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PubMedDate not supplied

[Termination of DNA transcription by a poly(dA).poly(dT) fragment inserted into pBR325 plasmid].

A recombinant DNA was constructed by inserting polynucleotide (dA).(dT) of 80-100 base pairs long into EcoRI site of the pBR325 plasmid DNA. Transcription of this DNA was studied in E. coli RNA polymerase system in vitro. Some transcripts obtained with the recombinant plasmid were shown to have poly(U) clusters at the 3'-ends. Obtained data indicate that poly(dA).poly(dT) sequence acts as a terminator of RNA synthesis. Orientation of this sequence in recombinant DNA was also established.

Base Composition↗
PubMed1982

Stereochemical course of polymerization catalyzed by avian myeloblastosis virus reverse transcriptase.

The Sp diastereomer of thymidine 5'-O-(1-thiotriphosphate) was polymerized by avian myeloblastosis virus reverse transcriptase using poly(A) . d(pT)10 as template-primer. Degradation of the template poly(A) by alkaline hydrolysis and isolation by gel chromatography gave a single-stranded poly(d(p(S)T)), a polymer of thymidine 5'-phosphorothioate. To determine the configuration of the phosphorothioate internucleotide linkage, this material was degraded by snake venom phosphodiesterase. Comparison of the rates of degradation by snake venom phosphodiesterase of poly(d(p(S)T)) prepared by reverse transcriptase and DNA polymerase I showed them to be very similar. Since it has been established earlier than the latter enzyme produces polymers with phosphorothioate linkages of the Rp configuration (Burgers, P. M. J., and Eckstein, F. (1979) J. Biol. Chem. 254, 6889-6893), it is concluded that the polymer produces by reverse transcriptase has the same stereochemistry. Further proof for this assignment comes from comparison by 31P nmr of this polymer with the diastereomers of synthetic 5'-O-thymidyl 3'-O-thymidyl phosphorothioate. The chemical shift observed for the polymer was identical with that of the Rp isomer of 5'-O-thymidyl 3'-O-thymidyl phosphorothioate. Avian myeloblastosis virus reverse transcriptase therefore polymerizes deoxynucleoside 5'-triphosphates with inversion of configuration at the alpha-phosphorus. This result indicates that direct nucleophilic attack by the 3-hydroxyl group of the growing polymer on the alpha-phosphoryl group occurs without formation of a covalent enzyme intermediate.

Avian Leukosis Virus↗
PubMedDate not supplied

[Effect of the protein product of phage f1, gene 5, on heat denaturation of synthetic polynucleotides].

The influence of phage f1 gene 5 protein on melting of the synthetic polynucleotides has been investigated, using UV-spectroscopy. In our experiments we have varied the proteins concentration. It has been shown, that the protein lowers the melting temperature of the studied polynucleotides (d/A--Tn dAndTn, rAndTn, rAn.r n, dAn.rn). The melting temperatures and the shapes of melting curves of various polynucleotides differ when the same protein concentrations are used. We have shown that the protein binds to the double-stranded polynucleotides, containing ribo-ribo-, deoxyribo-ribo-chains. The difference in melting temperatures and shapes of melting curves was explained using the data about the differences in the secondary structure of these polynucleotides. Only for d/A-Tn renaturation was observed after sample cooling. It may reflect the single-stranded hairpin structure of this polynucleotide.

Bacteriophages↗
PubMed1980

Mechanism of primer-template-dependent conversion of dNTP leads to dNMP by T5 DNA polymerase.

T5 DNA polymerase catalyzes both 5' leads to 3' polymerization and 3' leads to 5' hydrolysis in a processive fashion. This knowledge has been utilized to obtain evidence indicating that the enzyme has a single primer-template binding site which can function as either polymerase or exonuclease, perhaps with the cooperation of additional or different side groups. Template-dependent conversion of dNTP leads to dNMP was observed with an excess of either primer-template or enzyme. With primer-template excess, practically all the enzymes were functional as polymerase; with enzyme excess, all primer-templates were extended during the first cycle of catalysis. These observations suggest that turnover takes place at the points of chain growth. Evidence is also provided which demonstrates that the enzyme is capable of switching its direction of catalysis from 3' leads to 5' to 5' leads to 3' without leaving the primer-template. A clear correspondence between the relative amount of hydrolysis of a terminally labeled residue on the primer and the relative amount of turnover suggests that (a) the probability of hydrolysis of a given type of residue in contact with the "active site" is constant, and (b) during each turnover episode enzyme usually takes only one step in the 3' leads to 5' direction. A simple probabilistic model of turnover is discussed.

DNA-Directed DNA Polymerase↗
PubMed1980

Deoxyuridine triphosphate pools after polyoma virus infection.

The synthesis of polyoma DNA in virus-infected 3T6 mouse fibroblasts is discontinuous with the intermediate formation of short Okazaki fragments. Hydroxyurea, an inhibitor of the enzyme ribonucleotide reductase, inhibits polyoma DNA synthesis, as measured by incorporation of radioactive thymidine. In the inhibited state, almost all incorporation occurs into short fragments. We investigated to what extent formation of short DNA fragments might be the result of incorporation of deoxyuridine triphosphate (dUTP) into DNA, followed by excision and repair reactions. We devised a sensitive enzymatic method for measuring dUTP in cell extracts which allows the determination of the dUTP pool when this pool amounts to between 0.1 and 2% of the dTTP pool. No dUTP was detected in growing mouse fibroblasts. After infection with polyoma virus cell extracts contained 0.4% dUTP (of dTTP) at the peak of DNA synthesis. Addition of hydroxyurea at this point led to a disappearance of dUTP. We conclude that dUTP incorporation can contribute only minimally to the generation of short fragments during polyoma DNA synthesis.

Animals↗
PubMed1982

Template-prime-dependent turnover of (Sp)-dATP alpha S by T4 DNA polymerase. The stereochemistry of the associated 3' goes to 5'-exonuclease.

T4 DNA polymerase converts (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1-18O2]triphosphate) to 2'-deoxyadenosine 5'-O-[18O]-phosphorothioate in the presence of poly(d(A-T).poly(d(A-T)) template-primer. Control experiments involving either omitting the poly(d(A-T)).poly(d(A-T) template-primer or employing the (Rp)-2'-deoxyadenosine 5'-O-(1-thiotriphosphate) diastereomer showed no reaction. It is assumed, therefore, that this conversion as in the P--O case involves incorporation of the thionucleotide into the poly(d(A-T)) followed by hydrolysis resulting from the 3' goes to 5'-exonuclease activity. The 2'-deoxyadenosine 5'-O-[18O] phosphorothioate was converted to (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1-18O]triphosphate), with no change in the configuration at P alpha by using the coupled adenylate kinase-pyruvate kinase enzyme system. A 31P NMR spectrum of the product showed that the 18O was entirely in the nonbridging position, indicating an overall retention in the net turnover process (i.e. incorporation followed by excision). Since the incorporation process involves an inversion of configuration around the phosphorus (Romaniuk, P. J., and Eckstein, F. (1982) J. Biol. Chem. 257, 7684-7688), it must be inferred that the 3' goes to 5'-exonuclease activity of T4 polymerase proceeds with inversion of configuration at the phosphorus atom, most simply via a direct displacement mechanism. This finding represents the first example of phosphodiester hydrolysis catalyzed by an exonuclease that does not involve a covalent phosphoryl-enzyme intermediate (Knowles, J. R. (1980) Annu. Rev. Biochem. 49, 877-919).

DNA-Directed DNA Polymerase↗
PubMed1983

Pure yeast RNA polymerase B (II) initiates transcription at specific points on supercoiled yeast DNA.

Pure yeast RNA polmymerase B (II) can selectively initiate abortive transcription on a supercoiled recombinant plasmid DNA carrying yeast DNA in the presence of low concentrations of ribonucleoside triphosphates and Mn2+. Five major products ranging between 60 and 150 nucleotides were characterized by hybridization. Three of them originate from the vector pBR322 and two from the yeast DNA insert. Based on a RNA primer extension reaction with recombinant M13 DNAs as template, a method allowing the mapping of the short abortive RNA products has been developed. An initiation site within the yeast DNA insert has thus precisely been mapped. The DNA sequence in this region was determined and showed several relevant features. The in vitro initiation site is preceded by a potential TATATATA box at -40 base pairs and at -105 by the sequence GTTAATCT similar to the consensus sequence GCTCAATCT usually found around 80 base pairs upstream from the cap site. Large blocks of alternated purine pyrimidine residues are found in this region as for several known yeast promotors. The 5' end of the RNA initiated from this site contains several potential signals for the initiation of translation. The possibility that a B to Z transition of DNA could be important for the interaction of the RNA polymerase with its template is discussed.

Base Sequence↗
PubMed1977

Characterization of a new reverse transcriptase of possibly cellular origin in the chicken system.

The properties of an RNA-dependent DNA polymerase, which occurs ubiquitously in the allantoic fluid of uninfected, leukosis-virus-free eggs, are described. It is shown that the enzyme can synthesize faithful transcripts from natural RNA (globin mRNA). With biochemical and immunological methods, the enzyme can be clearly distinguished from the reverse transcriptases of the known chicken RNA tumour viruses and therefore seems to be a member of a so far unknown class of chicken polymerases. Our data show that in the chicken system reverse transcriptase can occur without connection to the replication of RNA tumour viruses and without relationship to the induction of malignancy.

Animals↗
PubMed1977

On the fidelity of DNA replication. Effect of metal activators during synthesis with avian myeloblastosis virus DNA polymerase.

The effect of metal activators on the fidelity of DNA synthesis has been examined. Using the DNA polymerase from avian myeloblastosis virus, the accuracy of Co2+-, M2+-, and Ni2+-activated DNA synthesis was determined with different polynucleotide templates. With poly[d(A-T)] as the template, the error frequency for dCMP incorporation was 1:1400, 1:1100, and 1:600 for Mg2+, Co2+, and Mn2+, respectively, at maximally activating concentrations. The error frequency was invariant with respect to [Mg2+] but increased with greater than activating concentrations of Co2+ and Mn2+. This increase resulted from differential rates of complementary and noncomplementary nucleotide incorporation. The enhanced error frequency was nonspecific as it occurred with all polynucleotide templates and with all noncomplementary deoxy- and ribonucleotides which were tested. Nearest neighbor analyses of the reaction products indicated that the noncomplementary deoxynucleotides were incorporated as single base substitutions. The fidelity of Ni2+-activated DNA synthesis was invariant with respect to [Ni2+] and was similar to that obtained using Mg2+. During DNA synthesis with Mg2+, the addition of Co2+, Mn2+, or Ni2+ resulted in a decrease in the fidelity of DNA synthesis. The relationship between decreases in the fidelity of DNA synthesis and metal mutagenesis, or carcinogenesis, or both, is considered.

Avian Myeloblastosis Virus↗
PubMed1987

O-Alkyl deoxythymidines are recognized by DNA polymerase I as deoxythymidine or deoxycytidine.

The O2- and O4-methyldeoxythymidine triphosphates (O-alkyl dTTP) can be used to substitute for dTTP in Escherichia coli DNA polymerase I (Pol I)-catalysed synthesis of poly[deoxyadenosine-deoxythymidine] (dA-dT). When incorporated into the polynucleotide, no detectable perturbation of structure occurred with even 20% O-methyldeoxythymidine in place of dT. However, on replication of such polymers with Pol I, significant amounts of deoxyguanosine triphosphate (dGTP) were incorporated, as well as high levels of deoxyadenosine triphosphate (dATP), indicating tautomer-like behaviour. Higher homologues, such as O4-ethyl (e4) dTTP or O4-isopropyl (ip4) dTTP, could also replace dTTP, but with lower efficiency. Nevertheless, their presence, like O4-methyl (m4) dT substitutions, caused transitions as well as inhibiting enzyme digestion with a variety of 3' nucleases, particularly to the 3'----5' exonuclease activity (proofreading) of polymerases. Further proof of mutagenicity comes from site-directed experiments placing m4dT or e4dT in place of dT at position 587 in am3 of phi X174, in which all revertants sequenced had A----G transitions. This implies that, since m4dT and e4dT are poorly repaired in eukaryotes, it is likely that they will remain in the DNA and lead to effects on enzyme activity, as well as mutations which contribute to the carcinogenicity of N-nitroso compounds.

Alkylation↗
PubMed1987

Catalytic activities of synthetic octadeoxyribonucleotides as coenzymes of poly(ADP-ribose) polymerase and the identification of a new enzyme inhibitory site.

The catalytic activity of highly purified poly(ADP-ribose) polymerase was determined at constant NAD+ concentration and varying concentrations of sDNA or synthetic octadeoxyribonucleotides of differing composition. The coenzymic activities of deoxyribonucleotides were compared in two ways: graphic presentation of the activation of poly(ADP-ribose) polymerase in the presence of a large concentration range of deoxyribonucleotides and by calculating kD values for the deoxyribonucleotides. As determined by method i, auto-mono-ADP-ribosylation of the enzyme protein at 25 nM NAD+ was maximally activated at 1:1 octamer/enzyme molar ratios by the octadeoxyribonucleotide derived from the regulatory region of SV40 DNA (duplex C). At a 0.4:1 sDNA/enzyme ratio, sDNA was the most active coenzyme for mono-ADP-ribosylation. At 200 microM NAD+, resulting in polymer synthesis and with histones as secondary polymer acceptors, duplex C was the most active coenzyme, and the octamer containing the steroid hormone receptor binding consensus sequence of DNA was a close second, whereas sDNA exhibited an anomalous biphasic kinetics. sDNA was effective on mono-ADP-ribosylation at a concentration 150-200 -times lower than on polymer formation. When comparison of deoxyribonucleotides was based on method ii (kD values), by far the most efficiently binding coenzyme for both mono and polymer synthesis was sDNA, followed by duplex C, with (dA-dT)8 exhibiting the weakest binding. The synthetic molecule 6-amino-1,2-benzopyrone (6-aminocoumarin) competitively inhibited the coenzymic function of synthetic octadeoxyribonucleotides at constant concentration of NAD+, identifying a new inhibitory site of poly(ADP-ribose) polymerase.

ADP Ribose Transferases↗
PubMed1990

Molecular recognition in the minor groove of the DNA helix. Studies on the synthesis of oligonucleotides and polynucleotides containing 3-deaza-2'-deoxyadenosine. Interaction of the oligonucleotides with the restriction endonuclease EcoRV.

An improved procedure for the preparation of 3-deaza-2'-deoxyadenosine (d3CA) is described which is suitable for the synthesis of gram quantities of this analogue. Using phosphoramidite chemistry d3CA has been incorporated into the Eco RV restiction endonuclease recognition sequence (underlined) present in the self-complementary dodecamer d(GACGATATCGTC). The modified oligonucleotides have been thoroughly characterised by nucleoside composition analysis, circular dichroism and thermal melting studies. Studies with Eco RV show that incorporation of d3CA into either the central or outer dA-dT base-pair results in a substantial reduction in the rate of cleavage. The two-step conversion of d3CA to 3-deaza-2'-deoxyadenosine-5'-O-triphosphate (d3CATP) via the 5'-O-tosylate is also described. d3CATP is not a substrate in the poly[d(AT)].poly[d(AT)] primed polymerisation for either E. coli DNA polymerase I or Micrococcus luteus DNA polymerase. In a more detailed kinetic analysis d3CATP was shown to be a competitive inhibitor of E. coli DNA polymerase I with respect to dATP.

Adenosine Triphosphate↗