Y) was placed opposite a uracil in duplex DNA (i.e., a U/Y pair), thereby preorganizing the target base in an extrahelical conformation [Jiang, Y. L., et al. (2001) J. Biol. Chem. 276, 42347-54]. The pyrene wedge completely rescued the large catalytic defects resulting from removal of the natural Leu191 wedge, presumably mimicking the pushing and plugging function of this group. Here we employ the pyrene rescue method in combination with transient kinetic approaches to assess the functional roles of six conserved enzymatic groups of UDG that have been implicated in the "pinch, push, plug, and pull" base-flipping mechanism (see the preceding paper in this issue). We find that a U/Y base pair increases the apparent second-order rate constant for damaged site recognition by L191G pushing mutation by 45-fold as compared to a U/A pair, thereby fully rescuing the kinetic effects of the mutation. Remarkably, the U/Y pair also allows L191G to proceed through the conformational docking step that is severely comprised with the normal U/A substrate, and allows the active site of UDG to clamp around the extrahelical base. Thus, pyrene also fulfills the plugging role of the Leu191 side chain. Preorganization of uracil in an extrahelical conformation by pyrene allows diffusion-controlled damage recognition by all of these base-flipping mutants, and allows the UDG conformational change to proceed as rapidly as the rate of uracil flipping with the natural U/A base pair. Thus, the pyrene wedge substrate allows UDG to recognize uracil by a lock-and-key mechanism, rather than the natural induced-fit mechanism. Unnatural pyrene base pairs may provide a general strategy to promote site-specific targeting of other enzymes that recognize extrahelical bases.

Amino Acid Substitution↗
PubMed2004

Recognition of an unnatural difluorophenyl nucleotide by uracil DNA glycosylase.

The DNA repair enzyme uracil DNA glycosylase (UDG) utilizes base flipping to recognize and remove unwanted uracil bases from the genome but does not react with its structural congener, thymine, which differs by a single methyl group. Two factors that determine whether an enzyme flips a base from the duplex are its shape and hydrogen bonding properties. To probe the role of these factors in uracil recognition by UDG, we have synthesized a DNA duplex that contains a single difluorophenyl (F) nucleotide analogue that is an excellent isostere of uracil but possesses no hydrogen bond donor or acceptor groups. By using binding affinity measurements, solution (19)F NMR, and solid state (31)P[(19)F] rotational-echo double-resonance (REDOR) NMR measurements, we establish that UDG partially unstacks F from the duplex. However, due to the lack of hydrogen bonding groups that are required to support an open-to-closed conformational transition in UDG, F cannot stably dock in the UDG active site. We propose that F attains a metastable unstacked state that mimics a previously detected intermediate on the uracil-flipping pathway and suggest structural models of the metastable state that are consistent with the REDOR NMR measurements.

Amino Acid Substitution↗