guanosine-triphosphate and thymidine-5--triphosphate

The distance-dependent based sensing mechanism, such as fluorescence resonance energy transfer (FRET) and surface plasmon resonance (SPR) absorption of gold nanoparticles, has been used widely in visual detection. In this work, we report another distance-dependent detection method for nucleoside triphosphates (NTPs) based on carbon dots (CDs) (

Topics: Adenosine Triphosphate; Carbon; Cytidine Triphosphate; Guanosine Triphosphate; Limit of Detection; Quantum Dots; Singlet Oxygen; Spectrophotometry; Thymine Nucleotides

We designed an aptasensor for the detection of adenosine triphosphate (ATP) based on chemiluminescence resonance energy transfer (CRET). An adenosine aptamer was cut into two pieces of ssDNA, which were attached to quantum dots (QDs) and horse radish peroxidase (HRP), respectively. They could reassemble into specific structures in the presence of ATP and then decrease the distance of HRP and QDs. ATP detection can be easily realized according to the fluorescent intensity of QDs, which is excited by CRET between luminol and QDs. Results show that the concentration of ATP is linear relation with the fluorescent intensity of the peak of QDs emission and the linear range for the linear equation is from 50 μM to 231 μM and the detection limit was 185 nM. When the concentration of ATP was 2 mM, the efficiency of CRET is 13.6%. Good specificity for ATP had been demonstrated compared to thymidine triphosphate (TTP), cytidine triphosphate (CTP) and guanosine triphosphate (GTP), when 1 mM of each was added, respectively. This method needs no external light source and can avoid autofluorescence and photobleaching, and ATP can be detected selectively, specifically, and sensitively in a low micromolar range, which means that the strategy reported here can be applicable to the detection of several other target molecules.

Topics: Adenosine Triphosphate; Aptamers, Nucleotide; Biosensing Techniques; Cytidine Triphosphate; Fluorescence Resonance Energy Transfer; Guanosine Triphosphate; Luminescence; Quantum Dots; Thymine Nucleotides

The multifunctional protein encoded by gene 4 of bacteriophage T7 (gp4) provides both helicase and primase activity at the replication fork. T7 DNA helicase preferentially utilizes dTTP to unwind duplex DNA in vitro but also hydrolyzes other nucleotides, some of which do not support helicase activity. Very little is known regarding the architecture of the nucleotide binding site in determining nucleotide specificity. Crystal structures of the T7 helicase domain with bound dATP or dTTP identified Arg-363 and Arg-504 as potential determinants of the specificity for dATP and dTTP. Arg-363 is in close proximity to the sugar of the bound dATP, whereas Arg-504 makes a hydrogen bridge with the base of bound dTTP. T7 helicase has a serine at position 319, whereas bacterial helicases that use rATP have a threonine in the comparable position. Therefore, in the present study we have examined the role of these residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity. Our results show that Arg-363 is responsible for dATP, dCTP, and dGTP hydrolysis, whereas Arg-504 and Ser-319 confer dTTP specificity. Helicase-R504A hydrolyzes dCTP far better than wild-type helicase, and the hydrolysis of dCTP fuels unwinding of DNA. Substitution of threonine for serine 319 reduces the rate of hydrolysis of dTTP without affecting the rate of dATP hydrolysis. We propose that different nucleotides bind to the nucleotide binding site of T7 helicase by an induced fit mechanism. We also present evidence that T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.

Topics: Adenosine Triphosphate; Amino Acid Substitution; Arginine; Bacteriophage T7; Crystallography, X-Ray; Cytidine Triphosphate; DNA; DNA Helicases; DNA-Directed DNA Polymerase; Guanosine Triphosphate; Hydrolysis; Kinetics; Mutant Proteins; Nucleotides; Plasmids; Protein Binding; Protein Structure, Quaternary; Protein Structure, Secondary; Substrate Specificity; Thymine Nucleotides

The genome of the mesophilic Gram-negative bacterium Burkholderia thailandensis contains an open reading frame (i.e. the Bth_I1158 gene) that has been annotated as a putative ribokinase and PFK-B family member. Notably, although the deduced amino acid sequence of the gene showed only 29% similarity to the recently identified nucleoside kinase from hyperthermophilic archaea Methanocaldococcus jannaschii, 15 of 17 residues reportedly involved in the catalytic activity of M. jannaschii nucleoside kinase were conserved. The gene was cloned and functionally overexpressed in Rhodococcus erythropolis, and the purified enzyme was characterized biochemically. The substrate specificity of the enzyme was unusually broad for a bacterial PFK-B protein, and the specificity extended not only to purine and purine-analog nucleosides but also to uridine. Inosine was the most effective phosphoryl acceptor, with the highest k(cat)/K(m) value (80 s(-1).mm(-1)) being achieved when ATP served as the phosphoryl donor. By contrast, this enzyme exhibited no activity toward ribose, indicating that the recombinant enzyme was a nucleoside kinase rather than a ribokinase. To our knowledge, this is the first detailed analysis of a bacterial nucleoside kinase in the PFK-B family.

Topics: Adenosine Triphosphate; Bacterial Proteins; Burkholderia; Catalysis; Cations, Divalent; Cloning, Molecular; Enzyme Stability; Guanosine Triphosphate; Hydrogen-Ion Concentration; Inosine Triphosphate; Kinetics; Molecular Weight; Phosphofructokinases; Phosphotransferases; Recombinant Proteins; Substrate Specificity; Thymine Nucleotides

The Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (dNK; EC 2.7.1.145) has a high turnover rate and a wide substrate range that makes it a very good candidate for gene therapy. This concept is based on introducing a suicide gene into malignant cells in order to activate a prodrug that eventually may kill the cell. To be able to optimize the function of dNK, it is vital to have structural information of dNK complexes. In this study we present crystal structures of dNK complexed with four different nucleoside analogs (floxuridine, brivudine, zidovudine and zalcitabine) and relate them to the binding of substrate and feedback inhibitors. dCTP and dGTP bind with the base in the substrate site, similarly to the binding of the feedback inhibitor dTTP. All nucleoside analogs investigated bound in a manner similar to that of the pyrimidine substrates, with many interactions in common. In contrast, the base of dGTP adopted a syn-conformation to adapt to the available space of the active site.

Topics: Adenosine Diphosphate; Animals; Antimetabolites; Bromodeoxyuridine; Cytarabine; Cytidine Triphosphate; Drosophila melanogaster; Drosophila Proteins; Feedback; Floxuridine; Guanosine Triphosphate; Hydrogen Bonding; Inhibitory Concentration 50; Kinetics; Models, Chemical; Models, Molecular; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Protein Structure, Secondary; Structure-Activity Relationship; Thymine Nucleotides; X-Ray Diffraction; Zalcitabine; Zidovudine

Gallic acid (GA) is a naturally occurring polyhydroxyphenolic compound and an excellent free radical scavenger. In this study, we examined its cytotoxic and biochemical effects on the human HL-60 promyelocytic leukemia cell line. GA caused a significant imbalance of deoxynucleosidetriphosphate (dNTP) pool sizes, indicating ribonucleotide reductase inhibition. Moreover, GA induced dose-dependent apoptosis in HL-60 cells (80microM GA led to the induction of apoptosis in 39% of cells) and attenuated progression from G0/G1 to the S phase of the cell cycle (60microM GA doubled the number of cells in G0/G1 phase from 22 to 44% when compared to untreated controls). We further determined IC(50) values of 3.5 and 4.4nM for the inhibition of cyclooxygenases I and II, respectively. When cells were simultaneously treated with GA and trimidox, another inhibitor of RR, highly synergistic growth inhibitory effects could be observed. Taken together, we identified novel biochemical effects of GA which could be the basis for further preclinical and in vivo studies.

Topics: Adenosine Triphosphate; Apoptosis; Benzamidines; Cell Cycle; Cell Proliferation; Cell Survival; Cyclooxygenase Inhibitors; Cytidine Triphosphate; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gallic Acid; Guanosine Triphosphate; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Molecular Structure; Prostaglandin-Endoperoxide Synthases; Ribonucleotide Reductases; Thymine Nucleotides

The basis for mutations at A:T base pairs in immunoglobulin hypermutation and defining how AID interacts with the DNA of the immunoglobulin locus are major aspects of the immunoglobulin mutator mechanism where questions remain unanswered. Here, we examined the pattern of mutations generated in mice deficient in various DNA repair proteins implicated in A:T mutation and found a previously unappreciated bias at G:C base pairs in spectra from mice simultaneously deficient in DNA mismatch repair and uracil DNA glycosylase. This suggests a strand-biased DNA transaction for AID delivery which is then masked by the mechanism that introduces A:T mutations. Additionally, we asked if any of the known components of the A:T mutation machinery underscore the basis for the paucity of A:T mutations in the Burkitt lymphoma cell lines, Ramos and BL2. Ramos and BL2 cells were proficient in MSH2/MSH6-mediated mismatch repair, and express high levels of wild-type, full-length DNA polymerase eta. In addition, Ramos cells have high levels of uracil DNA glycosylase protein and are proficient in base excision repair. These results suggest that Burkitt lymphoma cell lines may be deficient in an unidentified factor that recruits the machinery necessary for A:T mutation or that AID-mediated cytosine deamination in these cells may be processed by conventional base excision repair truncating somatic hypermutation at the G:C phase. Either scenario suggests that cytosine deamination by AID is not enough to trigger A:T mutation, and that additional unidentified factors are required for full spectrum hypermutation in vivo.

Topics: Adenosine Triphosphate; Animals; Burkitt Lymphoma; Cell Line, Tumor; Cytidine Deaminase; Cytidine Triphosphate; DNA Mismatch Repair; DNA Repair Enzymes; Guanosine Triphosphate; Humans; Mice; Mutation; Nucleotides; Somatic Hypermutation, Immunoglobulin; Thymine Nucleotides

Efforts to correlate genetic variations with phenotypic differences are intensifying due to the availability of high-density maps of single nucleotide polymorphisms (SNPs) and the development of high throughput scoring methods. These recent advances have led to an increased interest for improved multiplex preparations of genetic material to facilitate such whole genome analyses. Here we propose a strategy for the parallel amplification of polymorphic loci based on a reduced set of nucleotides. The technique denoted Tri-nucleotide Threading (TnT), allows SNPs to be amplified via controlled linear amplification followed by complete removal of the target material and subsequent amplification with a pair of universal primers. A dedicated software tool was developed for this purpose and variable positions in genes associated with different forms of cancer were analyzed using sub-nanogram amounts of starting material. The amplified fragments were then successfully scored using a microarray-based PrASE technique. The results of this study, in which 75 SNPs were analyzed, show that the TnT technique circumvents potential problems associated with multiplex amplification of SNPs from minute amounts of material. The technique is specific, sensitive and can be readily adapted to equipment and genotyping techniques used in other research laboratories without requiring changes to the preferred typing method.

Topics: Cytidine Triphosphate; Genome, Human; Genomics; Genotype; Guanosine Triphosphate; Humans; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Software; Thymine Nucleotides

The thymidine kinase encoded by Epstein-Barr virus (EBV TK) is an important target for antiviral therapy and the treatment of EBV-associated malignancies. Through computer-assisted alignment with other human herpesviral TK proteins, EBV TK was shown to contain a conserved ATP-binding motif as for the other TK enzymes. To investigate functional roles of three highly conserved residues (G294, K297, T298) within this region, site-directed mutagenesis was employed to generate various mutants. The TK enzyme activity and ATP-binding ability of these mutant TK enzymes were determined and compared with EBV wild-type TK (wtTK). Mutant G294V lost its ATP-binding ability and was inactive in enzyme activity assay. As the enzyme activity of G294A was reduced to 20% of that of wtTK, the K(m) for ATP binding of G294A was 48.7 microM as compared with 30.0 microM of EBV wtTK. These results suggested that G294 participates in ATP binding and contributes to maintenance of structure. EBV TK mutants K297E, K297Q, and K297R lost their ATP-binding ability and enzyme activity. However, K297R was shown to have a preference for usage of GTP (K(m): 43.0 microM) instead of ATP (K(m): 87.6 microM) as the phosphate donor. This implies that, in addition to nucleotide binding, K297 was involved in the selection of phosphate donor. While EBV TK mutant T298S retained approximately 80% of wtTK enzyme activity, T298A lost its enzyme activity, suggesting that a hydroxyl group at this position is important for the enzyme activity. Interestingly, T298A retained its ATP-binding ability, suggesting a role of T298 in the catalytic process but not in the coordination of ATP. This study demonstrated that amino acid residues G294, K297, and T298 in the ATP-binding motif of EBV TK enzyme are essential for the enzymatic activity but are involved in different aspects of its action.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Binding Sites; Conserved Sequence; Cytidine Triphosphate; Glycine; Guanosine Triphosphate; Herpesvirus 4, Human; Humans; Lysine; Magnesium; Manganese; Molecular Sequence Data; Mutagenesis, Site-Directed; Sequence Alignment; Sequence Homology, Amino Acid; Threonine; Thymidine Kinase; Thymine Nucleotides; Zinc

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

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides into deoxyribonucleotides, which constitute the precursor pools used for DNA synthesis and repair. Imbalances in these pools increase mutational rates and are detrimental to the cell. Balanced precursor pools are maintained primarily through the regulation of the RNR substrate specificity. Here, the molecular mechanism of the allosteric substrate specificity regulation is revealed through the structures of a dimeric coenzyme B12-dependent RNR from Thermotoga maritima, both in complexes with four effector-substrate nucleotide pairs and in three complexes with only effector. The mechanism is based on the flexibility of loop 2, a key structural element, which forms a bridge between the specificity effector and substrate nucleotides. Substrate specificity is achieved as different effectors and their cognate substrates stabilize specific discrete loop 2 conformations. The mechanism of substrate specificity regulation is probably general for most class I and class II RNRs.

Topics: Adenosine Triphosphate; Allosteric Site; Amino Acid Sequence; Binding Sites; Catalysis; DNA; DNA Repair; Evolution, Molecular; Guanosine Triphosphate; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Ribonucleotide Reductases; Sequence Homology, Amino Acid; Substrate Specificity; Thermotoga maritima; Thymine Nucleotides

The bacterial tRNALys-specific anticodon nuclease is known as a phage T4 exclusion system. In the uninfected host cell anticodon nuclease is kept latent due to the association of its core protein PrrC with the DNA restriction-modification endonuclease EcoprrI. Stp, the T4-encoded peptide inhibitor of EcoprrI activates the latent enzyme. Previous in vitro work indicated that the activation by Stp is sensitive to DNase and requires added nucleotides. Biochemical and mutational data reported here suggest that Stp activates the latent holoenzyme when its EcoprrI component is tethered to a cognate DNA substrate. Moreover, the activation is driven by GTP hydrolysis, possibly mediated by the NTPase domain of PrrC. The data also reveal that Stp can be replaced as the activator of latent anticodon nuclease by certain pyrimidine nucleotides, the most potent of which is dTTP. The activation by dTTP likewise requires an EcoprrI DNA substrate and GTP hydrolysis but involves a different form of the latent holoenzyme/DNA complex. Moreover, whereas Stp relays its activating effect through EcoprrI, dTTP targets PrrC. The activation of the latent enzyme by a normal cell constituent hints that anticodon nuclease plays additional roles, other than warding off phage T4 infection.

Topics: Amino Acid Sequence; Bacterial Proteins; Bacteriophage T4; DNA Restriction Enzymes; DNA, Bacterial; Enzyme Activation; Enzyme Activators; Escherichia coli; Escherichia coli Proteins; Gene Deletion; Genes, Bacterial; Genetic Complementation Test; Guanosine Triphosphate; Molecular Sequence Data; Mutagenesis, Site-Directed; Pyrimidine Nucleotides; Ribonucleases; RNA, Transfer, Amino Acyl; Site-Specific DNA-Methyltransferase (Adenine-Specific); Thymine Nucleotides; Viral Nonstructural Proteins; Viral Proteins

Topics: Adenosine Triphosphate; Animals; Cytidine Triphosphate; DNA; DNA Fingerprinting; DNA Primers; DNA, Fungal; Electrophoresis, Polyacrylamide Gel; Fungi; Guanosine Triphosphate; Insecta; Nucleotides; Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Thymine Nucleotides

DNA polymerase activity is essential for replication, recombination, repair, and mutagenesis. All DNA polymerases studied so far from any biological source synthesize DNA by the Watson-Crick base-pairing rule, incorporating A, G, C, and T opposite the templates T, C, G, and A, respectively. Non-Watson-Crick base pairs would lead to mutations. In this report, we describe the ninth human DNA polymerase, Pol(iota), encoded by the RAD30B gene. We show that human Pol(iota) violates the Watson-Crick base-pairing rule opposite template T. During base selection, human Pol(iota) preferred T-G base pairing, leading to G incorporation opposite template T. The resulting T-G base pair was less efficiently extended by human Pol(iota) compared to the Watson-Crick base pairs. Consequently, DNA synthesis frequently aborted opposite template T, a property we designated the T stop. This T stop restricted human Pol(iota) to a very short stretch of DNA synthesis. Furthermore, kinetic analyses show that human Pol(iota) copies template C with extraordinarily low fidelity, misincorporating T, A, and C with unprecedented frequencies of 1/9, 1/10, and 1/11, respectively. Human Pol(iota) incorporated one nucleotide opposite a template abasic site more efficiently than opposite a template T, suggesting a role for human Pol(iota) in DNA lesion bypass. The unique features of preferential G incorporation opposite template T and T stop suggest that DNA Pol(iota) may additionally play a specialized function in human biology.

Topics: Base Pairing; DNA Polymerase iota; DNA Replication; DNA-Directed DNA Polymerase; Genetic Code; Guanosine Triphosphate; Humans; Kinetics; Mutagenesis; Substrate Specificity; Templates, Genetic; Thymine Nucleotides

The potassium conductance of the basolateral membrane (BLM) of proximal tubule cells is a critical regulator of transport since it is the major determinant of the negative cell membrane potential and is necessary for pump-leak coupling to the Na+,K+-ATPase pump. Despite this pivotal physiological role, the properties of this conductance have been incompletely characterized, in part due to difficulty gaining access to the BLM. We have investigated the properties of this BLM K+ conductance in dissociated, polarized Ambystoma proximal tubule cells. Nearly all seals made on Ambystoma cells contained inward rectifier K+ channels (gammaslope, in = 24.5 +/- 0.6 pS, gammachord, out = 3.7 +/- 0.4 pS). The rectification is mediated in part by internal Mg2+. The open probability of the channel increases modestly with hyperpolarization. The inward conducting properties are described by a saturating binding-unbinding model. The channel conducts Tl+ and K+, but there is no significant conductance for Na+, Rb+, Cs+, Li+, NH4+, or Cl-. The channel is inhibited by barium and the sulfonylurea agent glibenclamide, but not by tetraethylammonium. Channel rundown typically occurs in the absence of ATP, but cytosolic addition of 0. 2 mM ATP (or any hydrolyzable nucleoside triphosphate) sustains channel activity indefinitely. Phosphorylation processes alone fail to sustain channel activity. Higher doses of ATP (or other nucleoside triphosphates) reversibly inhibit the channel. The K+ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does not alleviate the inhibition of millimolar doses of ATP. We conclude that this K+ channel is the major ATP-sensitive basolateral K+ conductance in the proximal tubule.

Topics: Adenosine Triphosphate; Ambystoma; Animals; Barium; Biological Transport; Cations; Cells, Cultured; Cytidine Triphosphate; Diazoxide; Diuretics; Electric Conductivity; Glyburide; Guanosine Triphosphate; Hypoglycemic Agents; Inosine Triphosphate; Ion Channel Gating; Kidney Tubules, Proximal; Kinetics; Microvilli; Patch-Clamp Techniques; Potassium Channels; Sodium Chloride Symporter Inhibitors; Sodium-Potassium-Exchanging ATPase; Thallium; Thymine Nucleotides; Uridine Triphosphate

We have reported that the deoxycytidine analog 2',2'difluoro-2'-deoxycytidine (dFdCyd) is a potent radiosensitizer of HT29 human colon cancer cells probably through its effects on intracellular deoxyribonucleotide (dNTP) pools. Because dFdCyd has activity against pancreatic cancer in clinical trials, we wished to determine if dFdCyd would radiosensitize human pancreatic cancer cells.. We assessed the effect of dFdCyd on radiation sensitivity of two human pancreatic cancer cell lines, Panc-1 and BxPC-3. To begin to investigate the mechanism of sensitization, we determined the effect of dFdCyd on dNTP pools and cell cycle distribution.. We found that dFdCyd produced radiation enhancement ratios of 1.7-1.8 under noncytotoxic conditions in both cell lines. Sensitization was not associated with intracellular levels of 2',2'-difluoro-2'-deoxycytidine triphosphate, the cytotoxic metabolite of dFdCyd, but occurred when dATP pools were depleted below the level of approximately 1 micromolar. Although both cell lines showed substantial cell cycle redistribution after drug treatment, the flow cytogram of the BxPC-3 cells would not, by itself, be anticipated to result in increased radiation sensitivity.. These findings demonstrate that dFdCyd is a potent radiation sensitizer of human pancreatic cancer cells and support the development of a clinical protocol using combined dFdCyd and radiation therapy in the treatment of pancreatic cancer.

Topics: Adenosine Triphosphate; Cell Cycle; Cell Survival; Cytidine Triphosphate; Deoxycytidine; Drug Screening Assays, Antitumor; Gemcitabine; Guanosine Triphosphate; Humans; Pancreatic Neoplasms; Radiation-Sensitizing Agents; Thymine Nucleotides; Tumor Cells, Cultured

We have synthesized 3'-substituted-2'-deoxyribonucleotide-5'-triphosphates corresponding to A, T, G and C. The 3' position was esterified by a separate anthranylic derivative (3'-tag) giving specific fluorescent properties to each nucleotide (nt). These nt acted as substrates with several DNA polymerases leading to chain termination. Upon alkali or enzymatic treatment of the terminated DNA chain, free 3'-hydroxyl groups were recovered and found able to undergo chain extension when incubated with a mixture of dNTPs and a DNA polymerase. Because each tag has different fluorescent properties in itself, i.e., as a free acid, it theoretically is possible, after removal and characterization of the tag, to infer which nt has been inserted. Reiteration of the process can then be used to determine a nt sequence with a non-gel-based method amenable to automation.

Topics: Adenosine Triphosphate; Base Sequence; Cytidine Triphosphate; Deoxyribonucleotides; DNA-Directed DNA Polymerase; Fluorescent Dyes; Guanosine Triphosphate; Molecular Sequence Data; ortho-Aminobenzoates; RNA-Directed DNA Polymerase; Sequence Analysis, DNA; Thymine Nucleotides

1H- and 31P-n.m.r. have been used to study the interaction of the bacterial chemotaxis protein, CheY, with ATP and a variety of other phosphates in the presence and absence of bivalent metal ions. In the metal-bound conformation, CheY will bind nucleotide phosphates and phosphates in general, while in the metal-free conformation CheY loses its affinity for phosphates. In the presence of low concentrations of nitroxide-spin-labelled ATP (SL-ATP), specific proton resonances of metal-bound CheY are suppressed, indicating that ATP binds to a specific site on this metal-bound form of the protein. These studies also show that the same resonances are affected by the binding of SL-ATP and Mn2+, indicating that the phosphate- and metal-binding sites are close to each other and to Asp-57 (the site of phosphorylation in CheY). 1H- and 31P-n.m.r. studies using ATP, GTP, TTP, UTP, ADP, AMP and inorganic phosphates show that the binding is not specific for adenine, and does not involve the base directly, but is mediated primarily by the phosphate groups. Experiments with a phosphorylation mutant (Asp-13-->Asn) suggest that the observed phosphate binding and activation of CheY by phosphorylation may be related. Our results indicate that the conformational change and charge interactions brought about by the binding of a metal ion at the active site are required for CheY to interact with a phosphate. These studies also demonstrate the utility of spin-label-induced relaxation in conjunction with two-dimensional-n.m.r. measurements for exploring ligand-binding sites.

Topics: Adenine; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Bacterial Proteins; Binding Sites; Guanosine Triphosphate; Kinetics; Magnetic Resonance Spectroscopy; Membrane Proteins; Methyl-Accepting Chemotaxis Proteins; Nucleotides; Phosphates; Phosphoric Monoester Hydrolases; Phosphorus; Phosphorylation; Protein Binding; Protons; Sensitivity and Specificity; Thymine Nucleotides; Uridine Triphosphate

Deoxynucleoside 5'-triphosphates (dNTPs) can be determined in cell extracts by high performance liquid chromatography after prior selective degradation of ribonucleoside 5'-triphosphates with sodium periodate and methylamine. When the method is used for the evaluation of deoxynucleoside triphosphates in 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP)-containing cell extracts, an additional peak coeluting with thymidine triphosphate (dTTP) is observed. This peak is due to the formation of a carboxylic acid derivative of ara-CTP by periodate oxidation, and it can lead to considerable overestimation of dTTP. Formation of this peak can be avoided by using alkaline reaction conditions (pH 7.5) and by changing the sequence of addition of the reagents used in the periodation procedure. By employing this modified protocol, cellular dNTP and ara-CTP levels can be monitored in extracts of leukaemic blasts during cytosine arabinoside treatment in two separate HPLC runs.

Topics: Adenosine Triphosphate; Arabinofuranosylcytosine Triphosphate; Cell Line; Chromatography, High Pressure Liquid; Cytidine Triphosphate; Deoxyribonucleotides; Guanosine Triphosphate; Humans; Leukemia; Ribonucleotides; Thymine Nucleotides; Uridine Triphosphate

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.

Topics: Adenosine Triphosphate; Base Sequence; Binding, Competitive; Cytidine Triphosphate; DNA Helicases; Edetic Acid; Escherichia coli Proteins; Guanosine Triphosphate; HeLa Cells; Humans; Hydrogen-Ion Concentration; Molecular Sequence Data; Molecular Weight; Oligodeoxyribonucleotides; Phosphates; Potassium; Potassium Chloride; Potassium Compounds; RNA; Sodium Chloride; Substrate Specificity; Thymine Nucleotides; Uridine Triphosphate

Direct partition through ultrafiltration was applied to develop a method for the study of nucleotide binding to ribonucleotide reductase from Escherichia coli. The assay involved a 0.5- to 1-min centrifugation step where bound and unbound nucleotides are separated over an ultrafiltration membrane. No effects were seen due to hyperconcentration of protein at the membrane surface. The method was verified by measuring binding of dATP, ATP, dTTP, dGTP, and GDP at 25 and 4 degrees C with dissociation constants ranging from 0.1 to 80 microM. The results were in good agreement with earlier data obtained by other techniques and extend our knowledge in the case of ATP and dGTP binding at 25 degrees C.

Topics: Adenosine Triphosphate; Bacterial Proteins; Centrifugation; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Escherichia coli; Guanosine Triphosphate; Kinetics; Nucleotides; Protein Binding; Proteins; Ribonucleotide Reductases; Thymine Nucleotides; Ultrafiltration

E. coli conditional iron-containing ribonucleotide reductase (Fe-RR) mutant and wild type strains grew anaerobically under conditions when Fe-RR was absent or inhibited. Furthermore, a B12-independent, hydroxyurea-resistant RR activity, unaffected by monoclonal antibodies against either subunit B1 or B2 of Fe-RR, was partially purified from anaerobically grown mutant and wild-type E. coli. These findings indicate that E. coli has a second RR representative of a new class of RRs and that this is the first report where both in vivo and in vitro evidence is presented. It is probable that other facultative anaerobes also have two different RRs such that an optimal supply of deoxyribonucleotides is maintained under all growth conditions.

Topics: Anaerobiosis; Antibodies, Monoclonal; Escherichia coli; Guanosine Triphosphate; Hydroxyurea; Ribonucleotide Reductases; Thymine Nucleotides

Pertussis toxin is composed of an enzymatically active A subunit and a binding component (B oligomer). Both the holotoxin and the isolated A subunit have previously been shown to exhibit NAD glycohydrolase activity although the A subunit is more active on a molar basis than the holotoxin. We have investigated the mechanism by which ATP stimulates the activity of this toxin. Since dissociation of pertussis toxin subunits would result in increased NAD glycohydrolase activity, the ability of ATP to promote release of the A subunit from the B oligomer was examined. In the presence of the zwitterionic detergent 3-(3-cholamidopropyldimethyl)-1-ammonio)-propanesulfonate, concentrations of ATP as low as 1 microM promoted subunit dissociation. The concentration of ATP required for release of the A subunit was similar to that required for stimulation of NAD glycohydrolase activity. Both ATP and ADP promoted subunit dissociation and stimulated NAD glycohydrolase activity. In contrast, AMP and adenosine did not alter NAD glycohydrolase activity or affect subunit structure. The ability of ATP to decrease the affinity of the A subunit for the B oligomer may play a role in nucleotide stimulation of pertussis toxin activity.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Cholic Acids; Cytidine Triphosphate; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Macromolecular Substances; Molecular Weight; NAD+ Nucleosidase; Pertussis Toxin; Thymine Nucleotides; Virulence Factors, Bordetella

Topics: Adenosine Triphosphate; Brevibacterium; Cations, Divalent; DNA Replication; Enzyme Activation; Guanosine Triphosphate; Manganese; Micrococcus; Ribonucleotide Reductases; 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