deoxyguanosine-triphosphate and thymidine-5--triphosphate

Changes in dNTP pools have been studied by a number of investigators, in a wide range of cell types. The in vitro pertubations in dNTP pool levels induced, in particular, by deoxynucleosides which act as allosteric modulators, are not totally consistent with current 'in vitro models' of ribonucleotide reductase function. This problem has also been addressed by Henderson et al. (1980) who stress the profusion of such models. Possible explanations, apart from the technical problems of the range of different experimental conditions (e.g. concentration of modulator used, time of incubation, etc.) for the various cell lines include: Modulators presumably have unpredictable 'network' effects by inhibiting or stimulating many other enzymes involved in the de novo and salvage synthesis of purines and pyrimidines. It is possible there are two separate forms of ribonucleotide reductase, one specifically reducing CDP/UDP, the other ADP/GDP. This, in particular, would explain the lack of decrease in dCTP levels after elevation of the dATP pool. There may be variations in ribonucleotide function which in vivo are cell specific, e.g. in thymic-derived compared with non-T-cell types. Peculiarities of T-cells include: Their ability to elevate their dNTP pools on exposure to very low exogenous concentration of deoxynucleoside. This may reflect very low rates of dNTP catabolism. The biological response of T-cells to elevation of the dATP or dGTP pool is reflected by a G1 block compared to an S phase block in cell-cycle progression in non-T-cell lines. The possibility that, in thymic cells, ribonucleotide reduction is restricted to ADP/GDP while pyrimidine dNTPs are synthesized by salvage pathways. As well, possible variation in the pool localization of dNTPs depending on production by either de novo or salvage synthesis could produce dNTP pool changes not clearly in accord with in vitro models. Clearly, the solution to these problems (although not easy) requires systematic comparative study, using cells of various origin (particularly T vs non-T), of dNTP pool responses to deoxynucleoside modulators, with an attempt to explore the factors described above. However, in the detailed pursuit of such an analysis the concept, that these variations in the control of nucleotide metabolism in T and non-T-cell systems may reflect quite significant differences in growth control and cell-cycle progression, should not be lost.

Topics: Animals; Aziridines; Cells, Cultured; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Humans; Hydroxyurea; Ribonucleotide Reductases; Substrate Specificity; T-Lymphocytes; Thiosemicarbazones; Thymine Nucleotides

Topics: Animals; Antimetabolites; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleosides; Deoxyribonucleotides; DNA; Humans; Kinetics; Liver Neoplasms, Experimental; Models, Biological; Nucleic Acid Synthesis Inhibitors; Ribonucleoside Diphosphate Reductase; Thermodynamics; Thymidylate Synthase; Thymine Nucleotides

Tenofovir (TFV) disoproxil fumarate (TDF) and emtricitabine (FTC), two nucleos(t)ide analogs (NA), are coformulated as an anti-HIV combination tablet for treatment and preexposure prophylaxis (PrEP). TDF/FTC may have effects on the deoxynucleoside triphosphate (dNTP) pool due to their similar structures and similar metabolic pathways. We carried out a comprehensive clinical study to characterize the effects of TDF/FTC on the endogenous dNTP pool, from baseline to 30 days of TDF/FTC therapy, in both treatment-naive HIV-positive and HIV-negative individuals. dATP, dCTP, dGTP, and TTP were quantified in peripheral blood mononuclear cells (PBMC) with a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. Forty individuals (19 HIV-positive) were enrolled and underwent a baseline visit and then received TDF/FTC for at least 30 days. Longitudinal measurements were analyzed using mixed-model segmented linear regression analysis. The dNTPs were reduced by 14% to 37% relative to the baseline level within 3 days in both HIV-negative and HIV-positive individuals (P ≤ 0.003). These reductions persisted to various degrees at day 30. These findings indicate that dNTP pools are influenced by TDF/FTC therapy. This may alter cellular homeostasis and could increase the antiviral effect through a more favorable analog/dNTP ratio. Further work is needed to elucidate mechanisms, to evaluate the clinical significance of these findings, and to further probe differences between HIV-negative and HIV-positive individuals. (This study has been registered at ClinicalTrials.gov under identifier NCT01040091.).

Topics: Adult; Anti-HIV Agents; Case-Control Studies; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Emtricitabine; Female; HIV Infections; HIV-1; Humans; Leukocytes, Mononuclear; Linear Models; Male; Tenofovir; Thymine Nucleotides

Efficient ways to produce single-stranded DNA are of great interest for diverse applications in molecular biology and nanotechnology. In the present study, we selected T7 RNA polymerase mutants with reduced substrate specificity to employ an

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA-Directed RNA Polymerases; Fluorine; Substrate Specificity; Synthetic Biology; Thymine Nucleotides; Transcription, Genetic; Viral Proteins

The accumulation of mutations is frequently associated with alterations in gene function leading to the onset of diseases, including cancer. Aiming to find novel genes that contribute to the stability of the genome, we screened the Saccharomyces cerevisiae deletion collection for increased mutator phenotypes. Among the identified genes, we discovered MET7, which encodes folylpolyglutamate synthetase (FPGS), an enzyme that facilitates several folate-dependent reactions including the synthesis of purines, thymidylate (dTMP) and DNA methylation. Here, we found that Met7-deficient strains show elevated mutation rates, but also increased levels of endogenous DNA damage resulting in gross chromosomal rearrangements (GCRs). Quantification of deoxyribonucleotide (dNTP) pools in cell extracts from met7Δ mutant revealed reductions in dTTP and dGTP that cause a constitutively active DNA damage checkpoint. In addition, we found that the absence of Met7 leads to dUTP accumulation, at levels that allowed its detection in yeast extracts for the first time. Consequently, a high dUTP/dTTP ratio promotes uracil incorporation into DNA, followed by futile repair cycles that compromise both mitochondrial and nuclear DNA integrity. In summary, this work highlights the importance of folate polyglutamylation in the maintenance of nucleotide homeostasis and genome stability.

Topics: Cell Nucleus; Deoxyguanine Nucleotides; Deoxyuracil Nucleotides; DNA Damage; DNA, Fungal; Folic Acid; Gene Deletion; Gene Expression Regulation, Fungal; Genome, Fungal; Genomic Instability; Mitochondria; Mutation; Peptide Synthases; Saccharomyces cerevisiae; Thymine Nucleotides; Uracil

The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 10

Topics: Click Chemistry; Copper; Cycloaddition Reaction; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Deoxyuracil Nucleotides; HCT116 Cells; HEK293 Cells; Humans; K562 Cells; Rhodamines; Staining and Labeling; Thymine Nucleotides

Reactive oxygen species generate the genotoxic 8-oxoguanine (oxoG) and 8-oxoadenine (oxoA) as major oxidative lesions. The mutagenicity of oxoG is attributed to the lesion's ability to evade the geometric discrimination of DNA polymerases by adopting Hoogsteen base pairing with adenine in a Watson-Crick-like geometry. Compared with oxoG, the mutagenesis mechanism of oxoA, which preferentially induces A-to-C mutations, is poorly understood. In the absence of protein contacts, oxoA:G forms a wobble conformation, the formation of which is suppressed in the catalytic site of most DNA polymerases. Interestingly, human DNA polymerase η (polη) proficiently incorporates dGTP opposite oxoA, suggesting the nascent oxoA:dGTP overcomes the geometric discrimination of polη. To gain insights into oxoA-mediated mutagenesis, we determined crystal structures of polη bypassing oxoA. When paired with dGTP, oxoA adopted a syn-conformation and formed Hoogsteen pairing while in a wobble geometry, which was stabilized by Gln38-mediated minor groove contacts to oxoA:dGTP. Gln38Ala mutation reduced misinsertion efficiency ∼55-fold, indicating oxoA:dGTP misincorporation was promoted by minor groove interactions. Also, the efficiency of oxoA:dGTP insertion by the X-family polβ decreased ∼380-fold when Asn279-mediated minor groove contact to dGTP was abolished. Overall, these results suggest that, unlike oxoG, oxoA-mediated mutagenesis is greatly induced by minor groove interactions.

Topics: Adenine; Base Pairing; Deoxyguanine Nucleotides; DNA Polymerase beta; DNA-Directed DNA Polymerase; Humans; Kinetics; Mutagenesis; Mutation; Thymine Nucleotides

We report the design and elaboration of a selection protocol for importing a canonical substrate of DNA polymerase, thymidine triphosphate (dTTP) in Escherichia coli. Bacterial strains whose growth depend on dTTP uptake, through the action of an algal plastid transporter expressed from a synthetic gene inserted in the chromosome, were constructed and shown to withstand the simultaneous loss of thymidylate synthase and thymidine kinase. Such thyA tdk dual deletant strains provide an experimental model of tight nutritional containment for preventing dissemination of microbial GMOs. Our strains transported the four canonical dNTPs, in the following order of preference: dCTP > dATP ≥ dGTP > dTTP. Prolonged cultivation under limitation of exogenous dTTP led to the enhancement of dNTP transport by adaptive evolution. We investigated the uptake of dCTP analogues with altered sugar or nucleobase moieties, which were found to cause a loss of cell viability and an increase of mutant frequency, respectively. E. coli strains equipped with nucleoside triphosphate transporters should be instrumental for evolving organisms whose DNA genome is morphed chemically by fully substituting its canonical nucleotide components.

Topics: Bacterial Outer Membrane Proteins; Decitabine; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Directed Molecular Evolution; Escherichia coli; Escherichia coli Proteins; Microalgae; Microorganisms, Genetically-Modified; Mutation Rate; Peptide Hydrolases; Thymidine Kinase; Thymidylate Synthase; Thymine Nucleotides

DNA can be damaged by many compounds in our environment, and the resulting damaged DNA is commonly replicated by translesion synthesis (TLS) polymerases. Because the mechanism and efficiency of TLS are affected by the type of DNA damage, obtaining information for a variety of DNA adducts is critical. However, there is no structural information for the insertion of a dNTP opposite an O6-dG adduct, which is a particularly harmful class of DNA lesions. We used molecular dynamics (MD) simulations to investigate structural and energetic parameters that dictate preferred dNTP insertion opposite O6-benzyl-guanine (Bz-dG) by DNA polymerase IV, a prototypical TLS polymerase. Specifically, MD simulations were completed on all possible ternary insertion complexes and ternary -1 base deletion complexes with different Bz-dG conformations. Our data suggests that the purines are unlikely to be inserted opposite anti- or syn-Bz-dG, and dTTP is unlikely to be inserted opposite syn-Bz-dG, because of changes in the active site conformation, including critical hydrogen-bonding interactions and/or reaction-ready parameters compared to natural dG replication. In contrast, a preserved active site conformation suggests that dCTP can be inserted opposite either anti- or syn-Bz-dG and dTTP can be inserted opposite anti-Bz-dG. This is the first structural explanation for the experimentally observed preferential insertion of dCTP and misincorporation of dTTP opposite Bz-dG. Furthermore, we provide atomic level insight into why Bz-dG replication does not lead to deletion mutations, which is in contrast with the replication outcomes of other adducts. These findings provide a basis for understanding the replication of related O6-dG adducts.

Topics: Benzyl Compounds; Catalytic Domain; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA Adducts; DNA Damage; DNA Polymerase beta; DNA Repair; DNA Replication; Escherichia coli; Escherichia coli Proteins; Guanine; Hydrogen Bonding; Molecular Dynamics Simulation; Mutagenesis; Protein Structure, Secondary; Protein Structure, Tertiary; Thymine Nucleotides

MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients' quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway.

Topics: Animals; Deoxyguanine Nucleotides; DNA Replication; DNA, Mitochondrial; Female; Fibroblasts; Gene Expression Regulation; Humans; Membrane Proteins; Mice; Mitochondria, Liver; Signal Transduction; Thymine Nucleotides

Alkylation DNA lesions are ubiquitous, and result from normal cellular metabolism as well as from treatment with methylating agents and chemotherapeutics. DNA damage tolerance by translesion synthesis DNA polymerases has an important role in cellular resistance to alkylating agents. However, it is not yet known whether Escherichia coli (E. coli) DNA Pol IV (DinB) alkylation lesion bypass efficiency and fidelity in vitro are similar to those inferred by genetic analyses. We hypothesized that DinB-mediated bypass of 3-deaza-3-methyladenine, a stable analog of 3-methyladenine, the primary replication fork-stalling alkylation lesion, would be of high fidelity. We performed here the first kinetic analyses of E. coli DinB•RecA binary complexes. Whether alone or in a binary complex, DinB inserted the correct deoxyribonucleoside triphosphate (dNTP) opposite either lesion-containing or undamaged template; the incorporation of other dNTPs was largely inefficient. DinB prefers undamaged DNA, but the DinB•RecA binary complex increases its catalytic efficiency on lesion-containing template, perhaps as part of a regulatory mechanism to better respond to alkylation damage. Notably, we find that a DinB derivative with enhanced affinity for RecA, either alone or in a binary complex, is less efficient and has a lower fidelity than DinB or DinB•RecA. This finding contrasts our previous genetic analyses. Therefore, mutagenesis resulting from alkylation lesions is likely limited in cells by the activity of DinB•RecA. These two highly conserved proteins play an important role in maintaining genomic stability when cells are faced with ubiquitous DNA damage. Kinetic analyses are important to gain insights into the mechanism(s) regulating TLS DNA polymerases.

Topics: Adenine; Alkylation; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA Adducts; DNA Replication; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Kinetics; Mutagenesis; Rec A Recombinases; Thymine Nucleotides

The fidelity with which organisms replicate their chromosomal DNA is of considerable interest. Detailed studies in the bacterium Escherichia coli have indicated that the fidelity of leading- and lagging-strand DNA replication is not the same, based on experiments in which the orientation of certain mutational targets on the chromosome was inverted relative to the movement of the replication fork: different mutation rates for several base-pair substitutions were observed depending on this orientation. While these experiments are indicative of differential replication fidelity in the two strands, a conclusion whether leading or lagging strand is the more accurate depends on knowledge of the primary mispairing error responsible for the base substitutions in question. A broad analysis of in vitro base-pairing preferences of DNA polymerases led us to propose that lagging-strand is the more accurate strand. In the present work, we present more direct in vivo evidence in support of this proposal. We determine the orientation dependence of mutant frequencies in ndk and dcd strains, which carry defined dNTP pool alterations. As these pool alterations lead to predictable effects on the array of possible mispairing errors, they mark the strands in which the observed errors occur. The combined results support the proposed higher accuracy of lagging-strand replication in E. coli.

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA Replication; Escherichia coli; Lac Operon; Thymine Nucleotides

The fidelity of DNA replication requires an appropriate balance of dNTPs, yet the nascent leading and lagging strands of the nuclear genome are primarily synthesized by replicases that differ in subunit composition, protein partnerships and biochemical properties, including fidelity. These facts pose the question of whether imbalanced dNTP pools differentially influence leading and lagging strand replication fidelity. Here we test this possibility by examining strand-specific replication infidelity driven by a mutation in yeast ribonucleotide reductase, rnr1-Y285A, that leads to elevated dTTP and dCTP concentrations. The results for the CAN1 mutational reporter gene present in opposite orientations in the genome reveal that the rates, and surprisingly even the sequence contexts, of replication errors are remarkably similar for leading and lagging strand synthesis. Moreover, while many mismatches driven by the dNTP pool imbalance are efficiently corrected by mismatch repair, others are repaired less efficiently, especially those in sequence contexts suggesting reduced proofreading due to increased mismatch extension driven by the high dTTP and dCTP concentrations. Thus the two DNA strands of the nuclear genome are at similar risk of mutations resulting from this dNTP pool imbalance, and this risk is not completely suppressed even when both major replication error correction mechanisms are genetically intact.

Topics: Base Pair Mismatch; Base Sequence; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA Mismatch Repair; DNA Replication; Genomic Instability; Molecular Sequence Data; Mutagenesis; Mutation Rate; Organisms, Genetically Modified; Saccharomyces cerevisiae; Thymine Nucleotides

We used surface plasmon resonance (SPR) to characterize the binding interactions between the exonulease-free Klenow fragment (Kf-exo(-)) and unmodified and modified dG adducts derived from arylamine carcinogens: fluorinated 2-aminofluorene (FAF), 2-acetylaminofluorene (FAAF), and 4-aminobiphenyl (FABP). Tight polymerase binding was detected with unmodified dG and the correct dCTP. The discrimination of correct versus incorrect nucleotides was pronounced with K(D) values in the order of dCTP ≪ dTTP < dATP < dGTP. In contrast, minimal selectivity was observed for the modified templates with Kf-exo(-) binding tighter to the FAAF (k(off): 0.02 s(-1)) and FABP (k(off): 0.01 s(-1)) lesions than to FAF (k(off): 0.04 s(-1)).

Topics: 2-Acetylaminofluorene; Aminobiphenyl Compounds; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Adducts; DNA Polymerase I; Fluorenes; Kinetics; Surface Plasmon Resonance; Thymine Nucleotides

The mechanisms by which imbalanced dNTPs induce mutations have been well characterized within a test tube, but not in vivo. We have examined mechanisms by which dNTP imbalances induce genome instability in strains of Saccharomyces cerevisiae with different amino acid substitutions in Rnr1, the large subunit of ribonucleotide reductase. These strains have different dNTP imbalances that correlate with elevated CAN1 mutation rates, with both substitution and insertion-deletion rates increasing by 10- to 300-fold. The locations of the mutations in a strain with elevated dTTP and dCTP are completely different from those in a strain with elevated dATP and dGTP. Thus, imbalanced dNTPs reduce genome stability in a manner that is highly dependent on the nature and degree of the imbalance. Mutagenesis is enhanced despite the availability of proofreading and mismatch repair. The mutations can be explained by imbalanced dNTP-induced increases in misinsertion, strand misalignment and mismatch extension at the expense of proofreading. This implies that the relative dNTP concentrations measured in extracts are truly available to a replication fork in vivo. An interesting mutational strand bias is observed in one rnr1 strain, suggesting that the S-phase checkpoint selectively prevents replication errors during leading strand replication.

Topics: Amino Acid Substitution; Amino Acid Transport Systems, Basic; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; INDEL Mutation; Mutagenesis; Mutation; Ribonucleotide Reductases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Thymine Nucleotides

SAMHD1, an analogue of the murine interferon (IFN)-γ-induced gene Mg11 (ref. 1), has recently been identified as a human immunodeficiency virus-1 (HIV-1) restriction factor that blocks early-stage virus replication in dendritic and other myeloid cells and is the target of the lentiviral protein Vpx, which can relieve HIV-1 restriction. SAMHD1 is also associated with Aicardi-Goutières syndrome (AGS), an inflammatory encephalopathy characterized by chronic cerebrospinal fluid lymphocytosis and elevated levels of the antiviral cytokine IFN-α. The pathology associated with AGS resembles congenital viral infection, such as transplacentally acquired HIV. Here we show that human SAMHD1 is a potent dGTP-stimulated triphosphohydrolase that converts deoxynucleoside triphosphates to the constituent deoxynucleoside and inorganic triphosphate. The crystal structure of the catalytic core of SAMHD1 reveals that the protein is dimeric and indicates a molecular basis for dGTP stimulation of catalytic activity against dNTPs. We propose that SAMHD1, which is highly expressed in dendritic cells, restricts HIV-1 replication by hydrolysing the majority of cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA (cDNA) synthesis.

Topics: Allosteric Regulation; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Dendritic Cells; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; HIV-1; Humans; Hydrolysis; Models, Biological; Models, Molecular; Monomeric GTP-Binding Proteins; Myeloid Cells; Nucleoside-Triphosphatase; Protein Structure, Tertiary; Reverse Transcription; SAM Domain and HD Domain-Containing Protein 1; Thymine Nucleotides; Viral Regulatory and Accessory Proteins; Virus Replication

Human DNA polymerase iota is a lesion bypass polymerase of the Y family, capable of incorporating nucleotides opposite a variety of lesions in both near error-free and error-prone bypass. With undamaged templating purines polymerase iota normally favors Hoogsteen base pairing. Polymerase iota can incorporate nucleotides opposite a benzo[a]pyrene-derived adenine lesion (dA*); while mainly error-free, the identity of misincorporated bases is influenced by local sequence context. We performed molecular modeling and molecular dynamics simulations to elucidate the structural basis for lesion bypass. Our results suggest that hydrogen bonds between the benzo[a]pyrenyl moiety and nearby bases limit the movement of the templating base to maintain the anti glycosidic bond conformation in the binary complex in a 5'-CAGA*TT-3' sequence. This facilitates correct incorporation of dT via a Watson-Crick pair. In a 5'-TTTA*GA-3' sequence the lesion does not form these hydrogen bonds, permitting dA* to rotate around the glycosidic bond to syn and incorporate dT via a Hoogsteen pair. With syn dA*, there is also an opportunity for increased misincorporation of dGTP. These results expand our understanding of the versatility and flexibility of polymerase iota and its lesion bypass functions in humans.

Topics: Base Pair Mismatch; Base Pairing; Base Sequence; Benzopyrenes; Carcinogens, Environmental; Computer Simulation; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA Adducts; DNA Damage; DNA Polymerase iota; DNA-Directed DNA Polymerase; Humans; Hydrogen Bonding; Magnesium; Models, Molecular; Nucleic Acid Conformation; Thymine Nucleotides

Ribonucleotide reductase is the main control point of dNTP production. It has two subunits, R1, and R2 or p53R2. R1 has 5 possible catalytic site states (empty or filled with 1 of 4 NDPs), 5 possible s-site states (empty or filled with ATP, dATP, dTTP or dGTP), 3 possible a-site states (empty or filled with ATP or dATP), perhaps two possible h-site states (empty or filled with ATP), and all of this is folded into an R1 monomer-dimer-tetramer-hexamer equilibrium where R1 j-mers can be bound by variable numbers of R2 or p53R2 dimers. Trillions of RNR complexes are possible as a result. The problem is to determine which are needed in models to explain available data. This problem is intractable for 10 reactants, but it can be solved for 2 and is here for R1 and ATP.. Thousands of ATP-induced R1 hexamerization models with up to three (s, a and h) ATP binding sites per R1 subunit were automatically generated via hypotheses that complete dissociation constants are infinite and/or that binary dissociation constants are equal. To limit the model space size, it was assumed that s-sites are always filled in oligomers and never filled in monomers, and to interpret model terms it was assumed that a-sites fill before h-sites. The models were fitted to published dynamic light scattering data. As the lowest Akaike Information Criterion (AIC) of the 3-parameter models was greater than the lowest of the 2-parameter models, only models with up to 3 parameters were fitted. Models with sums of squared errors less than twice the minimum were then partitioned into two groups: those that contained no occupied h-site terms (508 models) and those that contained at least one (1580 models). Normalized AIC densities of these two groups of models differed significantly in favor of models that did not include an h-site term (Kolmogorov-Smirnov p < 1 x 10(-15)); consistent with this, 28 of the top 30 models (ranked by AICs) did not include an h-site term and 28/30 > 508/2088 with p < 2 x 10(-15). Finally, 99 of the 2088 models did not have any terms with ATP/R1 ratios >1.5, but of the top 30, there were 14 such models (14/30 > 99/2088 with p < 3 x 10(-16)), i.e. the existence of R1 hexamers with >3 a-sites occupied by ATP is also not supported by this dataset.. The analysis presented suggests that three a-sites may not be occupied by ATP in R1 hexamers under the conditions of the data analyzed. If a-sites fill before h-sites, this implies that the dataset analyzed can be explained without the existence of an h-site.

Topics: Adenosine Triphosphate; Catalytic Domain; Computer Simulation; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Humans; Least-Squares Analysis; Models, Biological; Nonlinear Dynamics; Protein Multimerization; Protein Subunits; Ribonucleotide Reductases; Substrate Specificity; Thymine Nucleotides

DNA analysis is an important technology with respect to diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, and it was applied to single-nucleotide polymorphism (SNP) analysis using one-base extension reaction. The principle of this method is as follows. A specific primer within each aliquot possessing a short 3' end of the base of interest was hybridized to the single-stranded DNA template. Subsequently, (exo-)Klenow DNA polymerase and one of either alpha-thio-dATP, dTTP, dGTP, or dCTP were added and incubated for 1 min. Pyrophosphate released by DNA polymerase is converted to ATP by pyruvate phosphate dikinase (PPDK), and the concentration of ATP is determined using the firefly luciferase reaction. This method, which does not require expensive equipment, can be used to rapidly monitor one point mutation in the gene.

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Diphosphates; DNA; Luciferases; Luminescent Measurements; Models, Biological; Polymorphism, Single Nucleotide; Pyruvate, Orthophosphate Dikinase; ras Proteins; Reproducibility of Results; Thionucleotides; Thymine Nucleotides; Tumor Suppressor Protein p53

We describe a technique to synthesize DNA homopolymers on a surface using surface-initiated enzymatic polymerization (SIEP) with terminal deoxynucleotidyl transferase (TdTase), an enzyme that repetitively adds mononucleotides to the 3'-end of oligonucleotides. The thickness of the synthesized DNA layer was found to depend on the deoxymononucleotide monomer, in the order of dATP > dTTP > dGTP approximately dCTP. In addition, the composition and the surface density of oligonucleotide initiators were also important in controlling the extent of DNA polymerization. The extension of single-stranded DNA chains by SIEP was further verified by their binding to antibodies specific to oligonucleotides. TdTase-mediated SIEP can also be used to grow spatially defined three-dimensional DNA structures by soft lithography and is a new tool for bioinspired fabrication at the micro- and nanoscale.

Topics: Antibodies; Antibody Specificity; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA Nucleotidylexotransferase; DNA, Single-Stranded; Microscopy, Atomic Force; Nanotechnology; Oligonucleotides; Polydeoxyribonucleotides; Spectrometry, X-Ray Emission; Surface Plasmon Resonance; Surface Properties; Thymine Nucleotides; Time Factors

Bulky carcinogen-DNA adducts commonly cause replicative polymerases to stall, leading to a switch to bypass polymerases. We have investigated nucleotide incorporation opposite the major adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the DinB family polymerase, Dpo4, using molecular modeling and molecular dynamics (MD) simulations. PhIP, the most prevalent heterocyclic aromatic amine formed by cooking of proteinaceous food, is mutagenic in mammalian cells and is implicated in mammary and colon tumors. Our results show that the dG-C8-PhIP adduct can be accommodated in the spacious major groove Dpo4 open pocket, with Dpo4 capable of incorporating dCTP, dTTP or dATP opposite the adduct reasonably well. However, the PhIP ring system on the minor groove side would seriously disturb the active site, regardless of the presence and identity of dNTP. Furthermore, the simulations indicate that dATP and dTTP are better incorporated in the damaged system than in their respective mismatched but unmodified controls, suggesting that the PhIP adduct enhances incorporation of these mismatches. Finally, bulky C8-dG adducts, situated in the major groove, are likely to impede translocation in this polymerase (Rechkoblit et al. (2006), PLoS Biol., 4, e11). However, N2-dG adducts, which can reside on the minor groove side, appear to cause less hindrance when in this position.

Topics: Binding Sites; Carcinogens; Computational Biology; Computer Simulation; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyguanosine; Deoxyribonucleotides; DNA; DNA Adducts; DNA Polymerase beta; Imidazoles; Models, Molecular; Motion; Mutagenesis; Nucleic Acid Conformation; Sulfolobus solfataricus; Thymine Nucleotides

Dividing cultured cells contain much larger pools of the four dNTPs than resting cells. In both cases the sizes of the individual pools are only moderately different. The same applies to mitochondrial (mt) pools of cultured cells. Song et al. [Song S, Pursell ZF, Copeland WC, Longley MJ, Kunkel TA, Mathews CK (2005) Proc Natl Acad Sci USA 102:4990-4995] reported that mt pools of rat tissues instead are highly asymmetric, with the dGTP pool in some cases being several-hundred-fold larger than the dTTP pool, and suggested that the asymmetry contributes to increased mutagenesis during mt DNA replication. We have now investigated this discrepancy and determined the size of each dNTP pool in mouse liver mitochondria. We found large variations in pool sizes that closely followed variations in the ATP pool and depended on the length of time spent in the preparation of mitochondria. The proportion between dNTPs was in all cases without major asymmetries and similar to those found earlier in cultured resting cells. We also investigated the import and export of thymidine phosphates in mouse liver mitochondria and provide evidence for a rapid, highly selective, and saturable import of dTMP, not depending on a functional respiratory chain. At nM external dTMP the nucleotide is concentrated 100-fold inside the mt matrix. Export of thymidine phosphates was much slower and possibly occurred at the level of dTDP.

Topics: Animals; Biological Transport, Active; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyuracil Nucleotides; Male; Mice; Mice, Inbred C57BL; Mitochondria, Liver; Thymidine Monophosphate; Thymine Nucleotides

To investigate the molecular basis for the selective utilization of nucleoside triphosphates complementary to templating bases, by RB69 DNA polymerase (RB69 pol), we constructed a set of mutants that we predicted would perturb the "floor" of the nascent base-pairing interface in the enzyme. We then determined the pre-steady-state kinetic parameters for the incorporation of complementary and noncomplementary dNTPs by the exo(-) form of RB69 pol and its mutants. We found that the Y567A mutant had the same K(d) and k(pol) values for incorporation of C versus G as the wild-type exo(-) enzyme; however, the k(pol)/K(d) ratio for G versus G incorporation with the Y567A mutant was 10 times higher than the k(pol)/K(d) efficiency of G versus G incorporation using the exo(-) RB69 pol. The reduced level of discrimination by the Y567A mutant against incorporation of mismatched bases was also seen with the Y391A mutant. Stopped-flow fluorescence was also employed to monitor rates of putative conformational changes with the exo(-) RB69 pol and its mutants using a primer-template complex containing 2-aminopurine. The rates of fluorescence changes were equal to or greater than the rates of the rapid chemical quench, indicating that we were monitoring a process occurring before or during the phosphoryl transfer reaction. We have interpreted our results within the context of the crystal structure of the RB69 pol ternary complex [Franklin, M. C., et al. (2001) Cell 105, 657-667].

Topics: Alanine; Amino Acid Substitution; Base Pair Mismatch; Binding Sites; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA-Directed DNA Polymerase; Enterobacter; Hydrogen Bonding; Kinetics; Nucleotides; Phenylalanine; Substrate Specificity; Thymine Nucleotides; Toluene; Tyrosine; Viral Proteins

8-OxoGua (8-oxo-7,8-dihydroguanine) is produced in nucleic acids as well as in nucleotide pools of cells, by reactive oxygen species normally formed during cellular metabolic processes. MutT protein of Escherichia coli specifically degrades 8-oxoGua-containing deoxyribo- and ribonucleoside triphosphates to corresponding nucleoside monophosphates, thereby preventing misincorporation of 8-oxoGua into DNA and RNA, which would cause mutation and phenotypic suppression, respectively. Here, we report that the MutT protein has additional activities for cleaning up the nucleotide pools to ensure accurate DNA replication and transcription. It hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP with a K(m) of 0.058 microM, a value considerably lower than that for its normal counterpart, dGDP (170 microM). Furthermore, the MutT possesses an activity to degrade 8-oxo-GDP to the related nucleoside monophosphate, with a K(m) value 8000 times lower than that for GDP. These multiple enzyme activities of the MutT protein would facilitate the high fidelity of DNA and RNA syntheses.

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA Replication; DNA, Bacterial; Escherichia coli Proteins; Guanine; Guanosine Triphosphate; Hydrolysis; Kinetics; Multienzyme Complexes; Phosphoric Monoester Hydrolases; Pyrophosphatases; RNA, Bacterial; Thymine Nucleotides; Transcription, Genetic

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) conducted in real time is a powerful tool for measuring messenger RNA (mRNA) levels in biological samples. Multiplex PCR is defined as the simultaneous amplification of two or more DNA (cDNA) targets in a single reaction vessel and may be carried out only using uniquely labeled probes for each target. Up to four genes can be detected in a multiplex 5' nuclease assay when using the appropriate instrument and the right combination of fluorophores. One of the more important advantages of multiplexing is a reduced sample requirement, which is especially important when sample material is scarce. Additional benefits are saving time on reaction setup and lower cost compared to singleplex reactions. Although multiplexing has several advantages over singleplex qRT-PCR, limited work has been done to show its feasibility. Few publications on four-color multiplex qRT-PCR have been reported, and to our knowledge no work has been done to explore the assay's limitations. In this paper, we report the first in-depth analysis of a four-gene multiplex qRT-PCR. To achieve a better understanding of the potential limitations of the qRT-PCR assay, we used in vitro transcribed RNA derived from four human genes. To emulate gene expression experiments, we developed a model system in which the in vitro transcripts were spiked with plant total RNA. This model allowed us to develop an artificial system closely resembling differential gene expression levels varying up to a million fold. We identified a single "universal" reaction condition that enabled optimal amplification in real time of up to four genes over a wide range of template concentrations. This study shows that multiplexing is a feasible approach applicable to most qRT-PCR assays performed with total RNA, independent of the expression levels of the genes under scrutiny.

Topics: Color; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA Primers; DNA Probes; DNA, Complementary; Gene Expression; Reverse Transcriptase Polymerase Chain Reaction; Taq Polymerase; Thymine Nucleotides

Equilibrium as well as pre-steady-state measurements were performed to characterize the molecular basis of DNA binding and nucleotide incorporation by the thermostable archaeal DinB homologue (Dbh) DNA polymerase of Sulfolobus solfataricus. Equilibrium titrations show a DNA binding affinity of about 60 nm, which is approximately 10-fold lower compared with other DNA polymerases. Investigations of the binding kinetics applying stopped-flow and pressure jump techniques confirm this weak binding affinity. Furthermore, these measurements suggest that the DNA binding occurs in a single step, diffusion-controlled manner. Single-turnover, single dNTP incorporation studies reveal maximal pre-steady-state burst rates of 0.64, 2.5, 3.7, and 5.6 s(-1) for dTTP, dATP, dGTP, and dCTP (at 25 degrees C), which is 10-100-fold slower than the corresponding rates of classical DNA polymerases. Another unique feature of the Dbh is the very low nucleotide binding affinity (K(d) approximately 600 mum), which again is 10-20-fold lower compared with classical DNA polymerases as well as other Y-family polymerases. Surprisingly, the rate-limiting step of nucleotide incorporation (correct and incorrect) is the chemical step (phosphoryl transfer) and not a conformational change of the enzyme. Thus, unlike replicative polymerases, an "induced fit" mechanism to select and incorporate nucleotides during DNA polymerization could not be detected for Dbh.

Topics: Archaeal Proteins; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA-Directed DNA Polymerase; Kinetics; Thymine Nucleotides

Replication forks often stall at undamaged or damaged template sites in Escherichia coli. Subsequent resumption of DNA synthesis occurs by replacing DNA polymerase III, which is bound to DNA by the beta-sliding clamp, with one of three damage-induced DNA polymerases II, IV, or V. The principal role of the beta clamp is to tether the normally weakly bound polmerases to DNA thereby increasing their processivities. DNA polymerase IV binds dNTP substrates with about 10-fold lower affinity compared with the other E. coli polymerases, which if left unchecked could hinder its ability to synthesize DNA in vivo. Here we report a new property for the beta clamp, which when bound to DNA polymerase IV results in a large increase in dNTP binding affinity that concomitantly increases the efficiency of nucleotide incorporation at normal and transiently slipped mispaired primer/template ends. Primer-template DNA slippage resulting in single nucleotide deletions is a biological hallmark of DNA polymerase IV infidelity responsible for enhancing cell fitness in response to stress. We show that the increased DNA polymerase IV-dNTP binding affinity is an intrinsic property of the DNA polymerase IV-beta clamp interaction and not an indirect consequence of an increased binding of DNA polymerase IV to DNA.

Topics: Base Pair Mismatch; Binding Sites; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA; DNA Polymerase beta; DNA Polymerase III; DNA Primers; Escherichia coli; Fluorescence Polarization; Frameshift Mutation; Kinetics; Oligodeoxyribonucleotides; Templates, Genetic; Thymine Nucleotides

DNA polymerase mu (Polmu), an X-family DNA polymerase, is preferentially expressed in secondary lymphoid tissues with yet unknown physiological functions. In this study, Polmu was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme had a lifetime of <20 min at 37 degrees C, but was stable for over 3 h at 25 degrees C in an optimized reaction buffer. The fidelity of human Polmu was thus determined using pre-steady-state kinetic analysis of the incorporation of single nucleotides into undamaged DNA 21/41-mer substrates at 25 degrees C. Single-turnover saturation kinetics for all 16 possible deoxynucleotide (dNTP) incorporations and for four matched ribonucleotide (rNTP) incorporations were measured under conditions where Polmu was in molar excess over DNA. The polymerization rate (k(p)), binding affinity (K(d)), and substrate specificity (k(p)/K(d)) are 0.006-0.076 s(-1), 0.35-1.8 microM, and (8-64) x10(-3) microM(-1) s(-1), respectively, for matched incoming dNTPs, (2-30) x 10(-5) s(-1), 7.3-135 microM, and (4-61) x 10(-7) microM(-1) s(-1), respectively, for mismatched incoming dNTPs, and (2-73) x 10(-4) s(-1), 45-302 microM, and (7-1300) x 10(-7) microM(-1) s(-1), respectively, for matched incoming rNTPs. The overall fidelity of Polmu was estimated to be in the range of 10(-3)-10(-5) for both dNTP and rNTP incorporations and was sequence-independent. The sugar selectivity, defined as the substrate specificity ratio of a matched dNTP versus a matched rNTP, was measured to be in the range of 492-10959. In addition to a slow and distributive DNA polymerase activity, Polmu was identified to possess a weak strand-displacement activity. The potential biological roles of Polmu are discussed.

Topics: Binding Sites; Buffers; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA; DNA Nucleotidylexotransferase; DNA Primers; DNA-Directed DNA Polymerase; Enzyme Activation; Enzyme Stability; Humans; Kinetics; Peptide Chain Elongation, Translational; Ribonucleotides; Substrate Specificity; Templates, Genetic; Thymine Nucleotides; Uridine Triphosphate

DNA polymerase mu (pol mu) is a member of the pol X family of DNA polymerases, and it shares a number of characteristics of both DNA polymerase beta (pol beta) and terminal deoxynucleotidyl transferase (TdT). Because pol beta has been shown to perform translesion DNA synthesis past cisplatin (CP)- and oxaliplatin (OX)-GG adducts, we determined the ability of pol mu to bypass these lesions. Pol mu bypassed CP and OX adducts with an efficiency of 14-35% compared to chain elongation on undamaged DNA, which is second only to pol eta in terms of bypass efficiency. The relative ability of pol mu to bypass CP and OX adducts was dependent on both template structure and sequence context. Since pol mu has been shown to be more efficient on gapped DNA templates than on primed single-stranded DNA templates, we determined the ability of pol mu to bypass Pt-DNA adducts on both primed single-stranded and gapped templates. The bypass of Pt-DNA adducts by pol mu was highly error-prone on all templates, resulting in 2, 3, and 4 nt deletions. We postulate that bypass of Pt-DNA adducts by pol mu may involve looping out the Pt-GG adduct to allow chain elongation downstream of the adduct. This reaction appears to be facilitated by the presence of a downstream "acceptor" and a gap large enough to provide undamaged template DNA for elongation past the adduct, although gapped DNA is clearly not required for bypass.

Topics: Catalysis; Cisplatin; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA Adducts; DNA Damage; DNA Primers; DNA-Directed DNA Polymerase; DNA, Single-Stranded; Guanine Nucleotides; Humans; Oligonucleotides; Organoplatinum Compounds; Oxaliplatin; Templates, Genetic; Thymine Nucleotides

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder associated with multiple mutations in mitochondrial DNA, both deletions and point mutations, and mutations in the nuclear gene for thymidine phosphorylase. Spinazzola et al. (Spinazzola, A., Marti, R., Nishino, I., Andreu, A., Naini, A., Tadesse, S., Pela, I., Zammarchi, E., Donati, M., Oliver, J., and Hirano, M. (2001) J. Biol. Chem. 277, 4128-4133) showed that MNGIE patients have elevated circulating thymidine levels and they hypothesized that this generates imbalanced mitochondrial deoxyribonucleoside triphosphate (dNTP) pools, which in turn are responsible for mitochondrial (mt) DNA mutagenesis. We tested this hypothesis by culturing HeLa cells in medium supplemented with 50 microM thymidine. After 8-month growth, mtDNA in the thymidine-treated culture, but not the control, showed multiple deletions, as detected both by Southern blotting and by long extension polymerase chain reaction. After 4-h growth in thymidine-supplemented medium, we found the mitochondrial dTTP and dGTP pools to expand significantly, the dCTP pool to drop significantly, and the dATP pool to drop slightly. In whole-cell extracts, dTTP and dGTP pools also expanded, but somewhat less than in mitochondria. The dCTP pool shrank by about 50%, and the dATP pool was essentially unchanged. These results are discussed in terms of the recent report by Nishigaki et al. (Nishigaki, Y., Marti, R., Copeland, W. C., and Hirano, M. (2003) J. Clin. Invest. 111, 1913-1921) that most mitochondrial point mutations in MNGIE patients involve T --> C transitions in sequences containing two As to the 5' side of a T residue. Our finding of dTTP and dGTP elevations and dATP depletion in mitochondrial dNTP pools are consistent with a mutagenic mechanism involving T-G mispairing followed by a next-nucleotide effect involving T insertion opposite A.

Topics: Base Pairing; Blotting, Southern; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA, Mitochondrial; Gastrointestinal Diseases; Gene Deletion; HeLa Cells; Humans; Mitochondria; Mitochondrial Encephalomyopathies; Point Mutation; Polymerase Chain Reaction; Thymidine; Thymine Nucleotides

Azodicarbonamide tested as an anti-HIV agent was reported to expulse zinc from viral zinc-cysteine factors and to inhibit calcium mobilization machinery. It has structural analogy with hydroxyurea that inhibits ribonucleotide reductase and could also act on this target. Azodicarbonamide was therefore tested for its capacity to modulate deoxyribonucleotides triphosphate pools alone or in combination with other agents in the lymphoblastic SUP-T1 cell line susceptible to HIV infection. The deoxyribonucleotides triphosphate were evaluated by an enzymatic assay using sequenase. Two hours exposure of SUP-T1 cells to 100 microM azodicarbonamide induced a 50% reduction of each deoxyribonucleotide triphosphate. Among other inhibitors of nucleotide metabolism (hydroxyurea, methotrexate and thymidine), hydroxyurea only reproduces the effect of azodicarbonamide. This suggests, but does not demonstrate directly, that azodicarbonamide inhibits ribonucleotide reductase activity. The combination of azodicarbonamide with each of these inhibitors affected particularly the dCTP pool. During this study it was also suggested that azodicarbonamide could interfere with thymidine phosphorylation. Thymidine phosphorylating activity was measured with 3H-thymidine as substrate. In acellular preparations, azodicarbonamide also non-competitively inhibits thymidine phosphorylating activity. This effect was not reproduced by hydroxyurea. Thus, in vitro azodicarbonamide decreases the intracellular pool of deoxyribonucleotide and thymidine phosphorylation.

Topics: Animals; Antineoplastic Agents; Azo Compounds; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Drug Interactions; Hydroxyurea; Immunosuppressive Agents; Methotrexate; Phosphorylation; Ribonucleotide Reductases; Thymidine; Thymine Nucleotides; Tumor Cells, Cultured

Topics: Avian Sarcoma Viruses; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA, Complementary; Drug Stability; Electrophoresis, Agar Gel; Half-Life; Hot Temperature; Hydrolysis; Magnesium; Moloney murine leukemia virus; Nucleic Acid Denaturation; RNA; RNA-Directed DNA Polymerase; Thymine Nucleotides; Transcription, Genetic; Viral Proteins

Octamer-primed sequencing is a directed DNA sequencing strategy that employs the use of a presynthesized octamer primer library. Together with electronic octamer sequencing technology (eOST) it provides a faster, less expensive way to obtain DNA sequence information and can be used as a valuable tool for gap closure in large-scale genomic sequencing. In this paper we discuss the effect of dGTP/TTP supplementation on octamer sequencing. We show that addition of 75 microM dGTP and 5 microM TTP can improve the sequencing success rate by increasing the length and accuracy of generated sequence information. We also discuss the effect of template base composition immediately downstream of the octamer primer on the outcome of octamer sequencing.

Topics: Base Composition; Base Sequence; Deoxyguanine Nucleotides; DNA; DNA Primers; Gene Library; Molecular Sequence Data; Molecular Weight; Nucleotides; Oligodeoxyribonucleotides; Sensitivity and Specificity; Sequence Analysis, DNA; Taq Polymerase; Templates, Genetic; Thymine Nucleotides; Time Factors

The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment of Escherichia coli Pol I (exo(-)) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene diol epoxide isomer [(+)-anti-BPDE] to N(2)-guanine (G*). Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5'-end. All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5'-.XGY., with X, Y = C and/or T). In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G, with A > G). Depending on X or Y, full lesion bypass differed by factors of approximately 1.5-5 ( approximately 0.6-3.0% bypass efficiencies). A downstream T flanking G on the 5'-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template-primer intermediates are particularly dominant ( approximately 5.0-6.0% efficiencies) with an upstream flanking C, while a 3'-flanking T lowers the levels of deletion products ( approximately 0.5-2.5% efficiencies). The kinetics of (1) single dNTP insertion opposite G* and (2) extension of the primer beyond G* by a single dNTP, or in the presence of all four dNTPs, with different 3'-terminal primer bases (Z) opposite G* were investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching approximately 90%) was found with Z = T in the case of sequences with 3'-flanking upstream C rather than T. These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4-6 bases from G*) with the 3'-terminal primer base and its 5'-flanking base. The latter depend on the base Y and on the base preferentially inserted opposite the adduct. Thus, downstream template sequences as well as the bases flanking G* influence DNA translesion synthesis.

Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Base Sequence; Carcinogens, Environmental; Catalysis; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyguanosine; DNA Damage; DNA Polymerase I; DNA Primers; DNA Replication; Guanine; Kinetics; Mutagenesis; Mutagens; Polydeoxyribonucleotides; Pyrimidine Nucleotides; Sequence Analysis, DNA; Templates, Genetic; Thymine Nucleotides

The ribonucleoprotein enzyme telomerase adds telomeric repeats to the ends of linear chromosomes. The Tetrahymena telomerase reverse transcriptase (TERT) protein and the telomerase RNA can be reconstituted into an active complex in vitro in rabbit reticulocyte lysates. We have probed the structure of the telomerase RNA in the reconstituted complex with RNases T1 and V1. Upon TERT binding to the RNA, sites of both protection and enhancement of cleavage were observed, suggesting potential protein-binding sites and conformational changes in the RNA. Especially prominent was a large region of RNase V1 protection in stem-loop IV. A number of loop IV mutants still bound TERT but showed drastic decreases in the level of telomerase activity and the loss of protein-dependent folding of the pseudoknot region of the telomerase RNA. The telomerase activity defect and the misfolding of the pseudoknot were partially separable, leading to the proposal of two functions for stem-loop IV: to aid in the folding of the pseudoknot and to function more directly in the active site of telomerase. Thus an RNA element far from the template makes a major contribution to Tetrahymena telomerase enzyme activity.

Topics: Animals; Base Sequence; Binding Sites; Deoxyguanine Nucleotides; DNA-Binding Proteins; Endoribonucleases; Enzyme Activation; Molecular Sequence Data; Nucleic Acid Conformation; Rabbits; RNA; RNA, Protozoan; Sequence Deletion; Telomerase; Templates, Genetic; Tetrahymena thermophila; Thymine Nucleotides

Studies were undertaken to assess the ability of human polymerase alpha (pol alpha) and polymerase gamma (pol gamma) to incorporate 2'-fluoro- and 2'-O-methyldeoxynucleotides into DNA. In vitro DNA synthesis systems were used to detect incorporation and determine K(m) and V(max) for 2'-FdATP, 2'-FdUTP, 2'-FdCTP, 2'-FdGTP, 2'-O-MedATP, 2'-O-MedCTP, 2'-O-MedGTP, 2'-O-MedUTP, dUTP, UTP, and FIAUTP, in addition to normal deoxynucleotides. Pol alpha incorporated all 2'-FdNTPs except 2'-FdATP, but not 2'-O-MedNTPs. Pol gamma incorporated all 2'-FdNTPs, but not 2'-O-MedNTPs. In general, 2'-fluorine substitution decreased V(max)/K(m) 2'-FdUTP. Because kinetics of insertion of pol alpha can be affected by the nature of the primer, we examined the ability of pol alpha to polymerize 2'-fluoro- and 2'-O-MedATP and dGTP when elongating a primer synthesized by DNA primase. Under these conditions, both 2'-FdATP and 2'-FdGTP were polymerized, but 2'-O-MedATP and 2'-O-MedGTP were not. Primase alone could not readily polymerize these analogs into RNA primers. Previous studies showed that 2'-deoxy-2'-fluorocytosine (2'-FdC) is incorporated by several non-human DNA polymerases. The current studies showed that human polymerases can polymerize numerous 2'-FdNTPs but cannot polymerize 2'-O-MedNTPs.

Topics: Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA Polymerase gamma; DNA Polymerase I; DNA Primase; DNA-Directed DNA Polymerase; Humans; Thymine Nucleotides; Uridine Triphosphate

Topics: Aging; Animals; Buffers; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA; Hot Temperature; Hydrogen-Ion Concentration; Indicators and Reagents; Magnesium Chloride; Mice; Polymerase Chain Reaction; Sodium Hydroxide; Solubility; Taq Polymerase; Thymine Nucleotides

DNA replication normally occurs with high fidelity, but certain "slippery" regions of DNA with tracts of mono-, di-, and trinucleotide repeats are frequently mutation hot spots. We have developed an in vitro assay to study the mechanism of dinucleotide repeat expansion. The primer-template resembles a base excision repair substrate with a single nucleotide gap centered opposite a tract of nine CA repeats; nonrepeat sequences flank the dinucleotide repeats. DNA polymerases are expected to repair the gap, but further extension is possible if the DNA polymerase can displace the downstream oligonucleotide. We report here that the wild type bacteriophage T4 DNA polymerase carries out gap and strand displacement replication and also catalyzes a dinucleotide expansion reaction. Repeat expansion was not detected for an exonuclease-deficient T4 DNA polymerase or for Escherichia coli DNA polymerase I. The dinucleotide repeat expansion reaction catalyzed by wild type T4 DNA polymerase required a downstream oligonucleotide to "stall" replication and 3' --> 5' exonuclease activity to remove the 3'-nonrepeat sequence adjacent to the repeat tract in the template strand. These results suggest that dinucleotide repeat expansion may be stimulated in vivo during DNA repair or during processing of Okazaki fragments.

Topics: Deoxyguanine Nucleotides; DNA Polymerase I; DNA Primers; DNA-Directed DNA Polymerase; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Models, Genetic; Repetitive Sequences, Nucleic Acid; Thymine Nucleotides; Time Factors; Viral Proteins

While investigating the basis for marked natural asymmetries in deoxyribonucleoside triphosphate (dNTP) pools in mammalian cells, we observed that culturing V79 hamster lung cells in a 2% oxygen atmosphere causes 2-3-fold expansions of the dATP, dGTP, and dTTP pools, whereas dCTP declines by a comparable amount. Others have made similar observations and have proposed that, because O(2) is required for formation of the catalytically essential oxygen-bridged iron center in ribonucleotide reductase, dCTP depletion at low oxygen tension results from direct or indirect effects upon ribonucleotide reductase. We have tested the hypothesis that oxygen limitation affects ribonucleotide specificity using recombinant mouse ribonucleotide reductase and an assay that permits simultaneous monitoring of the reduction of all four nucleotide substrates. Preincubation and assay of the enzyme in an anaerobic chamber caused only partial activity loss. Accordingly, we treated the enzyme with hydroxyurea, followed by removal of the hydroxyurea and exposure to atmospheres of varying oxygen content. The activity was totally depleted by hydroxyurea treatment and nearly fully regained by exposure to air. By the criterion of activities regained at different oxygen tensions, we found CDP reduction not to be specifically sensitive to oxygen depletion; however, GDP reduction was specifically sensitive. The basis for the differential response to reactivation by O(2) is not known, but it evidently does not involve varying rates of reactivation of different allosteric forms of the enzyme or altered response to allosteric effectors at reduced oxygen tension.

Topics: Animals; Cells, Cultured; Cricetinae; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Hydrogen-Ion Concentration; Hydroxyurea; Hypoxia; Mice; Models, Chemical; Nucleic Acid Synthesis Inhibitors; Oxygen; Recombinant Proteins; Ribonucleotide Reductases; Thymine Nucleotides; Time Factors

Telomerase is classified as one of the reverse transcriptases (RTs). To clarify whether l-enantiomers of natural 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) are recognized by human telomerase, a quantitative telomerase assay based on the "stretch PCR" method was developed and used for kinetic analysis of the inhibitory effects of these compounds on the enzyme. Among the four l-enantiomers of dNTPs, l-dTTP and l-dGTP inhibited telomerase activity and the others showed slight or no inhibitory effect. Lineweaver-Burk plot analysis showed that the inhibition modes of l-dTTP and l-dGTP were partially competitive (mixed type) and competitive with the corresponding substrate dNTP, respectively. However, the K(i) values of l-dTTP and l-dGTP (21 and 15 microM) were several times larger than the K(m) values (3-6 microM). These results suggest that the active site of telomerase is not able to discriminate strictly the chirality of dNTPs, although it is more discriminatory than HIV-1 RT.

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Humans; Kinetics; Stereoisomerism; Substrate Specificity; Telomerase; Thymine Nucleotides

Rare Ig and TCR coding joints can be isolated from mice that have a targeted deletion in the gene encoding the 86-kDa subunit of the Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). However in the coding joints isolated from Ku86-/- animals, there is an extreme paucity of N regions (the random nucleotides added during V(D)J recombination by the enzyme TdT). This finding is consistent with a decreased frequency of coding joints containing N regions isolated from C.B-17 SCID mice that express a truncated form of the catalytic subunit of the DNA-PK (DNA-PKCS). This finding suggests an unexpected role for DNA-PK in addition of N nucleotides to coding ends during V(D)J recombination. In this report, we establish that TdT forms a stable complex with DNA-PK. Furthermore, we show that DNA-PK modulates TdT activity in vitro by limiting both the length and composition of nucleotide additions.

Topics: Animals; Catalysis; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA Nucleotidylexotransferase; DNA-Activated Protein Kinase; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Humans; Mice; Mice, SCID; Nuclear Proteins; Oligonucleotides; Protein Serine-Threonine Kinases; Substrate Specificity; Thymine Nucleotides; Tumor Cells, Cultured

Telomerase, which synthesizes telomeric DNA in eukaryotic cells, is classified as a reverse transcriptase. To clarify the recognition of 2'-deoxyribonucleoside 5'-triphosphate (dNTP) chirality by telomerase, we studied the inhibitory effects of L-dGTP on HeLa cell telomerase activity using a quantitative 'stretch PCR' assay. L-dGTP had a weakly inhibitory effect (IC50 = 200 microM) in the presence of 10 microM dGTP. This effect was less obvious when the concentration of dGTP was higher. L-dTTP had a similar inhibitory effect. These findings suggest that telomerase may bind to L-dGTP and L-dTTP, and that the ability of telomerase to bind to dGTP or dTTP is changed.

Topics: Base Sequence; Deoxyguanine Nucleotides; DNA Primers; HeLa Cells; Humans; Kinetics; Polymerase Chain Reaction; Stereoisomerism; Telomerase; Thymine Nucleotides

The mutational properties of 5-formyl-2'-deoxyuridine 5'-triphosphate (5-CHO-dUTP) and 5-hydroxy-2'-deoxycytidine 5'-triphosphate (5-OH-dCTP), the major oxidatively damaged pyrimidine nucleotides derived from dTTP and dCTP, respectively, were analyzed by an in vivo assay. 5-CHO-dUTP and 5-OH-dCTP were directly incorporated into Escherichia coli , and their mutagenicities were evaluated by the chromosomal lacI forward mutation assay. The mutation frequencies increased, depending on the dose of these damaged nucleotides, indicating that these nucleotides were incorporated into E.coli and acted as mutagens in vivo . The mutagenicities of 5-CHO-dUTP and 5-OH-dCTP were comparable to that of 8-hydroxy-2'-deoxyguanosine 5'-triphosphate, a major form of dGTP oxidative damage. 5-CHO-dUTP induced G.C to A.T, A.T to G.C and G.C to T.A mutations, and 5-OH-dCTP elicited G.C to A.T, A.T to C.G and G.C to T.A mutations.

Topics: Bacterial Proteins; Deoxyadenine Nucleotides; Deoxycytidine; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyuracil Nucleotides; Deoxyuridine; DNA Mutational Analysis; DNA Repair; Escherichia coli; Escherichia coli Proteins; Genome, Bacterial; Lac Repressors; Models, Genetic; Mutagenesis; Nucleic Acid Hybridization; Oxidants; Point Mutation; Repressor Proteins; Sequence Analysis, DNA; Thymine Nucleotides

Mutations that confer resistance to nucleoside analogs do not cluster around the deoxynucleotide triphosphate (dNTP) binding site. Instead, these mutations appear to lie along the groove in the enzyme where the template-primer binds. Based on such structural data and on complementary biochemical analyses, it has been suggested that resistance to nucleoside analogs involves repositioning of the template-primer. We have prepared mutations in HIV-2 RT that are the homologs of mutations that confer resistance to nucleoside analogs in HIV-1 RT. Analysis of the behavior of HIV-2 RT mutants (Leu74Val, Glu89Gly, Ser215Tyr, Leu74Val/Ser215Tyr and Glu89Gly/Ser215Tyr) in vitro confirms the results obtained with HIV-1 RT: resistance is a function of the length of the template overhang. These analyses also suggest that the homolog in HIV-2 RT of one of the mutations that confers resistance to AZT in HIV-1 RT (Thr215Tyr) confers resistance by repositioning of the template-primer.

Topics: Deoxyguanine Nucleotides; Deoxyribonucleotides; Dideoxynucleotides; DNA Primers; DNA, Viral; Drug Resistance, Microbial; HIV Reverse Transcriptase; HIV-1; HIV-2; Humans; Models, Molecular; Mutation; Recombinant Proteins; Reverse Transcriptase Inhibitors; RNA-Directed DNA Polymerase; Templates, Genetic; Thymine Nucleotides

5-Fluorouracil (5-FU) is a chemotherapeutic agent known to retard embryonic growth and induce cleft palate and limb deformities. The predominant mechanism underlying its toxic action is thought to be inhibition of thymidylate synthetase (TS), and hence thymidine triphosphate (dTTP) synthesis, resulting in alteration of the balance of deoxynucleotide (dNTP) pools and disruption of DNA synthesis. Indeed, previously we demonstrated retarded cell-cycle progression concurrent with a 60% decrease in TS activity in rat whole embryos following maternal exposure to 40 mg/kg 5-FU on Gestational Day 14 and in the murine erythroleukemic cell (MELC) suspension culture following exposure to 5-25 microM 5-FU for 2 hr. In the study described herein, we used high-performance liquid chromatography (HPLC) to demonstrate in both of these model systems that 5-FU exposure results in similar patterns of dNTP perturbations: a prolonged decrease in dTTP and dGTP levels and an increase in dCTP and dATP. In addition, we used centrifugal elutriation to synchronize MELC in the phases of the cell cycle (G0/G1 and early S) most sensitive to 5-FU to investigate the ability of nucleoside supplementation to mitigate 5-FU-induced toxicity. Our data indicate that following a 2-hr exposure to 5-25 microM 5-FU, supplementation with 1-10 microM thymidine (TdR) for 24 hr partially reverses 5-FU-induced toxicity as evidenced by increased cellular proliferation and cell-cycle progression and amelioration of 5-FU-induced perturbations of protein synthesis and cellular membrane permeability compared to unsupplemented 5-FU-exposed cells. However, TdR concentrations >/=100 microM inhibited growth or were cytotoxic. In comparison, supplementation with 10 microM-10 mM of deoxycytidine (CdR) was not toxic, but effected a dose-dependent recovery from 5-FU-induced toxicity. At 1-100 microM, neither deoxyadenosine nor deoxyguanosine supplementation reduced 5-FU-induced toxicity; at higher concentrations, both purine nucleotides inhibited cell growth. Although these results support the hypothesis that 5-FU disrupts the MELC cell cycle by depleting dTTP (a perturbation that is reversible by TdR supplementation), they also indicate that CdR supplementation offers an additional recovery pathway.

Topics: Animals; Antimetabolites, Antineoplastic; Cell Cycle; Deoxycytidine; Deoxyguanine Nucleotides; Embryo, Mammalian; Female; Flow Cytometry; Fluorouracil; Leukemia, Erythroblastic, Acute; Mice; Pregnancy; Rats; Rats, Sprague-Dawley; Thymidine; Thymine Nucleotides

The RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase has been investigated using pre-steady-state kinetics under single turnover conditions. In contrast to previous estimates of low replication fidelity of HIV-1 reverse transcriptase, the present study finds the enzyme to be more highly discriminating when an RNA/DNA template-primer is employed as compared with the corresponding DNA/DNA template-primer. The basis of this selectivity is due to extremely slow polymerization kinetics for incorporation of an incorrect deoxynucleotide. The maximum rates for misincorporation (kpol) of dGTP, dCTP, and dTTP opposite a template uridine were 0.2, 0.03, and 0.003 s-1, respectively. The equilibrium dissociation constants (Kd) for the incorrect nucleotide opposite a template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP, respectively. These kinetic values provide fidelity estimates of 26 000 for discrimination against dGTP, 176 000 for dCTP, and 1 x 10(6) for dTTP misincorporation at this position. Similar observations were obtained when incorrect nucleotide misincorporation was examined opposite a template adenine. Thus in a direct comparison of RNA/DNA and DNA/DNA template-primer substrates, HIV-1 RT exhibits approximately a 10-60-fold increase in fidelity. This study augments our current understanding of the similarities and differences of catalytic activity of HIV-1 reverse transcriptase using RNA and DNA substrates. Moreover, these studies lend further support for a model for nucleotide incorporation by HIV-1 reverse transcriptase involving a two-step binding mechanism governed by a rate-limiting conformational change for correct incorporation.

Topics: Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA Primers; DNA Replication; DNA, Viral; Escherichia coli; HIV Reverse Transcriptase; HIV-1; Kinetics; Recombinant Proteins; Substrate Specificity; Thymine Nucleotides

For enzymatic activity, mouse ribonucleotide reductase must form a heterodimeric complex composed of homodimeric R1 and R2 proteins. Both substrate specificity and overall activity are regulated by the allosteric effectors ATP, dATP, dTTP, and dGTP, which bind to two different sites found on R1, the activity site and the substrate specificity site. We have used biosensor technique to directly observe the effects of these nucleotides on R1/R2 interactions. In the absence of effectors, positive cooperativity was observed with a Hill coefficient of 1.8 and a KD of 0.5 microM. In the presence of dTTP or dGTP, there was no cooperativity and subunit interaction was observed at a much lower R1 concentration. The highest R1/R2 affinity was in the presence of dATP or ATP with KDs of 0.05-0.1 microM. In all experiments, the molar stoichiometry between the subunits was close to 1:1. Our data support a model whereby binding of any of the effectors to the substrate specificity site promotes formation of the R1 dimer, which we believe is prerequisite for binding to the R2 dimer. Additional binding of either ATP (a positive effector) or dATP (a negative effector) to the activity site further increases R1/R2 association. We propose that binding of ATP or dATP to the activity site controls enzyme activity, not by changing the aggregation state of the R1/R2 proteins as proposed earlier, but rather by locally influencing the long range electron transport between the catalytic site of R1 and the tyrosyl free radical of R2.

Topics: Adenosine Triphosphate; Allosteric Regulation; Animals; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Kinetics; Mice; Protein Binding; Ribonucleotide Reductases; Thymine Nucleotides

The high error rates characteristic of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) are a presumptive source of the viral hypermutability that impedes prevention and therapy of acquired immunodeficiency syndrome (AIDS). We have analyzed two mutants of HIV-1 RT by conducting a comparative study of the accuracy of DNA synthesis. Each mutant bears a single amino acid substitution adjacent to the two aspartic acid residues at positions 185 and 186 in the highly conserved DNA polymerase active site. The first mutant, Met 184-->Leu (M184L), displays a marked reduction in both misinsertion and mispair extension, suggesting a fidelity of DNA synthesis significantly higher than that of the wild-type HIV-1 RT. The second mutant, Tyr 183-->Phe (Y183F), shows a decrease in mispair extension with no significant change in misincorporation. Thus, the overall pattern of error-proneness of DNA synthesis is: wild-type HIV-1 RT > Y183F > M184L. Taken together, it is possible that residues 183 and 184 contribute to the low fidelity of DNA synthesis characteristic of the reverse transcriptases of HIV-1, HIV-2 and possibly, of other lentiviruses. Our observations may bear on the nature of potential mutations responsible for resistance to the nucleoside analogs used in chemotherapy of AIDS.

Topics: Base Sequence; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA Replication; DNA, Viral; Electrophoresis, Polyacrylamide Gel; HIV Reverse Transcriptase; HIV-1; Humans; Kinetics; Molecular Sequence Data; Mutation; RNA-Directed DNA Polymerase; Thymine Nucleotides

DNA polymerase activity was assayed in hepatitis B virus (HBV) and core particles isolated from chronic producer lines. The particle-associated DNA polymerase activity, which was found to be limited to incorporation of only a few nucleotides, was inhibited by the 5'-triphosphates of nucleoside analogs. The 1-beta-L (1S,4R) and 1-beta-D (1R,4S) enantiomers of antiviral nucleoside analogs were compared for the ability to inhibit incorporation of natural nucleoside triphosphates into the viral DNA. Previously, both enantiomers of several analogs were found to be substrates for human immunodeficiency virus type 1 reverse transcriptase (HIV RT); the 1-beta-D enantiomers of some pairs were preferred as substrates. In contrast, the 1-beta-L enantiomers of all pairs tested were the more potent inhibitors of labeled substrate incorporation into hepatitis B virus DNA; the concentration required to inhibit the incorporation of the natural substrate by 50% was 6-fold to several hundred-fold lower than the concentration of the 1-beta-D enantiomer required for the same inhibitory effect. This preference for the 1-beta-L enantiomers was observed for both RNA-directed synthesis in core particles and DNA-directed synthesis in viral particles. The observed antiviral effect of the nucleoside analogs in cell culture seemed to be limited chiefly by their phosphorylation in cells.

Topics: Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA-Directed DNA Polymerase; DNA, Viral; Emtricitabine; Hepatitis B virus; Humans; Isotope Labeling; Nucleic Acid Synthesis Inhibitors; Nucleotides; Templates, Genetic; Thymine Nucleotides; Zalcitabine

The effects of deoxynucloside triphosphates (dNTPs) on the frequency of sister-chromatid exchange (SCE) was studied in human peripheral lymphocytes. Treatment with each dCTP and dTTP did not change the SCE frequency; however, dGTP caused a significant dose-dependent increase in SCE frequency, whereas dATP caused a significantly decreased in SCE frequency. The SCE-increasing effect of the treatment with dGTP could be totally reversed by treatment with equal concentrations of dATP or dCTP. Treatment with a mixture of four equal concentrations of dATP, dGTP, dCTP, dTTP did not alter the SCE frequency. These results suggest that the dNTP pool imbalance was a SCE-effecting factor in human lymphocytes, and that dGTP may be mainly responsible for this effect.

Topics: Bromodeoxyuridine; Cells, Cultured; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Dose-Response Relationship, Drug; Humans; Sister Chromatid Exchange; T-Lymphocytes; Thymine Nucleotides

Reverse transcription of HIV-1, without detergent or amphipathic peptide-induced permeability of the viral envelope, has been demonstrated to occur in the intact HIV-1 virion. In this report, we demonstrate that the amphipathic domains in the C terminus of the transmembrane glycoprotein (gp41) account for the natural permeability of the HIV-1 envelope to deoxyribonucleoside triphosphates, the substrates for DNA polymerization. In addition, nonphysiological deoxyribonucleoside triphosphates, such as 3'-azido-3'-deoxythymidine 5'-triphosphate and 3'-deoxythymidine 5'-triphosphate, can also penetrate the viral envelope, incorporate into, and irreversibly terminate reverse transcripts. As a result, viral infectivity is potently inhibited. Since the lentiviral envelope with these newly demonstrated characteristics can serve as a delivery pathway for anti-reverse transcription agents, we propose a unique strategy to prevent HIV-1 interand, possibly, intrahost transmission.

Topics: Animals; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; HIV Envelope Protein gp41; HIV Reverse Transcriptase; HIV-1; Humans; Mice; Permeability; Rats; Thymine Nucleotides; Transcription, Genetic; Virion

We have devised a single-step method that enables purification of HIV-1 recombinant reverse transcriptase directly from bacterial lysates in less than 2 h. Clarified lysates are applied to commercial Q- and S-matrix cartridge columns connected in series. The columns are washed with low-salt buffer to remove unbound protein, then the Q column is removed and reverse transcriptase is eluted from the S column using a salt gradient. The purification has been carried out with both medium-pressure and high-pressure chromatographic systems. Purifications are carried out at room temperature near neutral pH, providing enzyme with high DNA polymerase specific activity. A crucial aspect of the procedure is the use of Tris buffer, a buffer that is normally incompatible in cation-exchange methods. The method is applicable for the purification of the p51/p66 heterodimer and the p5l and p66 homodimer forms of reverse transcriptase. We have used this method to purify wild-type reverse transcriptase and several recombinant proteins containing mutations correlated with dideoxynucleoside drug resistance.

Topics: Blotting, Western; Chromatography, High Pressure Liquid; Deoxyguanine Nucleotides; Dimerization; DNA-Directed DNA Polymerase; Electrophoresis, Polyacrylamide Gel; Escherichia coli; HIV Reverse Transcriptase; Isoenzymes; Kinetics; Molecular Weight; Mutation; Oligodeoxyribonucleotides; Poly A; Polymerase Chain Reaction; Protein Conformation; Recombinant Proteins; Thymine Nucleotides

dCMP-deaminase-deficient V79/dC hamster cells have highly imbalanced deoxyribonucleoside triphosphate (dNTP) pools, i.e. a 17-fold larger dCTP pool, a slightly reduced dTTP and a very low dGTP pool, compared to dCMP-deaminase-proficient V79/p cells. Nevertheless, the two lines showed the same rates of spontaneous mutation at the hprt and ouabain-resistance loci. Analysis of spontaneous hprt mutations indicated an increase in misincorporation of C in V79/dC cells, although it was not statistically significant. When the dCTP pool was further increased fivefold by incubating V79/dC cells with cytidine, C misincorporation increased to 88%, but the mutation frequency remained unchanged. The dNTP pools of V79/dC cells were also altered by treatment with thymidine, or with thymidine plus deoxycytidine. After incubation with thymidine alone, the dCTP pool all but disappeared, whereas it maintained a normal level in the presence of deoxycytidine. In both cases dTTP rose to nmol amounts, and dGTP accumulated. Incubation with 10 mM thymidine was the only treatment that increased the mutation frequency; T misincorporation then accounted for 94% of the base substitutions. In the presence of deoxycytidine the cells had a dTTP/dCTP ratio of 0.04, but 86% of the base substitutions involved C misincorporation and most probably originated from G mis-incorporation caused by excess dGTP. Alterations of RNA splicing and hot spots for base substitutions varied with the imbalance, the latter showed "next-nucleotide effects". Our results suggest that the fidelity of DNA replication in V79 cells is only affected by large changes in the pool and is more sensitive to changes in dGTP than in dCTP or dTTP.

Topics: Animals; Cell Line; Cricetinae; Cytidine; DCMP Deaminase; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA Replication; Fibroblasts; Hypoxanthine Phosphoribosyltransferase; Mutation; Thymidine; Thymine Nucleotides

In order to determine the role of Tyr 766 of Escherichia coli DNA polymerase I in the catalysis of DNA synthesis, we investigated the properties of a Tyr 766-->Ser (Y766S) mutant of the Klenow fragment of E. coli DNA polymerase I. We found that the rates of incorporation of only dTTP but not the other dNTP substrates were affected in the reactions catalyzed by the mutant enzyme, when homopolymeric template-primers were used. The mutant enzyme exhibited a reduced rate of synthesis only with poly(rA)- or poly(dA)-directed reactions. Examination of the ability of the mutant and the wild-type enzymes to bind to dGTP and dTTP, as judged by UV-mediated cross-linking, indicated nearly identical binding efficiencies of both nucleotides. However, the ability of the mutant enzyme to bind to poly(rA).(dT)15 and poly(dA).(dT)15 was found to be significantly reduced as compared to the binding to heteropolymeric DNA. In order to further define the nature of template-mediated restriction on the catalytic activity of the mutant enzyme, its ability to copy DNA templates containing a stretch of AAAAA and ACACA sequences was compared. The results show that DNA synthesis catalyzed by the mutant enzyme is significantly retarded when it encounters the AAAAA region of the template but not the ACACA region. Product analysis of the reaction directed by the two template-primers showed that the mutant enzyme stalls/terminates synthesis upon encountering an AAAAA sequence in the template.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Base Sequence; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA; DNA Adducts; DNA Polymerase I; DNA Primers; Escherichia coli; Molecular Sequence Data; Mutation; Serine; Substrate Specificity; Thymine Nucleotides; Tyrosine; Ultraviolet Rays

The reduction of ribonucleotides is catalyzed by different enzymes in aerobic and anaerobic Escherichia coli, each with a different primary and quaternary structure. Here, we describe the allosteric regulation of the substrate specificity of the anaerobic ribonucleoside triphosphate reductase. The enzyme reduced ribonucleotides at a low basal rate. Reduction was stimulated up to 10-fold by an appropriate modulator (dGTP for ATP reduction, ATP for CTP and UTP reduction, and dTTP for GTP reduction). dGTP and dTTP inhibited the reduction of the "incorrect" substrate; dATP inhibited reduction of all four. From kinetic, effector binding, and competition experiments we conclude that the enzyme has two classes of sites, one that binds ATP and dATP and regulates pyrimidine ribonucleotide reduction ("pyrimidine site"), the other that binds dATP, dGTP, and dTTP and regulates purine ribonucleotide reduction ("purine site"). This model differs slightly from the model for the aerobic reductase, but the physiological consequences remain the same and explain how a single enzyme can provide a balanced supply of the four dNTPs. The similarity of a highly sophisticated control mechanism for the aerobic and anaerobic enzymes suggests that both arose by divergent evolution from a common ancestor, in spite of their different structures.

Topics: Adenosine Triphosphate; Allosteric Regulation; Anaerobiosis; Binding, Competitive; Deoxyguanine Nucleotides; Escherichia coli; Kinetics; Ribonucleotide Reductases; Substrate Specificity; Thymine Nucleotides

Human immunodeficiency virus type 1 (HIV-1) replication is shown to be sensitive to the intracellular concentration of deoxynucleoside triphosphate substrates. Addition of thymidine to established cell lines resulted in a dramatic reduction of virus production. The effect could be substantially alleviated by addition of deoxycytidine, which, alone, enhanced viral titers by a factor of 2 to 3. Hydroxyurea treatment abolished HIV-1 replication in peripheral blood mononuclear cells and could be reversed by deoxyadenosine. These data show that HIV-1 replication occurs under suboptimal DNA precursor conditions.

Topics: CD4 Antigens; Cells, Cultured; Deoxyadenine Nucleotides; Deoxycytidine; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; HIV-1; Humans; Proviruses; Thymidine; Thymine Nucleotides; Transcription, Genetic; Virus Replication

The kinetics and the fidelity of replication of the base pair 6-thioguanine (Gs)/5-methyl-2-pyrimidinone (Th) have been determined by using defined oligomers with the large Klenow fragment of Escherichia coli DNA polymerase I. The insertion efficiency, Vmax/Km (min-1 microM-1), of Th opposite Gs is 1.5 and the insertion efficiency of Gs opposite Th is 0.7. By comparison, the insertion efficiencies of C opposite G and G opposite C are 0.5 and 1.5. The insertion efficiency of the next base, A opposite T, is 2 times greater after the base pair Gs/Th than after G/C. The fidelity of replication with respect to thymine and adenine has misinsertion frequencies, or ratios of the insertion efficiency of the "wrong" base to the "right" base, of 7 x 10(-4) for T opposite Gs (T/Gs), 4 x 10(-6) for T/Th, and a maximum stable misinsertion frequency of 4 x 10(-4) for A/Th. No detectable elongation occurs after an A is inserted opposite a Gs. These values are similar to the misinsertion frequencies of G and C with T and A. The maximum stable misinsertion frequencies with G and C are 4 x 10(-2) for G/Th, 3 x 10(-2) -7 x 10(-3) for Gs/C, and 2.6 x 10(-1) for C/Gs, and the misinsertion frequency is < 1 x 10(-3) for Th/G. The kinetics results and molecular modeling suggest modifications to the Gs/Th base pair that may provide higher levels of fidelity of replication with respect to C and G.

Topics: Base Composition; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA Polymerase I; DNA Replication; Escherichia coli; Exodeoxyribonuclease V; Exodeoxyribonucleases; Kinetics; Mathematics; Models, Biological; Pyrimidinones; Templates, Genetic; Thioguanine; Thymine Nucleotides

Carbovir (the carbocyclic analog of 2'-3'-didehydro-2',3'-dideoxyguanosine) is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) replication. Assays were developed to assess the mechanism of inhibition by the 5'-triphosphate of carbovir of HIV-1 reverse transcriptase using either RNA or DNA templates that contain all four natural nucleotides. Carbovir-TP was a potent inhibitor of HIV-1 reverse transcriptase using either template with Ki values similar to that observed by AZT-TP, ddGTP, and ddTTP. The kinetic constants for incorporation of these nucleotide analogs into DNA by HIV-1 reverse transcriptase using either template were similar to the values seen for their respective natural nucleotides. In addition, the incorporation of either carbovir-TP or AZT-TP in the presence of dGTP or dTTP, respectively, indicated that the mechanism of inhibition by these two nucleotide analogs was due to their incorporation into the DNA resulting in chain termination. Carbovir-TP was not a potent inhibitor of DNA polymerase alpha, beta, or gamma, or DNA primase. Given the potent activity of carbovir-TP against HIV-1 reverse transcriptase and its lack of activity against human DNA polymerases, we believe that further evaluation of this compound as a potential drug for the treatment of HIV-1 infection is warranted.

Topics: Antiviral Agents; Base Sequence; Deoxyguanine Nucleotides; Dideoxynucleotides; DNA; DNA Polymerase I; DNA Polymerase II; DNA Polymerase III; HIV-1; Humans; In Vitro Techniques; Kinetics; Molecular Sequence Data; Reverse Transcriptase Inhibitors; RNA; Templates, Genetic; Thymine Nucleotides; Zidovudine

Peripheral blood lymphocytes of healthy volunteers cultured with phytohaemagglutinin in folate-deficient medium exhibit megaloblastic maturation with reduced intracellular folate content. We have employed this in vitro model for megaloblastic maturation to determine accompanying changes in cellular thymidylate cycle activities and deoxynucleotide levels. Folate-deficient cells exhibit a two-fold increase in thymidine kinase and thymidylate synthase activities. These increased activities were reduced to those of folate-replete cells by co-culture of folate-deficient cells with thymidine. Folate deficiency was accompanied by reduced cellular levels of thymidine triphosphate (TTP) and deoxyguanosine triphosphate (dGTP). Exogenous deoxyuridine produced no increase in the reduced levels of TTP of folate-deficient cells but effected a two-fold increase in cellular deoxycytidine triphosphate. Exogenous thymidine increased the reduced TTP levels of folate-deficient cells and corrected the reduced dGTP level; the increase in cellular TTP accompanying exogenous thymidine was more pronounced in folate-deficient cells. These in vitro findings are compatible with a block in de novo thymidylate synthesis and explain in part the reported in vivo changes for the deoxynucleotide pool in megaloblastic marrow cells due to folate or vitamin B12 deficiency.

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyuridine; Dose-Response Relationship, Drug; Folic Acid; Folic Acid Deficiency; Humans; Lymphocyte Activation; Lymphocytes; Nucleotides; Thymidine; Thymidylate Synthase; Thymine Nucleotides

V79 hamster cells were made resistant against hydroxyurea by continuous culture at stepwise increasing drug concentrations. Two cell lines were cloned, resistant to 0.4 mM (V79/H0.4) and 4 mM (V79/H4) hydroxyurea, with a fivefold and a 20-fold increase in soluble ribonucleotide reductase activity. We investigated how the increased amount of enzyme affected the in situ activity of ribonucleotide reductase and deoxyribonucleotide metabolism, in particular substrate cycles between pyrimidine deoxyribonucleosides and their 5'-phosphates. The in situ activity of the reductase was only moderately elevated (1.3-fold in V79/H4 cells). In the fully resistant line, the steady-state level of dATP was increased fourfold, and that of dTTP twofold. These nucleotides are negative allosteric effectors of the reductase and we propose that the increased pools inhibit the enzyme and thereby maintain the in situ activity of the reductase at only a slightly increased level. The surplus deoxyribonucleotides was excreted from the cells as thymidine and deoxycytidine via substrate cycles. The data support and extend our previous model for the regulation of deoxyribonucleotide synthesis via the allosteric properties of ribonucleotide reductase and substrate cycles that link salvage and degradation of deoxyribonucleotides.

Topics: Animals; Cell Line; Cricetinae; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA; Drug Resistance; Hydroxyurea; Thymine Nucleotides

Deoxyribonucleoside triphosphate (dNTP) levels were measured in wild type Neurospora and nine mutagen-sensitive mutants, at nine different genes. Eight of these mutants are sensitive to hydroxyurea and histidine and show chromosomal instability, a phenotype which could result from altered levels of dNTPs. Two patterns were seen. Five of the mutants had altered ratios of dNTPs, with relatively high levels of dATP and dGTP and low levels of dCTP, but changes in the dTTP/dCTP ratio did not correlate with changes in spontaneous mutation levels. During exponential growth all but two of the mutants had small but consistent increases in dNTP pools compared to wild type. DNA content per microgram dry hyphae was altered in several mutants but these changes showed no correlation with the dNTP pool alterations.

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA, Fungal; Genes, Fungal; Histidine; Hydroxyurea; Mutation; Neurospora; Neurospora crassa; Thymine Nucleotides

The enzymatic assay for deoxyribonucleoside triphosphates has been improved by using synthetic oligonucleotides of a carefully defined sequence as template primers for DNA polymerase. High backgrounds, which limit the sensitivity of the assay when calf thymus DNA or alternating copolymers are used as template primers, were eliminated with these oligonucleotide template primers. Sensitivity was further increased by designing the template primer to incorporate multiple labeled deoxyribonucleotides per limiting unlabeled deoxyribonucleotide. Each of several DNA polymerases exhibited unique reaction characteristics with the oligonucleotide template primers, which was attributed to the differing exonuclease activities associated with these various enzymes. Assay optimization therefore included matching the polymerase with the template primer to obtain the lowest background reaction and highest sensitivity. This modified assay is particularly well suited for keeping cell sample size to a minimum in experimental protocols which generate large numbers of data points or require careful timing of sampling. With this technique, we measured the levels of all four deoxyribonucleoside triphosphates in extracts from as few as 2 x 10(4) cultured cells.

Topics: Base Sequence; Cells, Cultured; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA-Directed DNA Polymerase; Humans; Micrococcus; Oligonucleotides; Perchlorates; Poly dA-dT; Reference Standards; Templates, Genetic; Thymine Nucleotides; Tritium

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Dose-Response Relationship, Drug; Humans; Nucleoside-Phosphate Kinase; Thymine Nucleotides; Tumor Cells, Cultured; Zidovudine

MOLT-4 (T-), RAJI (B-), and KM-3 (non-B-non-T-, common ALL) malignant lymphoblasts demonstrated significant differences in their activities of purine de novo synthesis (PDNS) and purine salvage pathway and in their cell-kinetic parameters. Incubations with concentrations of methotrexate (0.02 and 0.2 microM), which can be maintained during many hours in the oral maintenance therapy of acute lymphoblastic leukemia, indicated large differences between the three cell lines with respect to the inhibition of PDNS, depending on the concentration of methotrexate (MTX) and on the activities of the two pathways. These dose- and cell line-dependent differences corresponded to the perturbations of cell-kinetics and purine and pyrimidine (deoxy)ribonucleotide pools in the three cell lines. Exposure of MOLT-4 cells to 0.02 microM MTX resulted in an incomplete inhibition of DNA synthesis in early S phase, as shown by DNA-flow cytometry and increase of dCTP levels, which recovered spontaneously after 48 hr. Almost no impairment of RNA synthesis occurred (unbalanced growth). In RAJI cells, exposed to 0.02 microM MTX, DNA synthesis was delayed in the S phase, not arrested, and RNA synthesis was not impaired, also indicating an unbalanced growth pattern, which, however, did not recover in time. KM-3 cells were arrested in G1 phase and subsequently in early S phase after incubation with 0.02 microM MTX, and perturbations of ribonucleotides indicated a complete inhibition of RNA synthesis, resulting in a balanced growth pattern. Cytotoxicity was more pronounced in KM-3 cells. The reliability of the soft agar colony forming assay after low dose MTX treatment is discussed. Exposure of MOLT-4 and KM-3 cells to 0.2 microM MTX resulted in a complete inhibition of DNA synthesis, with cessation of cell progression through all parts of the cell cycle and arrest in G1 phase. RAJI cells showed an increasing accumulation of cells in G1 phase without complete cessation of cell cycle progression. Perturbations of ribonucleotide pools suggested an inhibition of RNA synthesis in all cell lines, indicating a balanced growth pattern in KM-3 cells and MOLT-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Cycle; Cell Survival; Deoxyguanine Nucleotides; Humans; Leukemia; Lymphocytes; Methotrexate; Polyglutamic Acid; Purines; Pyrimidines; RNA; Thymine Nucleotides; Tumor Cells, Cultured

Using permeable diploid human fibroblasts, we have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent Km values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 microM. For UV-induced DNA repair synthesis, the apparent Km values were substantially lower, ranging from 0.11 to 0.44 microM for AG1518 cells and from 0.06 to 0.24 microM for IMR-90 cells. Control experiments established that these values were not significantly influenced by nucleotide degradation during the permeable cell incubations or by the presence of residual endogenous nucleotides within the permeable cells. Recent data implicate DNA polymerase delta in UV-induced repair synthesis and suggest that DNA polymerases alpha and delta are both involved in semiconservative replication. We measured Km values for dGTP and dTTP for polymerases alpha and delta, for comparison with the values for replication and repair synthesis. Km values for polymerase alpha were 2.0 microM for dGTP and 5.0 microM for dTTP. For polymerase delta, the Km values were 2.0 microM for dGTP and 3.5 microM for dTTP. The deoxyribonucleotide Km values for DNA polymerase delta are much greater than the Km values for UV-induced repair synthesis, suggesting that when polymerase delta functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Line; Cell Membrane Permeability; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA; DNA Polymerase II; DNA Polymerase III; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Fibroblasts; Humans; Potassium Chloride; Thymine Nucleotides; Ultraviolet Rays

Using affinity purified human immunodeficiency virus (HIV) reverse transcriptase the reaction assay conditions were determined. The optimum incorporation of dTMP into the (rA)n(dT)10 template with HIV reverse transcriptase required 6 mM MgCl2 and 80 mM KCl. The template specificity of HIV reverse transcriptase is quite different from those of the human gamma-polymerase-associated reverse transcriptase or avian virus reverse transcriptase. The preferential inhibition of HIV reverse transcriptase as compared to human gamma-reverse transcriptase was observed with several nucleoside analog triphosphates. The Ki values for thymidine triphosphate analogs with HIV reverse transcriptase ranged from 5 to 13 nM with decreasing effectiveness for 3'-fluoro greater than 3'-amino greater than 2',3'-dideoxy greater than 3'-azido groups. This study provides information on the structure activity relationships of the triphosphate analogs inhibitory effects on HIV reverse transcriptase versus human gamma-polymerase-associated reverse transcriptase, and the possible mechanisms of action of 3' azido thymidine and the 2',3'-dideoxynucleosides, and also identifies other nucleoside analogs for possible development as inhibitors of HIV.

Topics: Deoxyguanine Nucleotides; HIV; Humans; Kinetics; Molecular Weight; Nucleotides; RNA-Directed DNA Polymerase; Structure-Activity Relationship; Substrate Specificity; Thymine Nucleotides

DNA precursor synthesis can be blocked specifically by the drug hydroxyurea (HU) which has therefore been used for anticancer therapy. High concentrations of HU, however, affect other processes than DNA synthesis; nevertheless, most studies on the biological action of HU have been made with concentrations at least one order of magnitude higher than those needed for cell-growth inhibition. In this study we characterized the effects of low concentrations of HU (i.e. concentrations leading to 50% inhibition of cell growth in 72 h) on cell cycle kinetics and nucleotide pools in mouse S49 cells with various defined alterations in DNA precursor synthesis. The effect of 50 microM HU on deoxyribonucleoside triphosphate pools was a 2-3-fold decrease in the dATP and dGTP pools, with no change in the dCTP pool and a certain increase in the dTTP pool. Addition of deoxycytidine or thymidine led to a partial reversal of the growth inhibition and cell-cycle perturbation caused by HU, and was accompanied by an increased level of the deoxyribonucleoside triphosphates. Addition of purine deoxyribonucleoside gave no protection, indicating that salvage of these nucleosides could not supply precursors for DNA synthesis in T-lymphoma cells. We observed a higher sensitivity to HU of cells lacking purine nucleoside phosphorylase or with a ribonucleotide reductase with altered allosteric regulation. Cells lacking thymidine kinase or deoxycytidine kinase were just as sensitive as wild-type cells.

Topics: Animals; Cell Cycle; Cell Line; Deoxyadenine Nucleotides; Deoxycytidine Kinase; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Hydroxyurea; Lymphoma; Mice; Mutation; Purine-Nucleoside Phosphorylase; Ribonucleotide Reductases; T-Lymphocytes; Thymidine Kinase; Thymine Nucleotides

Inhibition of cell growth and DNA synthesis by hydroxyurea is thought to occur via an effect on the enzyme ribonucleotide reductase leading to a block of deoxyribonucleotide synthesis. Earlier attempts to bypass such a block by delivering deoxyribonucleosides to the medium of cultured cells have given equivocal results. Complications arise in such experiments from the specificity of the phosphorylating enzymes since 3 of the 4 deoxyribonucleosides are substrates for the same enzyme, with widely differing Km values, and from allosteric effects exerted by deoxyribonucleotides. We simplify this situation by using a mutant hamster V79 line that lacks the enzyme dCMP deaminase. The cells contain a 20-fold enlarged dCTP pool and require thymidine for optimal growth. Concentrations of hydroxyurea (50 or 100 microM) that in short-term experiments inhibited DNA synthesis depleted the dATP pool without seriously affecting pyrimidine deoxyribonucleotide pools. The dATP pool could be restored by addition of deoxyadenosine but this depleted the dGTP pool. This depletion could be counteracted by the simultaneous addition of deoxyguanosine but then critically depended on the relative concentrations of the two purine deoxyribonucleosides, with optimal results at 1 microM deoxyadenosine + 100 microM deoxyguanosine. Under those conditions the inhibition of DNA synthesis by hydroxyurea was partially reversed.

Topics: Animals; Cell Line; Cricetinae; Deoxyadenine Nucleotides; Deoxyadenosines; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyguanosine; DNA; Hydroxyurea; Nucleotides; Purine Nucleosides; Thymine Nucleotides

The effects of 5-fluoro-2'-deoxyuridine (FdUrd) and 5,8-dideazaisofolic acid on the coordination of thymidylate synthase activity and DNA synthesis were examined in human CCRF-CEM leukemic cells following a continuous exposure to these agents. In logarithmically growing control tumor cells, the rate of in situ thymidylate synthase activity equaled the rate of DNA synthesis. However, in tumor cells incubated with growth-inhibitory concentrations of either FdUrd or 5,8-dideazaisofolic acid for 48 h, the rate of thymidylate synthase activity was between 15- and 17-fold greater than the rate of DNA synthesis. The loss in tumor cell viability of FdUrd-treated cells was temporally related to this prolonged dissociation of thymidylate biosynthesis from DNA biosynthesis. The dissociation of thymidylate from DNA biosynthesis in cells incubated with FdUrd was not closely related to thymidylate depletion. The intracellular concentrations and activities of thymidylate synthase were comparable in tumor cells incubated for 24 or 48 h with either a growth-inhibitory or non-growth-inhibitory concentration of FdUrd, indicating no direct relationship among these parameters. Indirect thymidylate depletion induced by the combination of 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine, hypoxanthine, and glycine inhibited in situ thymidylate synthase activity and DNA synthesis to an equal extent. In addition, the intracellular concentrations of all four deoxyribonucleoside 5'-triphosphates in tumor cells incubated with FdUrd for 48 h were between 1.3- and 3.1-fold greater than their respective concentrations in control cells, reflecting their decreased utilization in DNA synthesis in FdUrd-treated cells. These data indicated that inhibition of CCRF-CEM cell growth and DNA synthesis following a continuous exposure to cytostatic concentrations of either FdUrd or 5,8-dideazaisofolic acid resulted primarily from interference with thymidylate incorporation into DNA, and not simple blockade of thymidylate synthase.

Topics: Deoxyguanine Nucleotides; DNA; DNA Polymerase II; Dose-Response Relationship, Drug; Floxuridine; Folic Acid Antagonists; Humans; Leukemia; Pyrimethamine; Quinazolines; Thymidine Monophosphate; Thymidylate Synthase; Thymine Nucleotides

The B1 subunit of Escherichia coli ribonucleotide reductase is coded for by the nrdA gene, of determined structure. Protein B1 contains two types of allosteric binding sites. One type (h-sites) determines the substrate specificity while the other type (l sites) governs the overall activity. The effectors dGTP and dTTP bind only to the h-sites while dATP and ATP bind to both the h- and the l-sites. Protein B1 has been photoaffinity-labeled with radioactive dTTP and dATP using direct UV irradiation. Following tryptic digestion of labeled protein B1 only one peptide labeled with dTTP was found, while several peptides were labeled with dATP. One of the dATP-labeled peptides had chromatographic properties very similar to that labeled with dTTP and this peptide most likely forms part of the h-site of protein B1. Labeling of the l-site could not be conclusively shown since substantial non-specific labeling occurred with dATP. CNBr fragments of dTTP-labeled protein B1 were used to localize the region of nucleotide binding in the deduced primary structure of the nrdA gene. The dTTP label was further localized to a tryptic octapeptide with the sequence Ser-X-Ser-Gln-Gly-Gly-Val-Arg. The labeled amino acid was found at position 2, but the residue itself could not be directly identified. Unexpectedly, this sequence was not found in the earlier reported primary structure of the nrdA gene. However, a recent revised structure of the gene identifies the labeled residue as Cys-289 and fully confirms the rest of the peptide sequence. Thus the present result clearly defines one of the allosteric binding sites in ribonucleotide reductase.

Topics: Adenosine Triphosphate; Affinity Labels; Allosteric Regulation; Allosteric Site; Amino Acid Sequence; Bacterial Proteins; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Chromatography, Thin Layer; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Escherichia coli; Peptides; Ribonucleotide Reductases; Thymine Nucleotides

Hydroxyurea inactivates ribonucleotide reductase from mammalian cells and thereby depletes them of the deoxynucleoside triphosphates required for DNA replication. In cultures of exponentially growing 3T6 cells, with 60-70% of the cells in S-phase, 3 mM hydroxyurea rapidly stopped ribonucleotide reduction and DNA synthesis (incorporation of labeled thymidine). The pool of deoxyadenosine triphosphate (dATP) decreased in size primarily, but also the pools of the triphosphates of deoxyguanosine and deoxycytidine (dCTP) were depleted. Paradoxically, the pool of thymidine triphosphate increased. After addition of hydroxyurea this pool was fed by a net influx and phosphorylation of deoxyuridine from the medium and by deamination of intracellular dCTP. An influx of deoxycytidine from the medium contributed to the maintenance of intracellular dCTP. 10 min after addition of hydroxyurea, DNA synthesis appeared to be completely blocked even though the dATP pool was only moderately decreased. As possible explanations for this discrepancy, we discuss compartmentation of pools and/or vulnerability of newly formed DNA strands to nuclease action and pyrophosphorolysis.

Topics: Cells, Cultured; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA; Hydroxyurea; Ribonucleotide Reductases; Thymine Nucleotides

When Escherichia coli DNA polymerase I (Pol I) replicates a homopolymer, the excision/polymerization (exo/pol) ratio varies with enzyme and initiator concentration. The study of this effect in the case of poly(dA).oligo(dT) replication led us to propose a mnemonic model for Pol I, in which the 3' to 5' excision activity warms up when the enzyme is actively polymerizing, and cools down when it dissociates from the template. The model predicts that the exo/pol ratio must increase with processivity length and initiator concentration and decrease with enzyme concentration. It predicts also that contact of the enzyme with one template alters its excision efficiency towards another template. The exo/pol ratio and processivities of Pol I and its Klenow fragment were studied on four templates: poly(dA).(dT)10, poly(dT).(dA)10, poly(dC).(dG)10 and poly(dI).(dC)10. We show that the Klenow fragment is usually much less processive than Pol I and when this is the case it has a much lower exo/pol ratio. At equal processivity, the exo/pol ratios are nearly equal. Furthermore, many factors that influence processivity length (e.g. manganese versus magnesium, inorganic pyrophosphate, ionic strength) influence the exo/pol ratio in the same direction. The study of deaminated poly(dC) replication, where we followed incorporation and excision of both G and A residues, allowed us to assign the origin of the dNMP variations to changes in the 3' to 5' proof-reading activity of Pol I. Similarly, the lower dNMP turnover of the Klenow fragment observed with deaminated poly(dC) was specifically assigned to a decreased 3' to 5' exonuclease activity. The exo/pol ratio generally increased with initiator and decreased with enzyme concentration, in agreement with the model, except for poly(dI).oligo(dC), where it decreased with initiator concentration. However, by terminating chain elongation with dideoxy CTP, we showed directly that, even in this system, excision is relatively inefficient at the beginning of synthesis. Interaction of Pol I with poly(dA).(dT) or with poly(dC).(dG) modifies its exo/pol characteristics in the replication of poly(dI).(dC) and poly(dA).(dT), respectively. The Klenow enzyme is not sensitive to such influences and this correlates with its reduced processivity on the influencing templates. Our results reveal the existence of differences between Pol I and its Klenow fragment that are more profound than has been thought previously.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Base Composition; Deoxyadenine Nucleotides; Deoxycytidine Monophosphate; Deoxyguanine Nucleotides; DNA Polymerase I; DNA Replication; Escherichia coli; Kinetics; Models, Biological; Poly dA-dT; Templates, Genetic; Thymine Nucleotides

Herpes simplex viruses are known to induce virus specified ribonucleotide reductase (RR) in infected cells. RR is considered as a possible target for the development of antiviral agents. In this study, the role of RR in virus replication has been investigated. The sensitivity of RR to hydroxyurea (HU) from virus infected and uninfected HeLa S3 cells was similar with IC50 values of 0.12 and 0.14 mM. In the presence of 2 mM HU, and 10 microM tetrahydrouridine (THU), a cytidine deaminase inhibitor, the incorporation of [14C]cytidine into viral DNA was found to be inhibited by 95%; [32P]-incorporation into viral DNA under the same conditions was inhibited by 75%. The pool size of dCTP and dGTP was 50 and 70%, respectively, with no significant effect on dATP and dTTP pools in virus infected cells treated with 2 mM HU, as compared with virus infected cells receiving no drug treatment. HU at 2 mM could not inhibit HSV-2 yield by more than one log. These results suggest that virus RR is not an effective target for developing anti HSV-2 compounds.

Topics: Antiviral Agents; Cytidine; Deoxyguanine Nucleotides; DNA, Viral; HeLa Cells; Humans; Hydroxyurea; Phosphates; Ribonucleotide Reductases; Simplexvirus; Thymine Nucleotides

Topics: Bacteriophage phi X 174; Base Sequence; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Deoxyuracil Nucleotides; Diphosphates; DNA Replication; DNA-Directed DNA Polymerase; DNA, Viral; Intercalating Agents; Manganese; Mutation; Thymine Nucleotides

The analysis of deoxyribonucleoside 5'-triphosphates (dNTPs) in cell extracts by high-pressure liquid chromatography [C. Garrett , and D.V. Santi (1979) Anal. Biochem. 99, 268-273] requires the prior, selective degradation of ribonucleoside 5'-triphosphates ( rNTPs ) that are present in the extracts in large quantities. When this method was used for quantifying the dNTPs in mammalian cell extracts, the presence of an interfering peak in the HPLC between the peaks for dTTP and dATP was observed. This unwanted peak sometimes overlapped with that of dATP, depending on the pH of the eluant. It was found that the material which gave this peak was formed during the periodate oxidation of rNTPs in the presence of methylamine, and that it could be removed by changing the order of addition of the reagents in the procedure, i.e., the methylamine was added only after the excess periodate was decomposed, instead of adding it together with periodate, as given in the original procedure. Furthermore, an addition of deoxyguanosine to the reaction mixture was found to be effective in preventing the partial loss of dGTP in the oxidation procedure. By using this improved method, the dNTP contents of the extracts of Ehrlich ascites tumor cells have been measured in an accurate and reproducible manner. The analysis requires about 10(6) cells, and all four dNTPs can be quantified in 2.5 h, starting from the harvest of the cells.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Extracts; Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Male; Methylamines; Mice; Oxidation-Reduction; Periodic Acid; Ribonucleotides; Thymine Nucleotides; Tissue Extracts

Intracellular deoxyribonucleotide pools were examined before and after thymidine treatment in highly sensitive T-lymphoid cells, relatively resistant B-lymphoid cells and moderately sensitive melanoma cells. Among the 4 cell lines studied, proportions of the 4 deoxyribonucleotide pools varied appreciably while ribonucleotide profiles were similar. The ratio of dGTP to dCTP increased with sensitivity to thymidine. Increase in dTTP levels with increasing thymidine concentration was dependent on sensitivity of cells to thymidine and was accompanied by reduction in the dCTP pool. dGTP levels increased as did dTTP levels in all cells, while dATP pool expansion correlated with thymidine sensitivity. The results indicate an additional aspect of purine deoxyribonucleotide involvement in the growth inhibitory effects of thymidine.

Topics: B-Lymphocytes; Cell Line; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Humans; Melanoma; T-Lymphocytes; Thymidine; Thymine Nucleotides

Deoxyribonucleoside triphosphate (dNTP) pool levels were examined in synchronized and unsynchronized log phase cultures and in quiescent cultures of human diploid foreskin fibroblasts. dNTP levels were in good agreement with those previously published for human HeLa and lymphoblastic leukemia cells. dCTP and dGTP levels showed only a modest lowering in quiescent as compared to log-phase cells, but dATP and dTTP levels were reduced dramatically in quiescent cultures. Cells synchronized by serum starvation and assayed at the peak DNA synthetic phase (18-21 hr post release) showed substantially higher pools of all four dNTPs. Hydroxyurea treatment reduced only purine dNTPs in both log phase and confluent cells while increasing dTTP and dCTP pools. The effects of deoxynucleosides on dNTP pools were also examined and are discussed in light of current models regarding regulation of purified ribonucleotide reductase formulated from in vitro studies.

Topics: Cells, Cultured; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleosides; Deoxyribonucleotides; Diploidy; Fibroblasts; Humans; Hydroxyurea; Thymine Nucleotides

The activity of deoxyguanosine kinase purified from human placenta was regulated by various nucleotides. dTTP, an activator, only increased the Vmax value of the enzyme. The feedback inhibition by dGTP, dGDP and dGMP were competitive with respect to deoxyguanosine. Both the activation by dTTP and the inhibition by dGTP were reversible.

Topics: Deoxyguanine Nucleotides; Enzyme Activation; Female; Humans; Nucleotides; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Placenta; Thymine Nucleotides

Topics: Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Escherichia coli; Guanosine Diphosphate; Macromolecular Substances; Photochemistry; Ribonucleotide Reductases; Thymine Nucleotides

dNTP pools are quite low in immature oocytes of the starfish, expand during the 1-methyladenine-induced maturational process and thereafter reach a maximal level (approx. 35, 20, 15 and 5 fmoles/egg for dTTP, dCTP, dATP and dGTP, respectively) which is maintained in overmatured eggs. Maturing oocytes were inseminated at the stage just before extrusion of the first polar body and determination of dNTP pools during early embryogenesis showed the same expansion pattern as that of the 1-methyladenine-treated oocytes. Therefore, the increase in dNTP pools during early embryogenesis is dependent on 1-methyladenine (1-MA) but independent of fertilization. Aphidicolin, a specific inhibitor of eukaryotic DNA polymerase alpha, has no effect on dNTP pool size in 1-methyladenine-treated oocytes, but causes considerable expansion of dNTP pools in fertilized eggs which cleave achromosomally in the presence of the drug.

Topics: Animals; Aphidicolin; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Diterpenes; Female; Oocytes; Oogenesis; Ovum; Starfish; Thymine Nucleotides; Zygote

This study asks whether nuclear and mitochondrial DNA replication are supplied by distinct and separately regulated precursor pools. Using improved methodology for extraction and quantitation of deoxyribonucleoside triphosphate pools from HeLa cells, we have confirmed and extended earlier findings of Bogenhagen and Clayton ((1976) J. Biol. Chem. 251, 2938-2944). The four mitochondrial dNTP pools actually expanded following treatment with antimetabolites, even while total cellular pools of dTTP and dGTP are being severely depleted. Ribonucleoside triphosphates also accumulate in mitochondria after antimetabolite treatment. This confirms the idea of distinct regulatory mechanisms affecting precursor supplies for nuclear and mitochondrial DNA. Mitochondrial dNTP pools are larger, in relation to the cellular complement of mitochondrial DNA than are the whole cell pools in relation to the chromosomal DNA complement. Also, of the four dNTPs, the most sensitive to antimetabolite depletion is dGTP. This indicates that dGTP depletion may be more significant than previously realized as an element of the cytotoxic effects of methotrexate and 5-fluorodeoxyuridine.

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA; Floxuridine; HeLa Cells; Humans; Methotrexate; Mitochondria; Ribonucleotides; Thymine Nucleotides

The effect of acyclovir on the deoxyribonucleoside triphosphate pools of Vero cells infected with herpes simplex virus type 1 was examined. Deoxyguanosine triphosphate and deoxyadenosine triphosphate pool levels in infected cells treated with acyclovir increased dramatically compared with pool levels in untreated infected cels. The increases were due, at least in part, to inhibition of viral DNA polymerase activity which resulted in reduced utilization of the deoxyribonucleoside triphosphates. Differences of as much as 26 times were detected in the sensitivity of herpes simplex virus type 1 to inhibition by acyclovir with different Vero cell cultures. These results were due to differences in acyclovir triphosphate levels, not to differences in deoxyguanosine triphosphate levels.

Topics: Acyclovir; Animals; Antiviral Agents; Cell Line; Chlorocebus aethiops; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Guanine; Phosphonoacetic Acid; Simplexvirus; Thymine Nucleotides

The M1 subunit of ribonucleotide reductase contains two kinds of allosteric sites, the activity site and the specificity site, which regulate the overall catalytic activity and the substrate specificity of the enzyme, respectively. The effector nucleotides, dGTP and dTTP, bind only to the specificity site; dATP and ATP bind to both sites. Partially purified protein M1 was photolabeled specifically after UV irradiation in the presence of [32P]dATP. The labeling occurred exclusively at the allosteric specificity site as evidenced by 1) total inhibition of the labeling by dGTP and dTTP, 2) normal photoincorporation of [32P]dATP by mutant protein M1 molecules that lack a functional activity site, and 3) coidentity of one-dimensional peptide maps of protein M1 labeled with either [32P]dATP or [32P]dTTP. A mutant protein M1 that is resistant to normal regulation by dGTP and dTTP (indicating an alteration in the allosteric specificity site) showed normal photoincorporation of [32P]dATP (but not [32P]dTTP). This labeling was not inhibited by dGTP or dTTP. Our data suggest that this mutation has altered the binding of dGTP and dTTP but not dATP (or ATP) at the specificity site. Thus, by the combination of genetic and photolabeling techniques, two independent nucleotide binding interactions occurring within this one complex regulatory domain can be distinguished.

Topics: Affinity Labels; Allosteric Site; Animals; Binding Sites; Binding, Competitive; Cell Line; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Lymphoma, Non-Hodgkin; Mice; Mutation; Neoplasms, Experimental; Ribonucleotide Reductases; Thymine Nucleotides; Ultraviolet Rays

Current evidence suggests that distinct mechanisms exist to regulate precursor synthesis for nuclear and mitochondrial DNA replication. We tested this is mouse L cells by asking whether nuclear and mitochondrial DNAs become labeled to equivalent specific activities when provided with an exogenous nucleic acid precursor. Cells were grown in [32P]orthophosphate-containing medium long enough to bring all pools to equivalent specific activities. [6-3H]Uridine was added to the medium as a general pyrimidine precursor. At intervals, cells were harvested and nuclear and mitochondrial DNA was isolated. After enzymatic hydrolysis of each DNA fraction to deoxyribonucleoside 5'-monophosphates, these were separated by high performance liquid chromatography and the 3H/32P ratio in each pyrimidine was determined as an index of the specific activity of DNA pyrimidine residues. The dTMP residues in nuclear and mitochondrial DNA reached roughly equal specific activities and at comparable rates. However, dCMP residues in mitochondrial DNA reached maximal specific activities more rapidly than those in nuclear DNA, and the maximal values attained were nearly twice those seen either with the nuclear DNA dCMP residues or in the dTMP residues from either DNA. This indicates that the pathways leading to dCTP synthesis are organized so that mitochondria can use exogenous precursors more effectively than can the nucleus. The nature of this compartmentation is not clear, but it evidently involves one or more steps beyond the divergence point between pathways to dCTP and dTTP.

Topics: Animals; Cell Compartmentation; Cell Nucleus; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA; L Cells; Mice; Mitochondria; Phosphorus Radioisotopes; Thymine Nucleotides; Tritium; Uridine

The nature of the inhibition of Ehrlich tumor cell ribonucleotide reductase by combinations of agents directed at the non-heme iron-containing component and the effector-binding component was studied with the use of isobolograms. From these studies, it was determined that the combinations of pyrazoloimidazole (IMPY) and dialdehyde of inosine, IMPY and deoxyguanosine triphosphate (dGTP), IMPY and deoxyadenosine triphosphate (dATP), and IMPY and deoxythymidine triphosphate (dTTP) gave synergistic inhibition of cytidine diphosphate reductase. The combination of dATP and dGTP also gave synergistic inhibition. The combinations of hydroxyurea and IMPY, 4-methyl-5-aminoisoquinoline thiosemicarbazone (MAIQ) and IMPY, and dialdehyde of inosine and dialdehyde derivative of 5'-deoxyinosine gave antagonistic inhibition. Other combinations utilizing MAIQ and dATP, MAIQ and dGTP, MAIQ and dTTP, hydroxyurea and dGTP, and hydroxyurea and dTTP gave inhibition which was additive.

Topics: Aldehydes; Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Drug Interactions; Drug Synergism; Edetic Acid; Hydroxyurea; Imidazoles; Inosine; Isoquinolines; Mice; Pyrazoles; Ribonucleotide Reductases; Thiosemicarbazones; Thymine Nucleotides

In four patients with Thy-acute lymphoblastic leukaemia changes in blast cell deoxynucleoside triphosphate concentrations and, in three, changes in blast cell S-adenosyl homocysteine hydrolase activity were measured during treatment with 2' deoxycoformycin, a potent inhibitor of adenosine deaminase. These studies were aimed at identifying the molecular basis of cell killing by this drug. In three patients an increase in blast deoxyadenosine triphosphate (dATP) concentration occurred which was found to be temporally related to cell killing and was accompanied by decreased concentrations of the other three deoxyribonucleoside triphosphates. In the one patient with Thy-ALL who responded poorly to treatment, the increase in dATP concentration was delayed and was not accompanied by a fall in the concentrations of the other deoxyribonucleoside triphosphates. Progressive inactivation of blast cell S-adenosyl homocysteine hydrolase was found to occur in the three patients tested but was maximal only after a substantial reduction of peripheral blast cell count. These results show that 2' deoxycoformycin has a potent cytoreductive effect in Thy-ALL and suggest that the molecular basis of this toxicity is related both to the intracellular accumulation of dATP with inhibition of ribonucleotide reductase. Inactivation of S-adenosyl homocysteine hydrolase may be of importance as an additional mechanism.

Topics: Adenosine Deaminase; Adenosylhomocysteinase; Adolescent; Adult; Bone Marrow; Coformycin; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Humans; Hydrolases; Leukemia, Lymphoid; Leukocyte Count; Male; Pentostatin; Ribonucleosides; Thymine Nucleotides

The Thy- mutants of Chinese hamster ovary cells have a 5- to 10-fold elevated pool of deoxycytidine 5'-triphosphate (dCTP) and are auxotrophic for thymidine as an apparent consequence of a single mutation. thy is also a mutator gene, elevating the spontaneous rate of mutation 5- to 200-fold for at least two genetic markers. Previous experiments suggested that this mutator activity was caused by the elevated pool of dCTP in Thy- cells. To test this, the dCTP and deoxythymidine 5'-triphosphate (dTTP) pools were manipulated by altering the external concentration of thymidine in the growth medium. The rate of mutation at one genetic locus, ouabain resistance, was directly related to cellular dCTP content. At the highest level of dCTP the rate in one Thy- strain was approximately 200 times that of wild-type cells. However, the relationship between dCTP content and the rate of mutation at the ouabain locus was different for two mutator strains and wild-type cells. The rate of mutation at a second locus, thioguanine resistance, was increased approximately 10-fold over wild type regardless of the dCTP-dTTP pools. These experiments suggest that the mutator activity of thy is clearly related to dCTP content, but the dCTP level alone does not appear to be the cause of the mutator.

Topics: Animals; CHO Cells; Cricetinae; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Emetine; Female; Mutation; Ovary; Protein Synthesis Inhibitors; Thymidine; Thymine Nucleotides

The nucleoside triphosphate pools of Escherichia coli minicells are different from those in parental cells. The growth phase in which minicells accumulate significantly affects the pool sizes.

Topics: Adenosine Triphosphate; Cytidine Triphosphate; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Escherichia coli; Guanosine Triphosphate; Nucleotides; Thymine Nucleotides; Uridine Triphosphate

Topics: Animals; CHO Cells; Cricetinae; Cytidine; Deoxyadenine Nucleotides; Deoxycytidine; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA Damage; DNA Repair; DNA Replication; Dose-Response Relationship, Radiation; Intracellular Fluid; RNA; S Phase; Thymidine; Thymine Nucleotides