deoxyguanosine-triphosphate and 2--deoxyadenosine-triphosphate

Comparison of the recently solved structure of HIV-1 reverse transcriptase (RT)-DNA-dNTP ternary complex with the previously solved structure of RT-DNA binary complex suggests mechanisms by which the HIV-1 RT becomes resistant to nucleoside-analog inhibitors, drugs currently used in the treatment of AIDS.

Topics: Anti-HIV Agents; Binding Sites; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA, Viral; Forecasting; HIV Reverse Transcriptase; Macromolecular Substances; Models, Molecular

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

The genetic deficiencies of adenosine deaminase and purine nucleoside phosphorylase lead to blocks in the purine pathway. The intracellular accumulation of deoxynucleosides and deoxynucleotides is toxic to both dividing and nondividing lymphocytes via multiple mechanisms. T-lymphocytes are uniquely sensitive to purine-mediated cytotoxicity because of a functional imbalance of phosphorylating and dephosphorylating enzymatic activities. These inborn errors or purine metabolism are rare disorders. The study of these conditions, however, has uncovered unique enzymatic properties of lymphocytes and lymphocyte subclasses. A better understanding of the mechanisms of lymphocytotoxicity in these two purine enzyme defects may lead to better modes of therapeutic manipulation of the immune system.

Topics: Adenosine Deaminase; Cytotoxicity, Immunologic; Deoxyadenine Nucleotides; Deoxyadenosines; Deoxyguanine Nucleotides; Humans; Immunologic Deficiency Syndromes; Lymphocytes; Purine-Nucleoside Phosphorylase; Purine-Pyrimidine Metabolism, Inborn Errors; Purines

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

Studies were conducted to determine if there is a mechanistic basis for reports of suboptimal virologic responses and concerns regarding the safety of regimens containing the combination of tenofovir (TFV) disoproxil fumarate (TDF) and didanosine (ddI) by assessing the pharmacokinetic consequences of coadministration of these drugs on intracellular nucleotides. This was a prospective and longitudinal study in HIV-1-infected patients of adding either TDF or ddI to a stable antiretroviral regimen containing the other drug. Intracellular concentrations of the nucleotide analogs TFV diphosphate (TFV-DP) and ddATP and the endogenous purine nucleotides dATP and 2'-dGTP in peripheral blood mononuclear cells were measured. A total of 16 patients were enrolled into the two study arms and a study extension. Intracellular TFV-DP concentrations (median, 120 fmol/10(6) cells) and ddATP concentrations (range, 1.50 to 7.54 fmol/10(6) cells in two patients) were unaffected following addition of ddI or TDF to a stable regimen containing the other drug. While coadministration of ddI and TDF for 4 weeks did not appear to impact dATP or dGTP concentrations, cross-sectional analysis suggested that extended therapy with ddI-containing regimens, irrespective of TDF coadministration, may decrease dATP and ddATP concentrations. Addition of TDF or ddI to a stable regimen including the other drug, in the context of ddI dose reduction, did not adversely affect the concentration of dATP, dGTP, TFV-DP, or ddATP. The association between longer-term ddI therapy and reduced intracellular nucleotide concentrations and this observation's implication for the efficacy and toxicity of ddI-containing regimens deserve further study.

Topics: Adenine; Adult; Aged; Anti-HIV Agents; Chromatography, Liquid; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Didanosine; Dideoxynucleotides; Female; HIV Infections; Humans; Male; Middle Aged; Nucleotides; Organophosphonates; Tandem Mass Spectrometry; Tenofovir; Young Adult

In a phase I study, 24 patients with refractory leukemia received Triapine, a novel ribonucleotide reductase (RR) inhibitor, as a continuous intravenous infusion over 96 h beginning on days 1 and 15 or days 1 and 8. On the days 1 and 15 regimen, the starting dose was 120 mg/m(2) per day, and the maximum tolerated dose (MTD) was 160 mg/m(2) per day. Three of eight patients receiving 160 mg/m(2) per day in the first course, and one patient escalated to this dose in a second course, developed hepatic dose-limiting toxicity (DLT). For the days 1 and 8 regimen, the first 96 h infusion was administered at a fixed dose of 140 mg/m(2) per day. The dose of the second infusion beginning on day 8 was escalated from 120 to 160 mg/m(2) per day without observing DLT. No objective responses occurred. Over 70% of patients had a >50% reduction in white blood cell counts. The steady-state levels of Triapine were between 0.6 and 1 microM. As expected from the in vitro studies, at these plasma concentrations there was a decline in dATP and dGTP pools and a decrease in DNA synthetic capacity of the circulating leukemia cells. Based on these clinical, pharmacokinetic, and pharmacodynamic data, Triapine warrants further study in patients with hematologic malignancies.

Topics: Adult; Aged; Aged, 80 and over; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA, Neoplasm; Enzyme Inhibitors; Female; Humans; Infusions, Intravenous; Leukemia, Lymphoid; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukocyte Count; Male; Middle Aged; Pyridines; Ribonucleotide Reductases; Safety; Thiosemicarbazones

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 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

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

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 deoxyribonucleotide triphosphohydrolase SAMHD1 restricts lentiviral infection by depleting the dNTPs required for viral DNA synthesis. In cultured human fibroblasts SAMHD1 is expressed maximally during quiescence preventing accumulation of dNTPs outside S phase. siRNA silencing of SAMHD1 increases dNTP pools, stops cycling human cells in G1, and blocks DNA replication. Surprisingly, knock-out of the mouse gene does not affect the well being of the animals. dNTPs are both substrates and allosteric effectors for SAMHD1. In the crystal structure each subunit of the homotetrameric protein contains one substrate-binding site and two nonidentical effector-binding sites, site 1 binding dGTP, site 2 dGTP or dATP. Here we compare allosteric properties of pure recombinant human and mouse SAMHD1. Both enzymes are activated 3-4-fold by allosteric effectors. We propose that in quiescent cells where SAMHD1 is maximally expressed GTP binds to site 1 with very high affinity, stabilizing site 2 of the tetrameric structure. Any canonical dNTP can bind to site 2 and activate SAMHD1, but in cells only dATP or dTTP are present at sufficient concentrations. The apparent Km for dATP at site 2 is ∼10 μm for mouse and 1 μm for human SAMHD1, for dTTP the corresponding values are 50 and 2 μm. Tetrameric SAMHD1 is activated for the hydrolysis of any dNTP only after binding of a dNTP to site 2. The lower Km constants for human SAMHD1 induce activation at lower cellular concentrations of dNTPs thereby limiting the size of dNTP pools more efficiently in quiescent human cells.

Topics: Allosteric Regulation; Animals; Binding Sites; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Gene Expression Regulation, Enzymologic; Humans; Hydrolysis; Kinetics; Mice; Models, Molecular; Monomeric GTP-Binding Proteins; Nucleotidases; SAM Domain and HD Domain-Containing Protein 1

Surface plasmon resonance (SPR) was used to measure polymerase-binding interactions of the bulky mutagenic DNA lesions N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl (FABP) or N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) in the context of two unique 5'-flanking bases (CG*A and TG*A). The enzymes used were exo-nuclease-deficient Klenow fragment (Kf-exo(-)) or polymerase β (pol β). Specific binary and ternary DNA binding affinities of the enzymes were characterized at subnanomolar concentrations. The SPR results showed that Kf-exo(-) binds strongly to a double strand/single strand template/primer junction, whereas pol β binds preferentially to double-stranded DNA having a one-nucleotide gap. Both enzymes exhibited tight binding to native DNA, with high nucleotide selectivity, where the KD values for each base pair increased in the order dCTP ≪ dTTP ∼ dATP ≪ dGTP. In contrast to that for pol β, Kf-exo(-) binds tightly to lesion-modified templates; however, both polymerases exhibited minimal nucleotide selectivity toward adducted DNA. Primer steady-state kinetics and (19)F NMR results support the SPR data. The relative insertion efficiency fins of dCTP opposite FABP was significantly higher in the TG*A sequence compared to that in CG*A. Although Kf-exo(-) was not sensitive to the presence of a DNA lesion, FAAF-induced conformational heterogeneity perturbed the active site of pol β, weakening the enzyme's ability to bind to FAAF adducts compared to FABP adducts. The present study demonstrates the effectiveness of SPR for elucidating how lesion-induced conformational heterogeneity affects the binding capability of polymerases and ultimately the nucleotide insertion efficiency.

Topics: Base Pairing; Base Sequence; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Adducts; DNA Polymerase beta; DNA Polymerase I; Kinetics; Magnetic Resonance Spectroscopy; Oligonucleotides; Protein Binding; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Surface Plasmon Resonance

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

Bioconjugating single molecules of the Klenow fragment of DNA polymerase I into electronic nanocircuits allowed electrical recordings of enzymatic function and dynamic variability with the resolution of individual nucleotide incorporation events. Continuous recordings of DNA polymerase processing multiple homopolymeric DNA templates extended over 600 s and through >10,000 bond-forming events. An enzymatic processivity of 42 nucleotides for a template of the same length was directly observed. Statistical analysis determined key kinetic parameters for the enzyme's open and closed conformations. Consistent with these nanocircuit-based observations, the enzyme's closed complex forms a phosphodiester bond in a highly efficient process >99.8% of the time, with a mean duration of only 0.3 ms for all four dNTPs. The rate-limiting step for catalysis occurs during the enzyme's open state, but with a nearly 2-fold longer duration for dATP or dTTP incorporation than for dCTP or dGTP into complementary, homopolymeric DNA templates. Taken together, the results provide a wealth of new information complementing prior work on the mechanism and dynamics of DNA polymerase I.

Topics: Catalysis; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Polymerase I; Templates, Genetic

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

Alpha,beta-difluoromethylene deoxynucleoside 5'-triphosphates (dNTPs, N = A or C) are advantageously obtained via phosphorylation of corresponding dNDP analogues using catalytic ATP, PEP, nucleoside diphosphate kinase, and pyruvate kinase. DNA pol beta K(d) values for the alpha,beta-CF(2) and unmodified dNTPs, alpha,beta-NH dUTP, and the alpha,beta-CH(2) analogues of dATP and dGTP are discussed in relation to the conformations of alpha,beta-CF(2) dTTP versus alpha,beta-NH dUTP bound into the enzyme active site.

Topics: Adenosine Triphosphate; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; DNA Polymerase beta; DNA-Directed DNA Polymerase; Molecular Probes; Molecular Structure; Nucleoside-Diphosphate Kinase; Pyruvate Kinase; Stereoisomerism

Abasic sites are among the most abundant DNA lesions formed in human cells, and they present a strong block to replication. DNA polymerase iota (Poliota) is one of the few DNA Pols that does not follow the A-rule opposite an abasic site. We present here three structures of human Poliota in complex with DNAs containing an abasic lesion and dGTP, dTTP, or dATP as the incoming nucleotide. The structures reveal a mechanism of translesion synthesis across an abasic lesion that differs from that in other Pols. Both the abasic lesion and the incoming dNTPs are intrahelical and are closely apposed across a constricted active site cleft. The dNTPs partake in distinct networks of hydrogen bonds in the "void" opposite the lesion. These different patterns of hydrogen bonds, as well as stacking interactions, may underlie Poliota's small preference for insertion of dGTP over other nucleotides opposite this common lesion.

Topics: Binding Sites; Catalysis; Crystallography, X-Ray; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Damage; DNA Polymerase iota; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Humans; Hydrogen Bonding; Kinetics; Models, Molecular; Nucleic Acid Conformation; Nucleotides; Protein Binding; Recombinant Fusion Proteins

We studied the incorporation of the fluorescent cytidine analogues 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) by human DNA polymerase alpha and Klenow fragment of DNA polymerase I (Escherichia coli). These tricyclic nucleobases possess the regular hydrogen bonding interface of cytosine but are significantly expanded in size toward the major groove. Despite the size alteration, both DNA polymerases insert dtCTP and dtCoTP with remarkable catalytic efficiency. Polymerization opposite guanine is comparable to the insertion of dCTP, while the insertion opposite adenine is only approximately 4-11 times less efficient than the formation of a T-A base pair. Both enzymes readily extend the formed tC(o)-G and tC(o)-A base pairs and can incorporate at least four consecutive nucleotide analogues. Consistent with these results, both DNA polymerases efficiently polymerize dGTP and dATP when tC and tCo are in the template strand. Klenow fragment inserts dGTP with a 4-9-fold higher probability than dATP, while polymerase alpha favors dGTP over dATP by a factor of 30-65. Overall, the properties of tC(o) as a templating base and as an incoming nucleotide are surprisingly symmetrical and may be universal for A and B family DNA polymerases. This finding suggests that the aptitude for ambivalent base pairing is a consequence of the electronic properties of tC(o).

Topics: Base Pairing; Cytosine; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA Polymerase I; Escherichia coli Proteins; Geobacillus stearothermophilus; Humans; Nucleic Acid Conformation; Nucleic Acid Heteroduplexes; Oxazines; Phenothiazines

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

Enzymatic activity of DNA polymerase iota (Pol t) was analyzed in human uveal melanoma cell extracts, using an earlier elaborated approach. The Pol t activity was observed in seven out of eight malignant tumors, while it was absent in the normal uveal tract cells of the same patients. These findings serve as an additional confirmation of the Pol t oncogenic potential.

Topics: Aged; Biomarkers, Tumor; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA Polymerase iota; DNA-Directed DNA Polymerase; Electrophoresis, Polyacrylamide Gel; Eye; Female; Humans; Male; Melanoma; Middle Aged; Uveal Neoplasms

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

The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use.

Topics: Biotechnology; Catalysis; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Kinetics; Magnesium; Models, Chemical; Pyruvate Kinase

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

Despite the nontemplating nature of the abasic site, dAMP is often preferentially inserted opposite the lesion, a phenomenon commonly referred to as the "A-rule". We have evaluated the molecular mechanism accounting for this unique behavior using a thorough kinetic approach to evaluate polymerization efficiency during translesion DNA replication. Using the bacteriophage T4 DNA polymerase, we have measured the insertion of a series of modified nucleotides and have demonstrated that increasing the size of the nucleobase does not correlate with increased insertion efficiency opposite an abasic site. One analogue, 5-nitroindolyl-2'-deoxyriboside triphosphate, was unique as it was inserted opposite the lesion with approximately 1000-fold greater efficiency compared to that for dAMP insertion. Pre-steady-state kinetic measurements yield a kpol value of 126 s(-1) and a Kd value of 18 microM for the insertion of 5-nitroindolyl-2'-deoxyriboside triphosphate opposite the abasic site. These values rival those associated with the enzymatic formation of a natural Watson-Crick base pair. These results not only reiterate that hydrogen bonding is not necessary for nucleotide insertion but also indicate that the base-stacking and/or desolvation capabilities of the incoming nucleobase may indeed play the predominant role in generating efficient DNA polymerization. A model accounting for the increase in catalytic efficiency of this unique nucleobase is provided and invokes pi-pi stacking interactions of the aromatic moiety of the incoming nucleobase with aromatic amino acids present in the polymerase's active site. Finally, differences in the rate of 5-nitroindolyl-2'-deoxyriboside triphosphate insertion opposite an abasic site are measured between the bacteriophage T4 DNA polymerase and the Klenow fragment. These kinetic differences are interpreted with regard to the differences in various structural components between the two enzymes and are consistent with the proposed model for DNA polymerization.

Topics: Bacteriophage T4; Base Composition; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA Damage; DNA Polymerase I; DNA Replication; DNA-Directed DNA Polymerase; Hydrophobic and Hydrophilic Interactions; Inosine Monophosphate; Kinetics; Nucleic Acid Heteroduplexes; Thermodynamics; Viral Proteins

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

The Y family DNA polymerase yeast pol eta inserts pyrene deoxyribose monophosphate (dPMP) in preference to A opposite an abasic site, the 3'-T of a thymine dimer, and a normal T with almost equal efficiency. In contrast, pol A family polymerases such as Klenow fragment and T7 DNA polymerase only insert dPMP efficiently opposite an abasic site and the 3'-T of a thymine dimer but not opposite undamaged DNA. Pyrene nucleotide is also an efficient chain-terminating inhibitor of DNA synthesis by pol eta but not by Klenow fragment or T7 DNA polymerase. To better understand the origin of the efficiency and sequence specificity of dPMP insertion by pol eta, the kinetics of dPMP insertion opposite various templates have been determined. In one sequence context, the efficiency of dPMP insertion increases 4.6-fold opposite G < A << T < C, suggesting that the templating nucleotide modulates dPMP insertion efficiency by having to destack prior to dPTP binding. The efficiency of insertion of dPMP opposite T in the same sequence context increases 7-fold for primers terminating in G < A < C < T and is similar to that observed for nontemplated blunt-end extension, suggesting that stacking interactions between the pyrene and the primer terminus are also important. On heterogeneous templates, the average selectivity for dPMP insertion relative to the complementary dNMP decreases in the order of dAMP > dGMP > dTMP > dCMP, from a high of 5.8 when dAMP is to be inserted following a T to a low of 0.5 when dCMP is to be inserted following a C. The relative preference for dPMP insertion at a given site can be largely explained by the energetic cost of destacking the templating base and stacking of pyrene nucleotide relative to that of stacking and base pairing the complementary nucleotide. Thus, pyrene nucleotide represents a novel class of nucleotide-based chain-terminating DNA synthesis inhibitors whose base portion consists of a hydrophobic, non-hydrogen bonding, base-pair mimic.

Topics: Apurinic Acid; Base Composition; Base Sequence; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Dimerization; DNA Primers; DNA-Directed DNA Polymerase; Enzyme Inhibitors; Kinetics; Molecular Sequence Data; Nucleic Acid Conformation; Nucleic Acid Synthesis Inhibitors; Nucleotides; Oligodeoxyribonucleotides; Pyrenes; Saccharomyces cerevisiae Proteins; Sequence Analysis, DNA; Templates, Genetic; Thymine

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

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

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

A solution-phase steady-state polarization-based method for discriminating among the four DNA nucleotides labeled identically with tetramethylrhodamine is described and demonstrated. Labeled nucleotides were dissolved in buffered surfactant solutions. In room temperature 4.5 mM Triton X-100 solutions at neutral pH, the measured steady-state polarizations of tetramethylrhodamine-labeled dATP, dCTP, dGTP and dUTP were 0.261 +/- 0.003, 0.112 +/- 0.003, 0.288 +/- 0.003, and 0.147 +/- 0.003, respectively. A blind test of 40 samples showed no errors in classification based on polarization. The reproducibility obtained during this study demonstrates that the four dye-labeled nucleotides can be discriminated with more than 99.8% confidence.

Topics: Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyuracil Nucleotides; Fluorescence Polarization; Fluorescent Dyes; Molecular Structure; Nucleotides; Octoxynol; Rhodamines; Solutions; Surface-Active Agents

Renin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney as a complex of renin, so-called high molecular weight (HMW) renin. Our recent studies demonstrated that human RnBP is the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353]. We have purified recombinant human, rat, and porcine RnBPs expressed in Escherichia coli JM 109 cells. The purified recombinant RnBPs existed as dimers and inhibited porcine renin activity strongly. On the other hand, porcine renin inhibited recombinant GlcNAc 2-epimerase activities. The human GlcNAc 2-epimerase activity could not be detected in the absence of a nucleotide, whereas ATP, dATP, ddATP, ADP, and GTP enhanced the human GlcNAc 2-epimerase activity. Other nucleotides had no effect on human GlcNAc 2-epimerase activity. Rat and porcine GlcNAc 2-epimerases were activated by several nucleotides. Nucleotides that enhance the activity of GlcNAc 2-epimerases protect these enzymes against degradation by thermolysin. These results indicate that mammalian RnBPs have GlcNAc 2-epimerase activity and that nucleotides are essential for formation of the catalytic domain of the enzyme.

Topics: Adenosine Triphosphate; Animals; Carbohydrate Epimerases; Carrier Proteins; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Humans; Hydrolysis; Nucleotides; Rats; Recombinant Proteins; Renin; Swine; Thermolysin

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

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

(E)-2'-Deoxy-(fluoromethylene)cytidine (FMdC) is known as an inhibitor of ribonucleoside diphosphate reductase, a key enzyme in the de novo pathway of DNA synthesis. FMdC was tested as a modifier of radiation response in vitro on a human colon carcinoma cell line (WiDr), and the observed radiosensitization was confirmed on two human cervix cancer cell lines (C33-A and SiHa). Using the clonogenic assay, the effect ratio (ER) at a clinically relevant dose level of 2 Gy was 2.10 (50 nM FMdC), 1.70 (30 nM FMdC), and 1.71 (40 nM FMdC) for the three cell lines WiDr, C33-A, and SiHa, respectively. A more detailed analysis of the importance of timing and concentration of FMdC was done on the WiDr cell line alone, yielding an increased ER(2Gy) with increasing concentration and duration of exposure to the drug, ranging from 1.0 (6 h) to 1.8 (72 h) at 30 nM FMdC and from 1.2 (6 h) to 3.5 (24 h) at 300 nM. We investigated the effect of FMdC on the cellular deoxynucleotide triphosphate pool in WiDr cells and demonstrated a marked depletion of dATP and a significant rise of TTP levels. Cell cycle analysis showed early S-phase accumulation induced by FMdC alone, G2-M block induced by irradiation alone, and an increased accumulation of cells in G2-M if both modalities are used. Our data suggest that FMdC is a radiation response modifier in vitro on different cancer cell lines. The observed radiosensitization may in part be explained by alteration of the deoxynucleotide triphosphate pool, which is consistent with the effect of FMdC on ribonucleoside diphosphate reductase.

Topics: Antineoplastic Agents; Cell Cycle; Cell Division; Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; Deoxycytidine; Deoxyguanine Nucleotides; Humans; Radiation-Sensitizing Agents; Ribonucleoside Diphosphate Reductase; Tumor Cells, Cultured

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

Using HPLC methods, we measured the concentrations of nucleosides and nucleotides for a patient with no purine nucleoside phosphorylase (PNP; EC 2.4.2.1) enzymatic activity. Concentrations of inosine and guanosine were abnormally high in urine and plasma, whereas guanosine diphosphate (GDP) and guanosine triphosphate (GTP) concentrations in erythrocytes were depleted. The unusual presence of deoxyribonucleosides (deoxyinosine and deoxyguanosine) and deoxyribonucleotides (dGDP and dGTP) was also notable. Thus, HPLC represents an accurate and useful tool for the study of purine metabolic disorders.

Topics: Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Erythrocytes; Guanosine; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Infant; Inosine; Male; Purine-Nucleoside Phosphorylase; Purine-Pyrimidine Metabolism, Inborn Errors

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

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

Telomeres play an important role in chromosome organization and stability. Human telomerase is a terminal transferase that adds TTAGGG units onto the telomere end. In general, telomerase activity is not detected in normal somatic cells but is present in immortalized cells. Consequently, telomerase might be a selective target for cancer chemotherapy. Using cell-free biochemical telomerase assay, we have found that 7-deaza-2'-deoxyguanosine-5'-triphosphate (7-deaza-dGTP) and 7-deaza-2'-deoxyadenosine-5'-triphosphate (7-deaza-dATP) were potent telomerase inhibitors. The concentrations of inhibitors in which 50% of the telomerase activity was inhibited (IC50 values) were 11 and 8 microM for 7-deaza-dGTP and 7-deaza-dATP, respectively. Additional studies show that both 7-deaza-dGTP and 7-deaza-dATP were also incorporated into telomeric DNA by telomerase. However, incorporation of 7-deaza-dATP or 7-deaza-dGTP results in a telomeric ladder that is prematurely shortened. No difference in the number or position of pause sites were observed when 7-deaza-dATP was compared to dATP as substrates. On the other hand, both a shift and an increase in pause sites was observed when dGTP was replaced by 7-deaza-dGTP. Incorporation of 7-deaza nucleotides by telomerase may be used as a tool for the study of telomerase mechanism and function. In addition, this may be a novel approach in the design of new telomerase inhibitors.

Topics: Cells, Cultured; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA Primers; Humans; Kidney; Oligodeoxyribonucleotides; Purine Nucleotides; Ribonuclease, Pancreatic; Telomerase

Topics: Base Composition; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Nucleotidylexotransferase; DNA-Directed DNA Polymerase; Inosine Triphosphate; Plasmids; Sequence Analysis, DNA

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

We present a simple screening method for detecting a known point mutation, using only one 5'-biotinylated oligonucleotide primer, with its 3' end adjacent to the mutation site. In parallel reactions, an amplified DNA template encompassing the biotinylated oligonucleotide and mutation site undergoes 40 step-cycles of single nucleotide incorporation using Taq thermostable DNA polymerase and only one radioactive [alpha-32P]dNTP, specified by either the normal or mutant sequence. The oligonucleotides, now radioactively labelled at the 3' end according to the template sequence, are then trapped by streptavidin-coated magnetic beads, and the percent of radiolabel incorporated is determined directly by the Cerenkov method in a scintillation counter. The trapped-oligonucleotide nucleotide incorporation (TONI) assay has been used for the screening of a mitochondrial polymorphism, and has also been shown to distinguish the genotypes of hemoglobin A/C, A/A, A/S, and S/S. It is reproducible over at least a 100-fold range of radioisotope and a 10-fold range of oligonucleotide primer. This method is particularly useful for diagnosing mutations which do not produce alterations detectable by restriction enzyme analysis, since optimization of conditions is rarely necessary. In addition, it requires only a single oligonucleotide, and no electrophoretic separation of the allele-specific products. It thus represents an improved and simplified modification of the existing allele-specific primer extension methods (Kuppuswamy et al., Proc Natl Acad Sci USA 88:1143-1147, 1991; Sokolov, Nucl Acids Res 18:3671, 1989; Syvanen et al., Genomics 8:684-692, 1990).

Topics: Base Sequence; Deafness; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA, Mitochondrial; Genetic Testing; Hemoglobin A; Hemoglobin, Sickle; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Phosphorus Radioisotopes; Point Mutation; Polymerase Chain Reaction; Polymorphism, Genetic

We have previously proposed that DNA polymerase alpha-primase provides short RNA-DNA precursors below 40 nucleotides (DNA primers), several of which assemble into an Okazaki piece after intervening RNA has been removed and the gaps have been filled by DNA polymerase delta (or epsilon) (T. Nethanel, S. Reisfeld, G. Dinter-Gottlieb, and G. Kaufmann, J. Virol. 62:2867-2873, 1988; T. Nethanel and G. Kaufmann, J. Virol. 64:5912-5918, 1990). In this report, we confirm and extend these conclusions by studying the effects of deoxynucleoside triphosphate (dNTP) concentrations and the presence of ATP on the occurrence, dynamics, and configuration of DNA primers in simian virus 40 replicative intermediate DNA. We first show that these parameters are not significantly affected by a 10-fold increase in dNTP precursor concentrations. We then demonstrate that Okazaki piece synthesis can be arrested at the level of DNA primers by ATP depletion. The arrested DNA primers faced short gaps of 10 to 20 nucleotides at their 3' ends and were progressively chased into Okazaki pieces when ATP was restored. ATP could not be substituted in this process by adenosine-5'-O-(3-thiotriphosphate) or adenyl-imidodiphosphate. The chase was interrupted by aphidicolin but not by butylphenyl-dGTP. The results implicate an ATP-requiring factor in the switch between the two DNA polymerases engaged in Okazaki piece synthesis. They also suggest that the replication fork advances by small, DNA primer-size increments.

Topics: Adenosine Triphosphate; Aphidicolin; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyuracil Nucleotides; DNA; DNA Primase; DNA Replication; DNA, Single-Stranded; DNA, Viral; Nucleic Acid Conformation; RNA Nucleotidyltransferases; Simian virus 40

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

Rickettsia prowazekii, an obligate intracellular parasitic bacterium, was shown to have a ribonucleotide reductase that would allow the rickettsiae to obtain the deoxyribonucleotides needed for DNA synthesis from rickettsial ribonucleotides rather than from transport. In the presence of hydroxyurea, R. prowazekii failed to grow in mouse L929 cells or SC2 cells (a hydroxyurea-resistant cell line), which suggested that R. prowazekii contains a functional ribonucleotide reductase. This enzymatic activity was demonstrated by the conversion of ADP to dADP and CDP to dCDP, using (i) a crude extract of Renografin-purified R. prowazekii that had been harvested from infected yolk sacs and (ii) high-performance liquid chromatographic analysis. The rickettsial ribonucleotide reductase utilized ribonucleoside diphosphates as substrates, required magnesium and a reducing agent, and was inhibited by hydroxyurea. ADP reduction was stimulated by dGTP and inhibited by dATP. CDP reduction was stimulated by ATP and adenylylimido-diphosphate and inhibited by dATP and dGTP. These characteristics provided strong evidence that the rickettsial enzyme is a nonheme iron-containing enzyme similar to those found in mammalian cells and aerobic Escherichia coli.

Topics: Adenosine Diphosphate; Cells, Cultured; Cytidine Monophosphate; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Gene Expression Regulation, Bacterial; Hydroxyurea; Ribonucleotide Reductases; Ribonucleotides; Rickettsia prowazekii

We previously reported a double-stranded endonuclease from HeLa cells, endonuclease R (endo R), which specifically cleaves duplex DNA at sites rich in G.C base pairs. In this report we describe the purification of endo R to near homogeneity by conventional and affinity chromatography. The molecular mass of the active form of endo R is approximately 115-125 kDa. SDS-gel electrophoresis reveals a major protein species of 100 kDa. The enzyme requires Mg2+ as a cofactor and is equally active on closed circular and linear duplex DNA substrates that contain G-rich sequences. A 50% reduction in cleavage activity is observed with Ca2+ ions and no double-stranded cleavage occurs with Zn2+. Use of Mn2+ causes an altered specificity at low concentrations of enzyme or divalent metal ion and nonspecific degradation of the substrate at higher concentrations. Endo R is strongly inhibited by sodium or potassium chloride and exhibits a wide pH optimum of 6.0-9.0. The pI of the enzyme is between 6.5 and 7.0. A 2-fold stimulation is observed with the addition of dGTP or dATP but specific cleavage is inhibited by ATP at an equivalent concentration. Cleavage activity is competitively inhibited 10-fold more efficiently by single-stranded poly(dG)12 than by other DNA competitors. The ends of endo R cleavage products contain 5'-phosphate and 3'-hydroxyl groups, and a significant portion of these products were substrates for T4 DNA ligase. Endo R appears to be a previously uncharacterized mammalian endonuclease.

Topics: Base Composition; Binding, Competitive; Calcium; Cations, Divalent; Chromatography, Affinity; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; Electrophoresis, Polyacrylamide Gel; Endodeoxyribonucleases; HeLa Cells; Humans; Hydrogen-Ion Concentration; Magnesium; Manganese; Molecular Weight; Poly G; Potassium Chloride; Sodium Chloride; Substrate Specificity

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

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

Comparative inhibition kinetics with natural dNTP end products (dNp3) and new synthetic bisubstrate-type analogs, dNp4A (deoxynucleoside 5'-adenosine 5'''-P1,P4-tetraphosphate), have been studied with their target deoxynucleoside kinases from Lactobacillus acidophilus. Analysis of inhibition specificity, inhibition patterns, and Ki(app) under various conditions has revealed the following conclusions. Both dNTP and dNp4A bind to the active site of the corresponding kinase through multiple binding determinants. The deoxynucleoside moiety of dNTP fits optimally at the deoxynucleoside binding site and provides the basis for its inhibition specificity, whereas the triphosphate group interacts with the ATP binding site, reinforcing the affinity of the molecule as a potent end product inhibitor (Ki = 0.4-3 microM). The adenosine moiety of dNp4A does not contribute to the binding of this compound, whereas the tetraphosphate portion is the second binding determinant, just as in the model developed for dNTP. dNTP and dNp4A proved to be useful tools for distinguishing the kinetic mechanisms of kinases which follow sequential pathways, i.e. the rapid equilibrium Random Bi Bi for dCyd and dGuo kinases and the steady state Ordered Bi Bi mechanism for two dAdo kinases associated either with dCyd kinase or with dGuo kinase on different multifunctional proteins.

Topics: Adenosine Triphosphate; Binding Sites; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Kinetics; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Structure-Activity Relationship

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

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

An intact cell assay system based upon Tween-80 permeabilization was used to investigate the regulation of ribonucleotide reductase activity in Chinese hamster ovary cells. Models used to explain the regulation of the enzyme have been based upon work carried out with cell-free extracts, although there is concern that the properties of such a complex enzyme would be modified by extraction procedures. We have used the intact cell assay system to evaluate, within whole cells, the current model of ribonucleotide reductase regulation. While some of the results agree with the proposals of the model, others do not. Most significantly, it was found that ribonucleotide reductase within the intact cell could simultaneously bind the nucleoside triphosphate activators for both CDP and ADP reductions. According to the model based upon studies with cell-free preparations, the binding of one of these nucleotides should exclude the binding of others. Also, studies on intracellular enzyme activity in the presence of combinations of nucleotide effectors indicate that GTP and perhaps dCTP should be included in a model for ribonucleotide reductase regulation. For example, GTP has the unique ability to modify through activation both ADP and CDP reductions, and synergistic effects were obtained for the reduction of CDP by various combinations of ATP and dCTP. In general, studies with intact cells suggest that the in vivo regulation of ribonucleotide reductase is more complex than predicted from enzyme work with cell-free preparations. A possible mechanism for the in vivo regulation of ribonucleotide reductase, which combines observations of enzyme activity in intact cells and recent reports of independent substrate-binding subunits in mammalian cells is discussed.

Topics: Adenosine Triphosphate; Animals; Binding, Competitive; Cell Line; Chemical Phenomena; Chemistry; Cricetinae; Cricetulus; Cytidine Diphosphate; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Female; Guanosine Triphosphate; Nucleotides; Ovary; Ribonucleotide Reductases; Substrate Specificity

Enzyme inhibitors used to simulate the inherited immunodeficiency diseases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency, have been assessed in cultured human lymphocytes. Only 2'-deoxycoformycin (dCF) completely inhibited ADA in T and B cells at concentrations in excess of 5 microM. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and 8-amino guanosine (8-NH2GR) did not inhibit ADA or PNP completely at any concentration. Detailed metabolic experiments comparing viability and deoxynucleotide accumulation showed that B cell lines of malignant origin also accumulated high levels of dATP from 2'-deoxyadenosine (dAR), and dGTP from 2'-deoxyguanosine (dGR) as effectively as T cells--even without inhibitors, however, dAR reduced cell viability only when ADA was inhibited by dCF, whilst dGR was equally toxic with or without inhibitor, even to a line which accumulated no dGTP. These experiments indicate that cultured lymphocytes, using either EHNA or 8-NH2GR as enzyme inhibitor, are not valid models of the toxicity to the immune system in inherited ADA or PNP deficiency. They demonstrate that the ability to accumulate high levels of dATP or dGTP is not exclusive to T cells and that the in vitro toxicity of dAR or dGR could relate to the use of excess substrate and/or accumulation in different nucleotide, not deoxynucleotide pools.

Topics: Adenine; Adenosine Deaminase Inhibitors; B-Lymphocytes; Cell Line; Cell Survival; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Deoxyguanine Nucleotides; Deoxyguanosine; Dose-Response Relationship, Drug; Guanosine; Humans; Pentostatin; Purine-Nucleoside Phosphorylase; T-Lymphocytes

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

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

We have studied the effects of various immunosuppressive drugs on the growth of human-derived T (MOLT-4) and B (MGL-8) lymphoblasts. In addition, we have examined whether the lymphotoxic effect of any of these drugs could be attributed to inhibition of either adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP). Results indicated that 1-beta-D-arabinofuranosylcytosine (Ara-C), methotrexate and chlorambucil were four to seven times more toxic for T than for B cells, while azathioprine, 6-thioguanine, 6-mercaptopurine, and 5-fluorouracil were highly toxic for both T and B cells. Cyclophosphamide and oxisuran were lymphotoxic only at concentrations exceeding 300 microM. Deoxyadenosine (50 microM), deoxyguanosine (10 microM) and deoxycoformycin (10 microM) failed to enhance T cell toxicity when individually combined with each drug. None of the drugs tested inhibited T or B lymphoblast ADA or PNP activity. With the exception of Ara-C, neither dATP nor dGTP accumulated in T lymphoblasts incubated in the presence of any of the drugs. We conclude that the cell culture system used in this investigation is useful for identifying lymphotoxic and T cell-specific immunosuppressive agents. However, none of the drugs studied appeared to function as an inhibitor of, or a competitive substrate for, either ADA or PNP.

Topics: Adenosine Deaminase Inhibitors; B-Lymphocytes; Cells, Cultured; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Humans; Immunosuppressive Agents; Nucleoside Deaminases; Pentosyltransferases; Purine-Nucleoside Phosphorylase; T-Lymphocytes

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

Low ATP/ADP ratios have been reported consistently for nucleotide levels of mononuclear cells separated from peripheral blood by conventional techniques. We have established that these low values (mean 2.3:1) were not due to cell damage or poor viability, but resulted from heavy platelet contamination, which is unavoidable when heparinized blood is used. The results reflect the low ATP/ADP ratios (mean 1.6:1) characteristic of platelets. Platelet-free extracts from defibrinated blood had very high ATP/ADP ratios (mean 17.4:1). The initial finding of detectable amounts of deoxy-ATP and deoxy-GTP in mononuclear cells from children with two distinct inherited immunodeficiency disorders [adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency respectively] many have been due to contamination by nucleated erythrocytes as well as platelets in non-defibrinated preparations. Defibrination before nucleotide extraction of mononuclear cells from a patient with T-cell leukaemic/lymphoma treated with the ADA inhibitor deoxycoformycin enabled the demonstration of grossly raised deoxy-ATP levels relative to deoxy-ADP levels (ratio 16.1:1), associated with severe ATP depletion. This reciprocal relationship between ATP and dATP was found by us previously in the erythrocytes in inherited ADA deficiency. These findings underline the importance of extracts uncontaminated by platelets, or nucleated erythrocytes, in the evaluation of lymphocyte nucleotide levels in inherited or acquired immunodeficiency syndromes.

Topics: Adenosine Deaminase; Adenosine Deaminase Inhibitors; Adenosine Diphosphate; Adenosine Triphosphate; Blood Platelets; Cell Separation; Child; Coformycin; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Humans; Immunologic Deficiency Syndromes; Lymphocytes; Pentostatin; Purine-Nucleoside Phosphorylase

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