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PubMed1989

Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. II. Phosphorylation by phage T4 polynucleotide kinase.

Phage T4 polynucleotide kinase (EC 2.7.1.78) proved incapable of catalyzing the phosphorylation of thymidylyl-(3'----5')-thymidine containing either a cis-syn-cyclobutane pyrimidine dimer (d-T less than p greater than T) or a 6-4'-[pyrimidin-2'-one]pyrimidine photoproduct (d-T[p]-T), and similarly the UV-modified compounds of (dT)3 bearing either photoproduct at their 5'-end (d-T less than p greater than TpT and d-T[p]TpT). In contrast, the 3'-structural isomers of these trinucleotides (d-TpT less than p greater than T and d-TpT[p]T) were phosphorylated at the same rate as the parent compound. These phosphorylatable lesion-containing oligonucleotides are quantitatively released from UV-irradiated poly(dA):poly(dT) by enzymatic hydrolysis with snake venom phosphodiesterase and alkaline phosphatase (Liuzzi, M., Weinfeld, M., and Paterson, M. C. (1989) J. Biol. Chem. 264, 6355-6363). By combining this digestion regimen with phosphorylation by polynucleotide kinase and [gamma-32P]ATP, pyrimidine dimers were quantitated at the fmol level following exposure of poly(dA):poly(dT) and herring sperm DNA to biologically relevant UV fluences. The rate of dimer induction in the synthetic polymer, approximately 10 dimers/10(6) nucleotides/Jm-2, was in close agreement with that obtained by conventional methods. Dimers were induced at one-fourth of this rate in the natural DNA. Further treatment of the phosphorylated oligonucleotides derived from irradiated herring sperm DNA with nuclease P1 released the labeled 5'-nucleotide, thus permitting analysis of the nearest-neighbor bases 5' to the lesions. We observed a ratio for pyrimidine-to-purine bases of almost 6:1, implicating tripyrimidine stretches as hotspots for UV-induced DNA damage.

Animals↗
PubMed1987

The sera of patients with Klebsiella infections contain a common anti-DNA idiotype (16/6) Id and anti-polynucleotide activity.

In view of recent reports linking Klebsiella pneumoniae with autoimmunity, we have examined the sera of 52 patients with urinary tract infection or septicaemia from this Gram-negative pathogen, for the presence of antibodies to DNA, polynucleotides, cardiolipin and a common anti-DNA idiotype 16/6. Up to 27% of these patients had anti-polynucleotide antibodies detectable, and in 37% the 16/6 idiotype was found. Absorption of the sera of two patients, with no DNA binding, against the Klebsiella polysaccharide K-30 induced a significant fall in both their anti-K30 antibody and 16/6 idiotype levels. Among 52 patients with other Gram negative infections a maximum of 17% and 19% respectively, had anti-DNA antibodies and the 16/6 idiotype present in their serum. In 37 normal controls, the rate of antibody and idiotype detection was 5% or less. The presence of autoantibodies in the serum of patients with Klebsiella infections may be the result of non-specific stimulation due to bacterial polyclonal activation. However, there might also be a specific stimulus triggered by idiotypic cross-reaction between autoantibodies and anti-Klebsiella antibodies.

Adult↗
PubMed1985

Antitemplate effect of polynucleotides and their hybrid complexes.

In continuation of efforts to correlate the antitemplate activities of modified polynucleotides with their structure, and to understand the factors governing both their potency and stability, a group of single-stranded poly(ribo- and deoxyribo-) nucleotides, and the "hybrid" double-stranded complexes were prepared and investigated. The double-stranded hybrid poly(A,hs5U).poly(dT) section was found to be more stable to murine blood nucleases than was the single-stranded poly(A,hs5U). In a comparative study as inhibitors of the DNA polymerase alpha from rat hepatoma, the results showed that the modified polynucleotides were more potent than the unmodified ones, in general, the polydeoxyribonucleotides were better antitemplates than their ribo counterparts and the poly(A70,hs5U30).poly(dT) hybrid was more active than either of the single-stranded components. Thus it is possible to increase the nuclease resistance of the modified polyribonucleotides by forming hybrid complexes with complementary polydeoxyribonucleotides, and at the same time, to augment their antitemplate activities.

Animals↗
PubMed1981

The stereochemical course of thiophosphoryl group transfer catalyzed by T4 polynucleotide kinase.

The stereochemical course of the phosphoryl transfer reaction catalyzed by T4 polynucleotide kinase has been determined using the chiral ATP analog, (Sp)-adenosine-5'-(3-thio-3-[18O]triphosphate). T4 polynucleotide kinase catalyzes the transfer of the gamma-thiophosphoryl group of (Sp)-adenosine-5'-(3-thio-3-[18O]triphosphate) to the 5'-hydroxyl group of ApA to give the thiophosphorylated dinucleotide adenyl-5'-[18O]phosphorothioate-(3'-5')adenosine. A sample of adenyl-5'-[18O]phosphorothioate-(3'-5')adenosine was subjected to venom phosphodiesterase digestion. The resulting adenosine-5'-[18O]phosphorothioate was shown to have the Rp configuration, thus indicating that the thiophosphoryl transfer reaction occurs with overall inversion of configuration of phosphorus.

Adenine Nucleotides↗
PubMed1980

Purification and some properties of polynucleotide kinase from rat liver nuclei.

Polynucleotide kinase was purified from crude extracts of rat liver nuclei by affinity chromatography on DNA agarose. At optimal pH (5.5) and at saturating concentrations of ATP and DNA, the purified enzyme was found to express maximal activity in the presence of 0.10-0.15 M NaCl; higher salt concentrations inhibited the activity. At the optimal pH and NaCl concentration, the apparent KM for 5'-OH-DNA at 100 microM ATP was 46.2 microM and the apparent KM for ATP at 1 mM 5'-OH-DNA was 15.8 microM. Polynucleotide kinase was protected against heat inactivation by ATP as well as by 5'-OH-DNA at low and moderately high NaCl concentrations, which suggests that under these conditions the enzyme reacts according to a random reaction mechanism. Studies on the heat inactivation of the enzyme in the presence of 5'-OH- or 5'-P-DNA revealed the protection occurs only if 5'-OH-DNA is present, at NaCl concentrations permitting the enzyme to bind DNA.

Adenosine Triphosphate↗
PubMed1983

Interaction of purified rat liver glucocorticoid-receptor complexes with polynucleotides: strong base composition dependence.

The binding of 10(3)-fold purified rat liver glucocorticoid receptors to natural and synthetic polynucleotides has been studied. The receptors may bind to RNA as well as to DNA. The binding effectiveness of nucleic acids is strongly base composition dependent, with natural DNAs (double-stranded or denatured) greater than E. coli rRNA greater than poly(dA).poly(dT) greater than or equal to poly d(A,T,G)7 greater than or equal to poly(U) greater than poly(A).poly(U) greater than poly(A) approximately poly(C). The equilibrium (apparent) constants of receptor binding to a number of polynucleotides were calculated by a method proposed.

Animals↗
PubMed1982

Studies on the mechanism of Escherichia coli DNA polymerase I large fragment. Chain termination and modulation by polynucleotides.

Homopolymer replication systems and measurement of precise product chain length have been used to elucidate two new points about the mechanism of Escherichia coli DNA polymerase I large fragment: chain termination as a function of product chain length is multiphasic, and polynucleotides exert a secondary effect in the mechanism of this enzyme. During replication of (dT)800 or (dA)800 with short oligonucleotides as primer, DNA polymerase I large fragment was processive, catalyzing hundreds of dNMP incorporations during each cycle of binding to the template-primer, incorporation, and termination. Our observations indicated, however, that polynucleotides could terminate chain elongation and that this effect probably occurred through interaction at a secondary binding site on the enzyme. Thus, in the presence of higher levels of template-primer, early termination occurred and the relatively short product molecules could be resolved by gel electrophoresis. Incubation conditions were adjusted so that the number of product molecules at each chain length was equal to the actual number of termination events, and, therefore, the statistical chance for termination as a function of product chain length could be calculated. These termination probability values depended upon specific incubation conditions, such as dNTP level and whether the primer was a ribo- or deoxyribonucleotide, and interestingly, the values changed as the chain length of the product increased. For the first 5 to 10 dMP residues added to the primer, termination probability declined with each dNMP addition, but then remained constant for the addition of the next 20 to 40 dNMP residues. These results are discussed in the context of a kinetic model representing two stages of synthesis during the formation of each product molecule.

DNA Polymerase I↗
PubMed1976

The dynamics of development of cell resistance to viruses induced by synthetic polynucleotides.

The dynamics of interaction of complexes of synthetic polynucleotides (polyinosinic and polycitidylic acid--poly (rI)-poly (rC), and polyguanylic and polycytidylic acid--poly (rG)-poly (rC)) with cells as well as the dynamics of interferon accumulation and development of antiviral effect against some RNA viruses were studied in primary chick embryo cell (CEC) cultures. Four phases were observed in the development of the antiviral effect of synthetic polynucleotides: adsorption, increase, marked antiviral effect and waning. The duration and extent of the antiviral effect depended upon the activity and the dose of the preparation and less so upon virus type. At the same time, the dynamics of the development of the antiviral effect in early stages differed significantly depending on the virus model.

Adsorption↗
PubMed1994

Uracil DNA N-glycosylase distributively interacts with duplex polynucleotides containing repeating units of either TGGCCAAGCU or TGGCCAAGCTTGGCCAAGCU.

Uracil DNA N-glycosylase (UDG) has been used as a model enzyme to test a novel universal approach to discriminate between two possible enzymatic mechanisms of specific site location in DNA, processive (DNA-scanning mechanism) and distributive (random diffusion-mediated mechanism). Two double-stranded concatemeric polynucleotides of defined length (440-480 nucleotides) containing deoxyuridine at either every 10th or 20th nucleotide in the DNA chain were prepared by the ligation of self-complementary 10- or 20-mer oligodeoxyribonucleotides. Incubation of these polynucleotides with Escherichia coli UDG, followed by thermal breakage of the abasic sites, formed fragments that were multiples of either the 10- or the 20-mer. Since the processive and distributive mechanisms of uracil removal by UDG would be very different, the fragment distribution, generated at each time interval during the UDG reaction, should be unique. To show this, we developed a computer model illustrating both possible mechanisms of UDG functioning. The distribution of DNA fragments experimentally generated during the time course of the UDG reaction was compared with the results of the computer programs that modeled the distributive and processive mechanisms. The data indicated that uracil removal, catalyzed by UDG, is consistent with a distributive model.

Base Sequence↗
PubMed1994

Immune response to a carcinoembryonic antigen polynucleotide vaccine.

We have constructed a DNA plasmid encoding the full length complementary DNA for human carcinoembryonic antigen (CEA) driven by the cytomegalovirus early promoter/enhancer (plasmid DNA encoding human CEA) and demonstrated that this plasmid can function as a polynucleotide vaccine. This polynucleotide vaccine induced humoral and/or cellular immune responses specific for human CEA in all 5 immunized mice. Lymphoblastic transformation data with the use of enriched T-cell populations detected the presence of CEA-specific memory T-cells in 3 of 5 mice. Lymphocytes from 2 of 5 mice had interleukin 2/interleukin 4 release in response to CEA. CEA specificity was confirmed by the absence of reactivity to a control antigen and lack of CEA reactivity among mice vaccinated with a control plasmid encoding chloramphenicol acetyltransferase. Four of 5 mice vaccinated with plasmid DNA encoding human CEA demonstrated anti-CEA antibody responses. This immune response compared favorably with a positive control group of mice immunized with vaccinia-CEA by a dose and schedule previously shown to induce immunoprotection and therapy against a human CEA expressing syngeneic murine colon carcinoma model. Studies are ongoing to establish the construct, dose, and schedule to elicit optimal CEA-specific immune response as well as immunoprotection and therapy against human CEA expressing syngeneic murine adenocarcinoma models.

Animals↗
PubMed1983

Localization of 5' and 3' ends of the ribosome-bound segment of template polynucleotides by immune electron microscopy.

Poly(U) with an average chain length of 40-70 nucleotides was modified at the 5'- or 3'-terminal residues with 2,4-dinitrophenyl derivatives. The modified poly(U) was used to form 30S.poly(U) or 70S.poly(U).Phe-tRNA complexes. Localization of the 5' and 3' ends of the template polynucleotide on the 30S subunit and the 70S ribosome was performed by immune electron microscopy using antibodies against dinitrophenyl haptens. The 5' and 3' ends of poly(U) (putative entry and exit sites of the message) were found in the same region both on the 30S subunit and the 70S ribosome. They were located on the dorsal side of the 30S subunit between the head and the body near the groove bordering the side ledge (platform). Comparison of the size of this region with the possible length of the polynucleotide chain covered by the ribosome allowed us to suggest that the message makes a 'U-turn" (or forms a 'loop') as it passes through the ribosome.

Binding Sites↗
PubMed1987

The first step in the functional inactivation of the Escherichia coli polynucleotide phosphorylase messenger is a ribonuclease III processing at the 5' end.

The transcripts covering pnp, the gene encoding polynucleotide phosphorylase, are processed by ribonuclease III. In this study, it is shown that the steady state level of the pnp mRNA increased 11-fold in a ribonuclease III-deficient strain. The synthesis rate of this messenger is only slightly affected in the mutant strain whereas the half-life, which is 1.5 min in the wild type, is considerably increased to more than 40 min. Moreover, polynucleotide phosphorylase is 10-fold over-expressed in the mutant strain, which shows that unprocessed pnp mRNA is functional. The position of the ribonuclease III-sensitive site suggests that the sequence involved in the stabilization of the pnp mRNA is located at the 5' end of the message and that the RNase III processing triggers the decay of the transcripts downstream. A similar function for ribonuclease III in the processing of the messenger for the beta beta' subunits of RNA polymerase is proposed.

Endoribonucleases↗
PubMed1999

Photometric assay for polynucleotide phosphorylase.

Polynucleotide phosphorylase (PNPase) is a prokaryotic enzyme that catalyzes phosphorolysis of polynucleotides with release of NDPs. It is also believed to play a key role in turnover of prokaryotic transcripts, thus regulating gene expression. At the moment, only radioisotopic methods are available for assaying PNPase in crude extracts; these involve incubating [32P]phosphate and poly(A) in the presence of the enzyme, separating [32P]phosphate from [32P]ADP, and quantifying ADP by scintillation counting. Photometric assay using pyruvate kinase and lactate dehydrogenase as auxiliary enzymes is not feasible in crude extracts because of endogenous ATPase activities, which regenerate ADP from the ATP released by pyruvate kinase. Here, we present a simple photometric assay that uses a cyclic detection system which, due to the sequential action of pyruvate kinase and hexokinase, results in an exponential increase of ADP and glucose 6-phosphate. Glucose 6-phosphate is then revealed by a glucose-6-phosphate dehydrogenase reaction. Based on the theoretical model, a linear increase in absorbance is predicted as a function of the square of the reaction time, with a slope proportional to PNPase activity. Experimental data confirmed the theoretical predictions and showed that the assay was quantitative and unquestionably specific. We also devised a simple procedure for determining absolute enzyme activities (expressed in micromoles of product formed per minute) using exact amounts of pure PNPase as internal standards.

Adenosine Diphosphate↗
PubMed1980

Isolation of a polynucleotide phosphorylase mutant using a kanamycin resistant determinant.

Insertion in an episome of a kanamycine-resistant element (Tn5) at the polynucleotide phosphorylase gene level, results, after transduction into a wild strain, by the loss of activities specific to polynucleotide phosphorylase. A low phosphorolytic activity is nevertheless detectable in crude extracts, but no longer in extracts slightly purified after heat treatment at 54 degrees C. The part played by other enzymes in these activities is discussed. Bacterial growth is not affected by introduction of the mutation.

Coliphages↗
PubMed1987

Nucleic acid-like structures. II. Polynucleotide analogues as possible primitive precursors of nucleic acids.

Activated derivatives of purine-containing deoxynucleoside- diphosphates spontaneously oligomerize to produce pyrophosphate- linked oligodeoxynucleotide analogues. These analogues are of potential interest as models of primitive, polynucleotide precursors. The efficiency of oligomerization (ImpdGpIm and ImpdApIm much greater than ImpdIpIm) appears to reflect a combination of stacking forces and the specific geometric orientations of the stacked units. Under favorable conditions, chain lengths greater than 20 have been obtained for oligomers containing pdGp in the absence of a template. In the presence of a complementary template, the activated derivatives of pdGp and pdAp oligomerize much more extensively. An acyclo-analogue of G has also been shown to undergo template-directed oligomerization on pol (C). These observations suggest the possibility that primitive information transfer might have evolved in much simpler systems and that this function was taken over by polynucleotides at a later stage in evolution.

Deoxyribonucleosides↗
PubMed1979

Polynucleotides XLVII. Synthesis and properties of poly(2-methylthio- and 2-ethylthioadenylic acid). Formation of non-Watson-Crick type complexes.

Poly(2-methyl- and 2-ethylthioadenylic acid) were prepared by polymerization of corresponding diphosphates with Escherichia coli polynucleotide phosphorylase. These polynucleotides have relatively large hypochromicity of 30-35%. Acid titration of these polymers showed abrupt transition at pH 5.34-5.4, which may indicate that the introduction of alkylthio group at 2-position of adenine bases reduced their basicity. Thermal melting of these polymers showed no clear transition points at neutral pH, but in acidic media they have Tm values of 57 and 56 degrees C, somewhat lower than that of poly(A). Upon complex formation with poly(U), these poly(A) analogs showed only one poly(rs2A) . poly(U) type double-strand complexes, similar to that found in the case of poly(m2A) . poly(U).

Circular Dichroism↗
PubMed1983

Base sequence selectivity in binding of aromatic hydrocarbons with synthetic polynucleotides.

The interactions between DOBP (3) and calf thymus DNA as well as four synthetic polynucleotides, poly(dA-dT), polydA:polydT, poly(dG-dC), and polydG:polydC, were investigated by spectroscopic techniques. It was found that the binding of 3 with poly(dA-dT) is favored appreciably over other synthetic polynucleotides and DNA. The results suggest that the initial association of carcinogenic BPDE (2) with DNA may take place preferentially at certain specific base sequences in DNA.

Animals↗