adenosine-5--o-(3-thiotriphosphate) and 2--deoxyadenosine-triphosphate

Escherichia coli RecA protein forms a right-handed helical filament with DNA molecules and has an ATP-dependent activity that exchanges homologous strands between single-stranded DNA (ssDNA) and duplex DNA. We show that the RecA-ssDNA filamentous complex is an elastic helical molecule whose length is controlled by the binding and release of nucleotide cofactors. RecA-ssDNA filaments were fluorescently labelled and attached to a glass surface inside a flow chamber. When the chamber solution was replaced by a buffer solution without nucleotide cofactors, the RecA-ssDNA filament rapidly contracted approximately 0.68-fold with partial filament dissociation. The contracted filament elongated up to 1.25-fold when a buffer solution containing ATPgammaS was injected, and elongated up to 1.17-fold when a buffer solution containing ATP or dATP was injected. This contraction-elongation behavior was able to be repeated by the successive injection of dATP and non-nucleotide buffers. We propose that this elastic motion couples to the elastic motion and/or the twisting rotation of DNA strands within the filament by adjusting their helical phases.

Topics: Adenosine Triphosphate; Buffers; Deoxyadenine Nucleotides; DNA-Binding Proteins; DNA, Single-Stranded; Elasticity; Escherichia coli Proteins; Models, Biological; Rec A Recombinases

The crystal structures of Mycobacterium smegmatis RecA (RecA(Ms)) and its complexes with ADP, ATPgammaS, and dATP show that RecA(Ms) has an expanded binding site like that in Mycobacterium tuberculosis RecA, although there are small differences between the proteins in their modes of nucleotide binding. Nucleotide binding is invariably accompanied by the movement of Gln 196, which appears to provide the trigger for transmitting the effect of nucleotide binding to the DNA-binding loops. These observations provide a framework for exploring the known properties of the RecA proteins.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Binding Sites; Crystallography, X-Ray; Deoxyadenine Nucleotides; Models, Molecular; Mycobacterium smegmatis; Nucleotides; Protein Conformation; Rec A Recombinases

Transcription initiation by RNA polymerase II is a complex, multistep process that minimally involves transcription complex assembly, open complex formation, and promoter clearance. Hydrolysis of the beta--gamma phosphoanhydride bond of ATP has previously been shown to be required for open complex formation, as well as for the phosphorylation of the carboxy-terminal domain of the largest subunit of RNA polymerase II. The observation that ATP-dependent activation of transcription complexes can be blocked by ATP analogues that contain nonhydrolyzable beta--gamma phosphoanhydride bonds (such as ATPgammaS) was exploited to develop a functional kinetic assay for ATP-activated transcription complexes. Activated complexes on the promoter present in the long terminal repeat of the proviral DNA of mouse mammary tumor virus were defined as those that could productively initiate transcription in the presence of excess ATPgammaS. Activation is dependent on treatment of assembled preinitiation complexes with ATP (or dATP) prior to addition of ATPgammaS. At least 15-35% of the total number of preinitiation complexes present become activated within 2 min in the presence of (d)ATP, and activation appears to be rapidly reversible. The time course of formation of activated complexes in the presence of dATP is characterized by two kinetic phases: a rapid formation followed by a relatively slow decay. Activated complexes were estimated to form with a half-time of less than 1 min.

Topics: Adenosine Triphosphate; Cell Nucleus; Deoxyadenine Nucleotides; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Stability; Half-Life; HeLa Cells; Humans; Kinetics; Mammary Tumor Virus, Mouse; Promoter Regions, Genetic; RNA Polymerase II; Sarcosine; Transcription, Genetic; Transcriptional Activation

Adenosine tri- and diphosphate (ATP and ADP) and their structural analogues stimulate insulin secretion from the isolated perfused rat pancreas, an effect mediated by P2Y-purinoceptor activation. Concerning the base moiety of the nucleotide, it was previously shown that purine but not pyrimidine nucleoside triphosphates were active and that substitution on purine C2 with the 2-methylthio group greatly enhanced the potency. In this study, we further analyze the consequences of ribose and polyphosphate chain modifications. Modifications in 2' and 3' position on the ribose led to a decrease in insulin response when bulky substitutions were made: indeed, 2'-deoxy ATP was similar in activity to ATP, whereas arylazido-aminopropionyl ATP (ANAPP3) was weakly effective and trinitrophenyl ATP (TNP-ATP) was inactive. Substitution on the gamma phosphorus of the triphosphate chain led to a decrease (gamma-anilide ATP) or no change (gamma-azido ATP) in potency; the replacement of the bridging oxygen between beta and gamma phosphorus by a peroxide group did not significantly change the activity, whereas substitution by a methylene group completely abolished stimulation of insulin secretion. As for the phosphorothioate analogues, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) induced an insulin response similar to that produced by ATP, whereas adenosine-5'-O-(2-thiodiphosphate) (ADP beta S) was about 100-fold more potent than ATP, as previously shown. In conclusion, two structural features seem to have a strategic importance for increasing the insulin secretory activity of ATP analogues: substitution at the C2 position on the adenine ring of ATP and modifications of the polyphosphate chain at the level of the beta phosphorus.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Area Under Curve; Azides; Deoxyadenine Nucleotides; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; Pancreas; Polyphosphates; Rats; Receptors, Purinergic P2; Ribose; Structure-Activity Relationship; Thionucleotides

The functions of the 70 kDa heat-shock proteins (hsp70s) are regulated by their bound nucleotide. We previously observed major differences in the effect of bound ATP and ADP on the interaction of hsc70 (constitutive hsp70) with its protein substrates. In the present study, we investigated the interaction of protein substrates with nucleotide-free hsc70 and with hsc70 with bound ATP analogues. We found, first, that nucleotide-free hsc70 appeared to interact differently with different substrates. Specifically, nucleotide-free hsc70 behaved much more like hsc70-ATP than hsc70-ADP in that clathrin very rapidly bound to and dissociated from nucleotide-free hsc70 in contrast to its very slow binding to and dissociation from hsc70-ADP. On the other hand, nucleotide-free hsc70 behaved more like hsc70-ADP than hsc70-ATP in that cytochrome c peptide dissociated very slowly from nucleotide-free hsc70 compared to its rapid dissociation from hsc70-ATP. Second, binding of the ATP analogues AMP-PNP, dATP, and ATP gamma S to nucleotide-free hsc70 had very little further effect on the properties of the nucleotide-free hsc70. Therefore, previously observed effects of ATP analogues may have been due to removal of the bound ADP rather than to the presence of analogues.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Binding Sites; Carrier Proteins; Cattle; Clathrin; Cytochrome c Group; Deoxyadenine Nucleotides; HSC70 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; In Vitro Techniques; Kinetics; Nucleotides; Peptides; Protein Binding

Topics: Adenosine Triphosphate; Bacteriophage T4; Cross-Linking Reagents; Deoxyadenine Nucleotides; DNA Polymerase I; DNA Probes; DNA Replication; DNA-Binding Proteins; DNA-Directed DNA Polymerase; DNA, Viral; Escherichia coli; Indicators and Reagents; Photochemistry; Promoter Regions, Genetic; Trans-Activators; Transcription, Genetic; Ultraviolet Rays; Uridine Triphosphate; Viral Proteins

Eukaryotic initiation factor 2 (eIF-2) is shown to bind ATP with high affinity. Binding of ATP to eIF-2 induces loss of the ability to form a ternary complex with Met-tRNAf and GTP, while still allowing, and even stimulating, the binding of mRNA. Ternary complex formation between eIF-2, GTP, and Met-tRNAf is inhibited effectively by ATP, but not by CTP or UTP. Hydrolysis of ATP is not required for inhibition, for adenyl-5'-yl imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP, is as active an inhibitor; adenosine 5'-O-(thiotriphosphate) (ATP gamma S) inhibits far more weakly. Ternary complex formation is inhibited effectively by ATP, dATP, or ADP, but not by AMP and adenosine. Hence, the gamma-phosphate of ATP and its 3'-OH group are not required for inhibition, but the beta-phosphate is indispensible. Specific complex formation between ATP and eIF-2 is shown 1) by effective retention of Met-tRNAf- and mRNA-binding activities on ATP-agarose and by the ability of free ATP, but not GTP, CTP, or UTP, to effect elution of eIF-2 from this substrate; 2) by eIF-2-dependent retention of [alpha-32P]ATP or dATP on nitrocellulose filters and its inhibition by excess ATP, but not by GTP, CTP, or UTP. Upon elution from ATP-agarose by high salt concentrations, eIF-2 recovers its ability to form a ternary complex with Met-tRNAf and GTP. ATP-induced inhibition of ternary complex formation is relieved by excess Met-tRNAf, but not by excess GTP or guanyl-5'-yl imidodiphosphate (GMP-PNP). Thus, ATP does not act by inhibiting binding of GTP to eIF-2. Instead, ATP causes Met-tRNAf in ternary complex to dissociate from eIF-2. Conversely, affinity of eIF-2 for ATP is high in the absence of GTP and Met-tRNAf (Kd less than or equal to 10(-12) M), but decreases greatly in conditions of ternary complex formation. These results support the concept that eIF-2 assumes distinct conformations for ternary complex formation and for binding of mRNA, and that these are affected differently by ATP. Interaction of ATP with an eIF-2 molecule in ternary complex with Met-tRNAf and GTP promotes displacement of Met-tRNAf from eIF-2, inducing a state favorable for binding of mRNA. ATP may thus regulate the dual binding activities of eIF-2 during initiation of translation.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Binding Sites; Binding, Competitive; Deoxyadenine Nucleotides; Eukaryotic Initiation Factor-2; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Macromolecular Substances; Penicillium chrysogenum; Protein Biosynthesis; Rabbits; RNA, Double-Stranded; RNA, Messenger; RNA, Transfer, Amino Acyl

Bacteriophage-T4 and human type I DNA ligases were found capable of self-adenylating upon exposure to both ribo- and deoxyribo-[alpha-35S]thio-ATP. However, the joining reaction does not take place in the presence of the deoxyribotriphosphates. Enzyme adenylation is reversed in all cases by an excess of PPi, but the rate of reversion is lower with thio derivatives. Therefore thio derivatives can be used to study the adenylation of DNA ligases and to search for specific inhibitors of the first step of the ligation reaction. In addition we show that thio derivatives can be used to detect DNA ligase adenylation activity covalently bound to a solid matrix.

Topics: Adenosine Triphosphate; Antibiotics, Antineoplastic; Collodion; Deoxyadenine Nucleotides; DNA Ligases; HeLa Cells; Humans; Phosphates; T-Phages; Thionucleotides

The physical basis of ATP binding and activation of DNA polymerase III holoenzyme was studied by an ultraviolet irradiation cross-linking technique. ATP and dATP were photocrosslinked to the alpha, tau, gamma, and delta subunits of holoenzyme; photocrosslinking of dATP was competitively inhibited by ATP. No photocrosslinking was observed with GTP or CTP, nor did GTP, CTP, or UTP inhibit cross-linking of ATP. ADP and adenosine 5'-O-(3-thio)-triphosphate, both potent inhibitors of ATP activation of holoenzyme, inhibited cross-linking of ATP to tau, gamma, and delta subunits, but not to the alpha subunit, suggesting that one or more of these subunits are ATP (or dATP)-binding sites. Photocrosslinking of dTTP to the ATP-activated holoenzyme was exclusively to the epsilon subunit, the dnaQ ( mutD ) gene product; dCTP and dGTP were not photocrosslinked to any subunit. Binding of dTTP was enhanced by ATP, but by no other nucleotide (or deoxynucleotide). This binding of dTTP to epsilon, a subunit likely responsible for regulation of proofreading by the holoenzyme, may function in the control of the fidelity of replication.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Affinity Labels; Binding Sites; Deoxyadenine Nucleotides; DNA Polymerase III; DNA-Directed DNA Polymerase; Escherichia coli; Photochemistry; Thionucleotides; Thymine Nucleotides; Ultraviolet Rays