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PubMed2006

Purine and pyrimidine metabolism: a firm basis for a transformed society.

Purines and pyrimidines form the backbone of DNA and RNA. Hence, modification of purine and pyrimidine metabolism can have serious effects on normal functioning of a subject. These aspects formed the main topics for an International and a European Series of meetings, dedicated to the metabolism in man. In order to streamline the organization of these meetings the European Society was transformed to an International society: the Purine and Pyrimidine Society (www.ppsociety.org). This special issue of Nucleosides, Nucleotides, and, Nucleic, Acids highlights the last European meeting in Prague, focusing on inborn errors, cardiac diseases, inflammatory diseases, rheumatology, haematology, cancer, virology, genetic polymorphism, specific methodology, and, of course, metabolism. The meeting in Chicago in 2007 will be the first meeting of the Purine and Pyrimidine Society.

Animals↗

PubMed2004

Synthesis, 18F-radiolabelling and biological evaluations of C-6 alkylated pyrimidine nucleoside analogues.

Synthesis of pyrimidine derivatives with a side-chain attached to the C-6 of pyrimidine ring (6-14) is reported. Target compounds 8 and 12 were subjected to in vitro phosphorylation tests, determination of their binding affinities to herpes simplex virus (HSV-1) thymidine kinase (TK) and catalytic turnover constants. Fluorinated pyrimidine derivative 12 (40 microM) exhibited better binding affinity for HSV-1 TK than acyclovir (ACV, 170 microM) and ganciclovir (GCV, 48 microM). Catalytic turnover constant (k(cat)) of 12 (0.08 s(-1)) was close to the k(cat) values of ACV (0.10 s(-1)) and GCV (0.10 s(-1)). Furthermore, compounds 8 and 12 showed no cytotoxic effects in HSV-1 TK-transduced and non-transduced cell lines. Besides, compounds 8 and 12 did not exhibit antiviral or cytostatic activities against several viruses and malignant tumor cell lines that were evaluated. The new fluorinated pyrimidine derivative 16 that is phosphorylated by HSV-1 TK could be developed as non-toxic PET-tracer molecule. Thus, 18F labelling of the precursor 14 was performed by nucleophilic substitution using [18F] tetrabutylammonium fluoride as the fluorinating reagent.

Alkylation↗

PubMed1999

Regulated splicing of an alternative exon of beta-tropomyosin pre-mRNAs in myogenic cells depends on the strength of pyrimidine-rich intronic enhancer elements.

Alternative splicing of chicken beta-tropomyosin (beta-TM) pre-mRNAs ensures that in nonmuscle cells, only exon 6A is expressed, whereas in skeletal muscle, exon 6B is utilized preferentially. We have previously shown that efficient splicing of the nonmuscle exon 6A requires two pyrimidine-rich splicing enhancers (S4 and I5Y) that are present in the introns flanking exon 6A. Here, we examined the function of the S4 and I5Y elements by replacing them within beta-TM minigenes by other pyrimidine- and purine-rich sequence elements and analyzing splicing in transfected quail nonmuscle and muscle cells. Several features of these splicing regulatory elements were revealed by this study. First, a wide variety of pyrimidine-rich sequences can replace the intronic S4 splicing enhancer, indicating that pyrimidine composition, rather than sequence specificity, determines activity for this element. Second, one type of purine-rich sequence (GARn), normally found within exons, can also replace the S4 splicing enhancer. Third, the diverse elements tested exhibit differential activation of the splice sites flanking exon 6A and different positional constraints. Fourth, the strength of the S4 splicing enhancer is appropriately set to obtain proper regulation of the transition from exon 6A splicing in myoblasts to exon 6B splicing in myotubes, but this splicing regulatory element is not the target for cell-type-specific splicing factors.

Alternative Splicing↗

PubMed2001

Nitric oxide is involved in arsenite inhibition of pyrimidine dimer excision.

Arsenite is a human carcinogen reported to inhibit DNA repair. The binding of arsenite to functional thiol groups of DNA repair enzymes has in the past been suggested as a possible mechanism for the effect of arsenite on DNA repair. However, recent studies indicate that reactive oxygen species and nitric oxide are involved in arsenite toxicity. This research aims to elucidate the role of these possible mechanisms in the inhibition of UV-induced DNA repair by arsenite. As arsenite inhibits UV-DNA repair in Chinese hamster ovary cells, and this is a commonly used cell line for UV repair experiments, we used these cells to examine the effect of arsenite on the expression of UV-irradiated reporter genes. The T4 UV endonuclease V-incorporated comet assay was used to examine specifically the effect of arsenite on pyrimidine dimer excision. We showed that inhibition of UV-DNA repair by arsenite was suppressed by nitric oxide synthase inhibitors. Arsenite increased nitric oxide production and nitric oxide generators inhibited UV-DNA repair. The involvement of nitric oxide in the inhibition of pyrimidine dimer excision by arsenite was also confirmed in human fibroblasts. Investigation into the effect of oxidant modulators did not give a clear indication that reactive oxygen species are involved in arsenite inhibition of UV-DNA repair. Phenylarsine oxide, a strong thiol-reacting agent, did not inhibit pyrimidine dimer excision and also did not increase nitric oxide production. Our results show conclusively that nitric oxide is involved in the inhibition of pyrimidine dimer excision by arsenite. Reactive oxygen species and the binding of arsenite to functional thiol groups of DNA repair enzymes do not appear to be involved.

Animals↗

PubMed1983

Induction of pyrimidine dimers and unscheduled DNA synthesis in cultured mouse epithelial cells exposed to 254-nm- and u.v.-B radiation.

The induction and fate of pyrimidine dimers and unscheduled DNA synthesis were measured in u.v.-irradiated primary, newborn SENCAR mouse epithelial cells. Unscheduled DNA synthesis was induced in a dose responsive manner by two u.v. sources, a germicidal lamp (254 nm) and an FS40 sunlamp (280--400 nm). Using the endonuclease-sensitive site assay to detect pyrimidine dimer production and excision, we examined the response of the newborn mouse cells to both u.v. sources. We were unable to detect the removal of pyrimidine dimers with either of the two sources of u.v. The speculation is made that primary, newborn mouse epidermal cells excise u.v.-induced pyrimidine dimers to an extent below the level of detection of the endonuclease-sensitive site assay but to an extent sufficient to induce unscheduled DNA synthesis.

Animals↗

PubMed2004

Alterations in pyrimidine nucleotide metabolism as an early signal during the execution of programmed cell death in tobacco BY-2 cells.

Changes in pyrimidine metabolism were investigated during programmed cell death (PCD) of tobacco BY-2 cells, induced by a simultaneous increase in the endogenous levels of nitric oxide (NO) and hydrogen peroxide. The de novo synthesis of pyrimidine nucleotides was estimated by following the metabolic fate of the (14)C-labelled orotic acid, whereas the rates of salvage and degradation pathways were studied by measuring the respective incorporation of (14)C-labelled uridine and uracil under different treatments. Nucleic acid metabolism was also examined using labelled thymidine as a marker. The results show that specific alterations in the balance of pyrimidine nucleotide synthesis, which include a decreased rate of salvage activity of uracil and uridine and increased salvage activity of thymidine, represent a metabolic switch that establishes proper cellular conditions for the induction of PCD. In particular, a reduction in the utilization of uracil for salvage products occurs very early during PCD, before the appearance of typical cytological features of the death programme, thus representing an early metabolic marker for PCD. These changes are strictly associated with PCD, since they do not occur if NO or hydrogen peroxide are increased individually, or if actinomycin, which inhibits the death programme, is added into the medium in the presence of NO and hydrogen peroxide. The possible roles of these fluctuations in pyrimidine metabolism on the cellular nucleotide pool are discussed in relation to the induction of cell death.

Apoptosis↗

PubMed1998

Repair of cyclobutane pyrimidine dimers in unstimulated human mononuclear cells is deficient at very low fluences of ultraviolet B and is not enhanced by addition of deoxyribonucleosides.

Unstimulated human T lymphocytes are exquisitely sensitive to UVB irradiation. This hypersensitivity appears to relate to low deoxyribonucleotide pool sizes. They have also been reported to be defective in global excision of cyclobutane pyrimidine dimers, but such experiments may have been carried out at supralethal doses, where unrepaired excision breaks persist indefinitely. We use a T4 endonuclease Comet assay to show that removal of cyclobutane pyrimidine dimers is defective in the unstimulated mononuclear cell fraction (mainly T lymphocytes) even at sublethal fluences from an FS20 broad spectrum UVB lamp. Moreover, removal is not enhanced by addition of deoxyribonucleosides to the medium. Cells which are failing to remove cyclobutane pyrimidine dimers readily form fresh incision breaks in response to a second UVB fluence, indicating that they retain repair capacity and suggesting that removal of types of damage other than cyclobutane pyrimidine dimers is effective.

Adult↗

PubMed2001

Triplex formation by morpholino oligodeoxyribonucleotides in the HER-2/neu promoter requires the pyrimidine motif.

Triplex-forming oligonucleotides (TFOs) are good candidates to be used as site-specific DNA-binding agents. Two obstacles encountered with TFOs are susceptibility to nuclease activity and a requirement for magnesium for triplex formation. Morpholino oligonucleotides were shown in one study to form triplexes in the absence of magnesium. In the current study, we have compared phosphodiester and morpholino oligonucleotides targeting a homopurine-homopyrimidine region in the human HER2/neu promoter. Using gel mobility shift analysis, our data demonstrate that triplex formation by phosphodiester oligonucleotides at the HER-2/neu promoter target is possible with pyrimidine-parallel, purine-antiparallel and mixed sequence (GT)-antiparallel motifs. Only the pyrimidine-parallel motif morpholino TFO was capable of efficient triple helix formation, which required low pH. Triplex formation with the morpholino TFO was efficient in low or no magnesium. The pyrimidine motif TFOs with either a phosphodiester or morpholino backbone were able to form triple helices in the presence of potassium ions, but required low pH. We have rationalized the experimental observations with detailed molecular modeling studies. These data demonstrate the potential for the development of TFOs based on the morpholino backbone modification and demonstrate that the pyrimidine motif is the preferred motif for triple helix formation by morpholino oligonucleotides.

Base Sequence↗

PubMed1979

The relationship between pyrimidine dimers and replicating DNA in UV-irradiated human fibroblasts.

The relationship between pyrimidine dimers (measured as endonuclease-sensitive sites) and newly-synthesized DNA has been examined in several different ways, with the following results:- 1. After UV-irradiation of normal human fibroblasts the frequency of pyrimidine dimer sites in sections of DNA which have been synthesized immediately before the UV-irradiation is similar to that in the bulk DNA. 2. The frequency of pyrimidine dimer sites in the parental strands of replicating DNA in UV-irradiated normal human fibroblasts is similar to that in the bulk DNA. 3. In UV-irradiated XP variant cells the size of DNA synthesized in the presence of caffeine immediately after UV irradiation accurately corresponds with the average interdimer distance in the parental DNA. This suggests that in this experimental situation each pyrimidine dimer gives rise to a disocntinuity or a termination site in the daughter strand.

Cells, Cultured↗

PubMed2003

Formation of stable triplexes between purine RNA and pyrimidine oligodeoxyxylonucleotides.

Hybridization properties of oligodeoxyxylonucleotides (OXNs) built from pyrimidine monomers with an inverted 3'-OH group of the furanose have been studied using the gel mobility shift, UV melting and circular dichroism (CD) spectroscopy methods. Pyrimidine OXNs form triple helices with complementary purine RNA in which one OXN is parallel and another is antiparallel with respect to the RNA target. Surprisingly, no duplex formation between the pyrimidine OXNs and purine RNAs is detected. The modified triplexes are stable at pH 7. Their thermal stability depends on the number of C(G-C) triplets and, for G-rich RNA sequences, it is comparable with the stability of native DNA-RNA duplexes. The CD spectra of triplexes formed by OXNs with purine RNA targets are similar to spectra of A-type helices. A pyrimidine OXN having a clamp structure efficiently inhibits reverse transcription of murine pim-1 mRNA in vitro mediated by the Mo-MuLV reverse transcriptase.

Animals↗

PubMed1981

The mechanism of preferential synthesis of poly[r(purine)] in the transcription of poly[d(purine)] . poly[d(pyrimidine)] by T. thermophilus RNA polymerase.

The mechanism of preferential transcription on poly[d(purine)] . poly[d(pyrimidine)] was investigated using RNA polymerase of T. thermophilus HB8. Though the machinery for initiation is lacking, the core enzyme has the latent ability to synthesize poly[r(pyrimidine)] as well as poly[r(purine)]. The holoenzyme can synthesize poly[r(purine)] in the usual manner. Poly[r(pyrimidine)] synthesis by the holoenzyme is, however, forbidden. These results suggest that the sigma factor plays a crucial role in this preferential transcription, and that this preferential transcription may be useful as a model for the sense strand recognition. Various results led us to the hypothesis that the high affinity site for the poly[d(pyrimidine)] strands on the enzyme plays a very important role.

Adenosine Triphosphate↗

PubMed1988

Repair of DNA-containing pyrimidine dimers.

Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.

DNA↗

PubMed1999

Synthesis and anticancer activities of 4-oxobenzopyrano[2,3-d]pyrimidines.

Several 2-aryl-4-oxoxbenzopyrano[2,3-d]pyrimidines have previously been shown to exhibit in vivo antitumor activity in mice with P388 lymphocytic leukemia. In the present study, a series of novel substituted benzopyrano[2,3-d]pyrimidines have been prepared and tested for cytotoxic activity against a panel of cancer cell lines including the P388 lymphocytic leukemia cell line. The unsubstituted parent compound, some methoxylated derivatives and a cyclohexyl derivative all exhibited potent cytotoxic activity (IC50 values 0.3-0.64 microM). A number of derivatives, including the unsubstituted parent pyrimidine, were shown to cause a significant perturbation in cell cycle kinetics with an observed 2- to 3-fold increase in cells in the G2/M phase of the cell cycle. Furthermore, a polymethoxylated derivative, 2-(3,4,5-trimethoxyphenyl)-9-methoxy-4-oxo-2,3-dihydrobenzopyrano[ 2,3-d]pyrimidine 13, was shown to be selectively active against a number of human ovarian cell lines.

Animals↗

PubMed1983

Isolation and properties of strains of Micrococcus (Deinococcus) radiodurans unable to excise ultraviolet light-induced pyrimidine dimers from DNA: evidence for two excision pathways.

A mutant of Deinococcus (formerly Micrococcus) radiodurans (strain 302, mutant in mtcA) sensitive to both the lethal effect of mitomycin C and the mutagenic effect of simple alkylating agents, but having wild-type resistance to UV light, was treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine in an attempt to isolate strains deficient in the ability to excise UV-induced pyrimidine dimers. Three strains were isolated that were UV-sensitive, but had wild-type resistance to the lethal effect of methyl methanesulphonate and all were shown to be unable to excise pyrimidine dimers. The three strains UVS9, UVS25 and UVS78 had, in addition to the mutation in mtcA, mutations in loci designated uvsC, uvsD and uvsE, respectively. When the mutant mtcA gene was replaced by its wild-type allele in all three strains they became UV- and mitomycin C-resistant. On incubating the double mutants UVS9, UVS25 and UVS78 with wild-type DNA about 50% of the transformants selected for UV resistance were mitomycin C-sensitive and about 50% resistant depending on whether the mutant mtcA or the uvsC, D or E genes had been replaced by their wild-type alleles. Although strains mutant singly in uvsC, D or E were UV-resistant the rates of excision of pyrimidine dimers differed between them and was slower in all of them than in the wild-type and strain 302. The results indicate that wild-type D. radiodurans possesses two pathways for the excision of pyrimidine dimers and that mutational blocks in both must exist for the excisionless phenotype to be expressed.

DNA Repair↗

PubMed1976

Regulation of arginine and pyrimidine biosynthesis in Pseudomonas putida.

Repression of biosynthetic enzyme synthesis in Pseudomonas putida is incomplete even when the bacteria are growing in a nutritionally complex environment. The synthesis of four of the enzymes of the arginine biosynthetic pathway (N-acetyl-alpha-glutamokinase/N-acetylglutamate-gamma-semialdehyde dehydrogenase, ornithine carbamoyltransferase and acetylornithine-delta-transaminase) could be repressed and derepressed, but the maximum difference observed between repressed and derepressed levels for any enzyme of the pathway was only 5-fold (for ornithine carbamoyltransferase). No repression of five enzymes of the pyrimidine biosynthetic pathway (aspartate carbamoyltransferase, dihydro-orotase, dihydro-orotate dehydrogenase, orotidine-5'-phosphate pyrophosphorylase and orotidine-5'-phosphate decarboxylase) could be detected on addition of pyrimidines to minimal asparagine cultures of P. putida A90, but a 1-5- to 2-fold degree of derepression was found following pyrimidine starvation of pyrimidine auxotrophic mutants of P. putida A90. Aspartate carbamoyltransferase in crude extracts of P. putida A90 was inhibited in vitro by (in order of efficiency) pyrophosphate, CTP, UTP and ATP, at limiting but not at saturating concentrations of carbamoyl phosphate.

Aldehyde Oxidoreductases↗

PubMed2005

Functional analysis of the pyrimidine de novo synthesis pathway in solanaceous species.

Pyrimidines are particularly important in dividing tissues as building blocks for nucleic acids, but they are equally important for many biochemical processes, including sucrose and cell wall polysaccharide metabolism. In recent years, the molecular organization of nucleotide biosynthesis in plants has been analyzed. Here, we present a functional analysis of the pyrimidine de novo synthesis pathway. Each step in the pathway was investigated using transgenic plants with reduced expression of the corresponding gene to identify controlling steps and gain insights into the phenotypic and metabolic consequences. Inhibition of expression of 80% based on steady-state mRNA level did not lead to visible phenotypes. Stepwise reduction of protein abundance of Asp transcarbamoylase or dihydro orotase resulted in a corresponding inhibition of growth. This was not accompanied by pleiotropic effects or by changes in the developmental program. A more detailed metabolite analysis revealed slightly different responses in roots and shoots of plants with decreased abundance of proteins involved in pyrimidine de novo synthesis. Whereas in leaves the nucleotide and amino acid levels were changed only in the very strong inhibited plants, the roots show a transient increase of these metabolites in intermediate plants followed by a decrease in the strong inhibited plants. Growth analysis revealed that elongation rates and number of organs per plant were reduced, without large changes in the average cell size. It is concluded that reduced pyrimidine de novo synthesis is compensated for by reduction in growth rates, and the remaining nucleotide pools are sufficient for running basic metabolic processes.

Aspartate Carbamoyltransferase↗

PubMed1992

A cytokinetic approach to determine the range of O2-dependence of pyrimidine(deoxy)nucleotide biosynthesis relevant for cell proliferation.

In vitro cultured Ehrlich ascites tumour (EAT) cells were used because of the ease of their manipulation under different levels of hypoxia. They were used to clarify further the complex mechanism of oxygen-dependent cell proliferation. On reducing the oxygen concentration from 20% to lower levels (1-7%) an increase in the length of the population doubling time with concomitant reductions in protein, RNA and DNA content of cultures were observed. The incorporation of [14C]HCO3- into the RNA fraction of cells by de novo biosynthesis of uridine monophosphate (UMP) was reduced proportionally to the microenvironmental O2 tension. Uptake of this labelled precursor by cells in the presence of N-phosphonoacetyl-L-aspartate was found to be similarly inhibited. To correlate the reduction of cell growth under hypoxia with the functional pyrimidine supply, hypoxic cells were cultured in the presence of a balanced mixture of deoxynucleosides and/or uridine (100 microM deoxycytidine, 10 microM deoxyadenosine, 10 microM deoxyguanosine, 100 microM uridine). Above 3% O2 in the protective atmosphere, no improvement of growth parameters by the exogenous pyrimidinenucleotide precursors was obtained, whereas these compounds had a positive influence below this level. The increase in cell number was raised to about 60% of that of control cultures (20% O2) irrespective of the oxygen tension. In addition, when above 3% O2 the incorporation of HCO3- into RNA was comparable to that of controls, indicating that the pyrimidine de novo pathway is not a limiting factor in RNA biosynthesis. In conclusion, whereas at suboptimal O2 levels (5-7%) no correlation between pyrimidine metabolism and reduction of proliferation rate appears to exist, at low O2 concentrations (less than 3%) the rate of orotate/UMP production seems to be an important factor in the growth cessation of EAT cells; at critical O2 tensions (less than 1%) the lack of pyrimidine-deoxynucleosides substantially reduces cell cycle progression.

Animals↗

PubMed1994

De novo synthesis of pyrimidine nucleotides by Helicobacter pylori.

The incorporation of pyrimidine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in their synthetic pathways were investigated by radioactive tracer analysis and 31P nuclear magnetic resonance spectroscopy. The bacterium was found to take up aspartate and bicarbonate and to incorporate carbon atoms from these precursors into its genomic DNA. Orotate, an intermediate of de novo pyrimidine biosynthesis, and uracil and uridine, precursors for pyrimidine pathways, were also incorporated by the micro-organism. Radiolabelled substrates were used to assess the activities of aspartate transcarbamoylase, orotate phosphoribosyltransferase, orotidylate decarboxylase, CTP synthetase, uracil phosphoribosyltransferase, thymidine kinase and deoxycytidine kinase in bacterial lysates. The study provided evidence for the presence in H. pylori of an operational de novo pathway, and a less active salvage pathway for the biosynthesis of pyrimidine nucleotides.

Aspartate Carbamoyltransferase↗