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PubMed2003

Fluorescence studies of the interaction of pyrenylmethyl tributylphosphonium bromide with double-strand polynucleotides.

The interaction between pyren-1-ylmethyl tri-n-butylphosphonium bromide (PMTP), a water-soluble cationic pyrene derivative and the double-strand polynucleotides Poly[dA-dT], Poly[dA].Poly[dT], Poly[dG-dC] and Poly[dG].Poly[dC] was studied using UV-Vis absorption and fluorescence spectroscopy. The PMTP probe interacts with polynucleotides through both weak and intercalative binding, evidenced through changes in the absorption spectrum (hypochromicity and red shift). The two binding types were distinguished using time-resolved fluorescence, as the intercalative environment differs from that of the surface. Thymine and cytosine are more efficient quenchers of PMTP situated on the surface because of the higher proton accessibility to this region. In contrast, adenine does not quench PMTP fluorescence, whereas guanine residues are always very efficient quenching sites. Therefore, through the use of spectroscopic techniques, it was possible to obtain information concerning the partition of PMTP in each form of binding. In the heteropolymers, Poly[dA-dT] and Poly[dG-dC], PMTP exhibits a significant preference for intercalation in AT sequences, while with GC, the intercalation is lower. In the homopolymers, Poly[dA].Poly[dT] and Poly[dG].Poly[dC], the main mechanism of interaction is weak binding, but some base preferences are elucidated. Additionally, Poly[dA].Poly[dT] can be distinguished from Poly[dA-dT] through a direct energy-transfer process between several bound PMTP molecules.

Base Pairing↗

PubMed1972

Partial purification and properties of rat liver mitochondrial polynucleotide phosphorylase.

1. Polynucleotide phosphorylase was partially purified from the inner membrane of rat liver mitochondria. 2. The partially purified particulate enzyme catalyses phosphorolysis of poly(A), poly(C), poly(U) and RNA to nucleoside diphosphates. 3. It is devoid of nucleoside diphosphate-polymerization activity. 4. Variable amounts of ADP/P(i)-exchange activity are associated with the polynucleotide phosphorylase and are probably due to a different enzyme. 5. ADP is the preferred substrate for exchange, and little or no reaction occurs with other nucleoside diphosphates, but ATP/P(i)-exchange takes place at one-third the rate observed with ADP. 6. The partially purified enzyme is free from the phosphatases found in the crude mitochondrial inner membrane, but is associated with an endonuclease activity and some adenylate kinase activity; no cytidylate kinase activity analogous to the latter was detectable.

Adenosine Diphosphate↗

PubMed1973

Interaction of quinacrine mustard with mononucleotides and polynucleotides.

1. The interaction between quinacrine mustard and mononucleotides and polynucleotides was investigated by fluorimetry and absorbance spectrophotometry. 2. The fluorescence spectrum of quinacrine mustard is independent of the ionic strength and pH. The dependence of the quinacrine mustard fluorescence intensity on ionic strength, pH and anions is described. 3. The fluorescence intensity of quinacrine mustard was enhanced with the mononucleotide adenylic acid and polynucleotides such as poly(rA), poly(rU) and poly(rA,rU). 4. Quenching of the fluorescence intensity of quinacrine mustard occurred with the mononucleotide guanylic acid and with poly(rG) and poly(rC,rG). 5. The mononucleotide cytidylic acid or poly(rC) showed no effect on the fluorescence intensity of quinacrine mustard. 6. The interaction between the dye and native DNA species was also dependent on the presence of base-specific binding sites in the DNA. The higher the (G+C) content was in the native DNA tested the higher was the quenching effect on the fluorescence intensity of quinacrine mustard. 7. No interaction was found between the dye and methylated DNA. The binding between quinacrine mustard and apurinic DNA was confirmed to be in the phosphate groups of the purines.

Adult↗

PubMed1993

Differential effects of spermine and its analogues on the structures of polynucleotides complexed with ethidium bromide.

The interactions of spermine and polyamine analogues with synthetic polynucleotides of various base sequences complexed with ethidium bromide (EB) were investigated using measurements of fluorescence intensity and steady-state fluorescence polarization. Spermine and polyamine analogues displaced some but not all of the EB bound to poly(dA-dT).poly(dA-dT) or poly(dG-dC).poly(dG-dC), suggesting that polyamines may stabilize these polynucleotides in a conformation with reduced affinity for EB. Modifications of the aliphatic backbone of spermine have pronounced effects on its ability to displace EB from poly(dA-dT).poly(dA-dT) but not from poly-(dG-dC).poly(dG-dC). Spermine and some but not all of the polyamine analogues caused fluorescence depolarization when they interacted with the complex of EB and poly(dA-dT).poly-(dA-dT). Neither spermine nor any of the analogues, however, induced fluorescence depolarization in the complex of EB with poly(dG-dC).poly(dG-dC) or poly(dA).poly(dT). This suggests that spermine and some spermine analogues induce structural changes specific to alternating A-T sequences.

Ethidium↗

PubMed2000

Polynucleotide phosphorylase functions both as a 3' right-arrow 5' exonuclease and a poly(A) polymerase in Escherichia coli.

In vitro, polynucleotide phosphorylase of Escherichia coli can both synthesize RNA by using nucleotide diphosphates as precursors and exonucleolytically degrade RNA in the presence of inorganic phosphate. However, because of the high in vivo concentration of inorganic phosphate in exponentially growing cells, it has been assumed that the enzyme works exclusively as an exonuclease. Here we demonstrate that, contrary to this prediction, polynucleotide phosphorylase not only synthesizes long, highly heteropolymeric tails in vivo, but also accounts for all of the observed residual polyadenylylation in poly(A) polymerase I deficient strains. In addition, the enzyme is responsible for adding the C and U residues that are found in poly(A) tails in exponentially growing cultures of wild type E. coli.

Base Sequence↗

PubMed1976

Alteration of polynucleotide secondary structure by ribosomal protein S1.

Ribosomal 30S protein S1 causes disruption of the secondary structure of certain pyrimidine-containing polynucleotides. Helical poly(U), poly(C, U), and neutral and acidic poly(C) are stoichiometrically converted by S1 to structures indistinguishable from their partially or completely thermally denatured forms, as revealed by circular dichroism. Of the several double- and triple-stranded helical polynucleotides tested that contain one polypurine strand and at least one polypyrimidine strand, only the conformation of the DNA.RNA hybrid, poly(A)-poly(dT), is perturbed. In the presence of S1, this hybrid undergoes a transition to a new structure that has a circular dichroism spectrum unlike either the native or thermally denatured forms. Intercalated ethidium bromide is released from poly(A)-poly(dT) by S1, confirming the occurrence of a conformational rearrangement. The translation inhibitor, autintricarboxylic acid, completely inhibits the action of S1 on polypyrimidines, but has no effect on the conformational perturbation of poly(A(-poly(dT). The possible relation between these observations and the biological function of protein S1 is discussed.

Circular Dichroism↗

PubMed1999

Molecular cloning of the human gene, PNKP, encoding a polynucleotide kinase 3'-phosphatase and evidence for its role in repair of DNA strand breaks caused by oxidative damage.

Mammalian polynucleotide kinases catalyze the 5'-phosphorylation of nucleic acids and can have associated 3'-phosphatase activity, predictive of an important function in DNA repair following ionizing radiation or oxidative damage. The sequences of three tryptic peptides from a bovine 60-kDa polypeptide that correlated with 5'-DNA kinase and 3'-phosphatase activities identified human and murine dbEST clones. The 57.1-kDa conceptual translation product of this gene, polynucleotide kinase 3'-phosphatase (PNKP), contained a putative ATP binding site and a potential 3'-phosphatase domain with similarity to L-2-haloacid dehalogenases. BLAST searches identified possible homologs in Caenorhabditis elegans, Schizosaccharomyces pombe, and Drosophila melanogaster. The gene was localized to chromosome 19q13.3-13.4. Northern analysis indicated a 2-kilobase mRNA in eight human tissues. A glutathione S-transferase-PNKP fusion protein displayed 5'-DNA kinase and 3'-phosphatase activities. PNKP is the first gene for a DNA-specific kinase from any organism. PNKP expression partially rescued the sensitivity to oxidative damaging agents of the Escherichia coli DNA repair-deficient xth nfo double mutant. PNKP gene function restored termini suitable for DNA polymerase, consistent with in vivo removal of 3'-phosphate groups, facilitating DNA repair.

Amino Acid Sequence↗

PubMed1998

A study of metalloporphyrin-polynucleotide interactions by microcalorimetry and circular dichroism.

In this paper we examine the interactions of Calf Thymus DNA and the model polynucleotides poly(dA).poly(dT), poly(dAdT)2 and poly(dG.dC)2 with a group of metalloporphyrins derived from the freebase porphyrin tetrakis(4-N-methylpyridyl)porphine, H2(TMpy-P4), by means of ultraviolet absorption spectroscopy, circular dichroism spectroscopy and microcalorimetry. We have studied the interactions of the copper, cobalt, nickel and zinc derivatives of H2(TMpy-P4) in addition to the free base porphyrin itself. We have found strong evidence for an external self-stacking interaction of the Cu(TMpy-P4) and Zn(TMpy-P4) derivatives with poly(dA).poly(dT) and poly(dAdT)2 even at low concentrations of porphyrin, and all of the porphyrin derivatives studied appear to display such a self-stacking in interaction with poly(dA.dT)2 at sufficiently high ratios of porphyrin to polynucleotide.

Animals↗

PubMed1981

Polyspecific monoclonal lupus autoantibodies reactive with both polynucleotides and phospholipids.

Hybridomas the produce anti-DNA autoantibodies were prepared from spleen cells of unimmunized MRL/1 mice, a strain that spontaneously develops severe systemic lupus erythematous (SLE). Reactivities of these monoclonal antibodies with a wide range of polynucleotides prompted tests of their reactions with phospholipids which, like polynucleotides, contain diester-linked phosphate groups in their backbones. In competitive radioimmunoassays, cardiolipin, phosphatidic acid, and phosphatidyl glycerol blocked the binding of these hybridoma antibodies to denatured DNA. These phospholipids also specifically inhibited the reaction between a hybridoma antibody and a site-specific anti-idiotypic antibody. The antinuclear reaction of one of these antibodies was specifically inhibited by cardiolipin. This same antibody prolonged the activated partial thromboplastin time in a manner characteristic of a lupus anticoagulant, presumably by binding to phospholipid in the test system. The polyspecific reactivity of a single molecular species of lupus autoantibody suggests that some of the diverse serological abnormalities of SLE may be a result of the binding of certain autoantibodies to a phosphodiester-containing epitope that is present in diverse biological molecules.

Animals↗

PubMed1986

A human monoclonal macroglobulin with specificity for alpha(2----8)-linked poly-N-acetyl neuraminic acid, the capsular polysaccharide of group B meningococci and Escherichia coli K1, which crossreacts with polynucleotides and with denatured DNA.

We have described an IgM antibody from a patient with macroglobulinemia specifically reacting with poly-alpha(2----8)N-acetyl neuraminic acid (NeuNAc) the capsular polysaccharide of two important human pathogens, group B meningococcus and E. coli K1. This antibody has a narrowly defined specificity in its interactions with polysaccharides, being unable to bind poly-alpha(2----9)NeuNAc or alternating poly-alpha(2----8)alpha(2----9)NeuNAc. However, it shows interesting crossreactivity with seemingly unrelated polynucleotides and denatured DNA, supporting the hypothesis that charged groups with a given spacing may determine the specificity of antigen-antibody interactions on otherwise dissimilar molecular structures. Despite the crossreactivity with denatured DNA and polynucleotides, the antibody does not appear to have adverse effects in the patient. The antibody protects newborn rats against E. coli K1 infection, as well as the standard horse antiserum H46, and one would expect it to prove useful in humans as an adjunct to antibiotic therapy in infections with group B meningococcus and E. coli K1. We have attempted to clone the antibody-producing cells from peripheral blood, and have shown that the relevant cells are present and can be cultured.

Aged↗

PubMed1989

Ring-opened 7-methylguanine nucleotides are resistant to nuclease P1 digestion and good substrates to polynucleotide kinase.

Dimethyl sulfate was used to prepare 7-methyl-2'-deoxy-guanosine 3'-monophosphate (7-methyl-dGMP), which was ring-opened in alkali to 2'-deoxy-N5-methyl-N5-formyl-2,5,6-triamino-4-oxopyrimidine 3'-monophosphate (ROM-dGMP). ROM-dGMP was not dephosphorylated by nuclease P1 in contrast to normal deoxynucleotides. It was efficiently 5'-phosphorylated by T4 polynucleotide kinase. When methylated DNA was alkali-treated and digested with micrococcal nuclease, spleen phosphodiesterase and nuclease P1, ROM-dGMP was formed and this was labeled with [gamma-32P]-ATP in the presence of polynucleotide kinase. Ring-opening and P1 treatment appear methods of choice for 32P-post-labeling of 7-alkylguanines in DNA.

Deoxyguanine Nucleotides↗

PubMed2002

Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme.

T4 polynucleotide kinase (Pnk), in addition to being an invaluable research tool, exemplifies a family of bifunctional enzymes with 5'-kinase and 3'-phosphatase activities that play key roles in RNA and DNA repair. T4 Pnk is a homotetramer composed of a C-terminal phosphatase domain and an N-terminal kinase domain. The 2.0 A crystal structure of the isolated kinase domain highlights a tunnel-like active site through the heart of the enzyme, with an entrance on the 5' OH acceptor side that can accommodate a single-stranded polynucleotide. The active site is composed of essential side chains that coordinate the beta phosphate of the NTP donor and the 3' phosphate of the 5' OH acceptor, plus a putative general acid that activates the 5' OH. The structure rationalizes the different specificities of T4 and eukaryotic Pnk and suggests a model for the assembly of the tetramer.

Amino Acid Sequence↗

PubMed1995

Polynucleotides compensate for impaired T-dependent antibody production induced in C57B1/6 mice by a nucleotide-free diet both in vivo and in vitro, but a mononucleotide-nucleoside mixture is effective only in vivo.

Actions of nucleotides on in vitro humoral immune responses were studied in mice fed a nucleotide-free diet, a nucleotide-free diet plus a mononucleotide-nucleoside mixture or a nucleotide-free diet plus yeast RNA (polynucleotides). Cultured spleen cells from mice fed a nucleotide-free diet produced fewer numbers of antibody-secreting cells in response to a T-dependent antigen, compared with those from controls fed nucleotide-supplemented diets. Immunoglobulin M concentrations in these supernatants were significantly lower in cultured cells from mice fed the nucleotide-free diet or the nucleotide-free diet plus the mononucleotide/nucleoside mixture compared with concentrations in cells from mice fed the nucleotide-free diet plus RNA. Concanavalin A-potentiated cytokine (interleukin-4 and interleukin-5) production by purified T helper cells was also lower in cultured cells from mice fed a nucleotide-free diet than in those from mice fed nucleotide-supplemented diets. In vivo supplementation with the mononucleotide/nucleoside mixture restored impaired in vitro antibody and concanavalin A-potentiated cytokine production in mice fed a nucleotide-free diet. However, addition of RNA to the culture enhanced antibody production in spleen cells from mice of all diet groups. Supplementing the culture with RNA did not enhance mitogen-potentiated cytokine production. This in vitro action of RNA was retained after the removal of oligonucleotides (molecular weight < 1000), but was reduced by modification of bases and cleavage of phosphodiester bonds of RNA. Thus the in vitro action of RNA is mainly attributed to polynucleotides, indicating their potential role in modulation of local humoral immune responses in the body.

Animals↗

PubMed1983

Derivatization of unprotected polynucleotides.

A simple and efficient method for attaching amines to the terminal 5'-phosphate of unprotected oligonucleotides or nucleic acids in aqueous solution is described. The method is applicable to low molecular-weight amines, polypeptides, or proteins. The terminal 5'-phosphate of an oligonucleotide or nucleic acid reacts with a water-soluble carbodiimide in imidazole buffer at pH 6 to give good yields of the 5'-phosphorimidazolide. Exposure of the phosphorimidazolide to amine-containing molecules in aqueous solution results in the production of a wide range of stable phosphoramidates in high yield. The exposure of polynucleotides to carbodiimide does not result in significant breakage of phosphodiester bonds or damage to nucleoside bases. The biological activity of a drug resistant plasmid is not affected. The direct condensation of polynucleotides with amines in 1-methylimidazole buffer is also possible. However, it is not a satisfactory preparative method if the ligand is sensitive to carbodiimide.

Adenosine Triphosphate↗

PubMed1975

Metal ion-mediated specific interactions between nucleic acid bases of polynucleotides and amino acid side chains of polypeptides.

Interactions between copolypeptides containing Glu and Tyr residues and polynucleotides can be mediated through divalent metal ions such as Zn-2+ and Ci-2+. Circular dichroism studies show that the binding of metal ion - polypeptide complexes to poly(A) induces an unstacking of adenine bases. Fluorescence investigations demonstrate that Tyrosine - Adenine interactions result from the formation of ternary complexes polypeptide-Zn-2 plus-polynucleotide.

Calcium↗

PubMed1990

Anti-DNA antibodies induced by BK virus inoculations. Demonstration of the specificities for eukaryotic dsDNA and synthetic polynucleotides.

The ability of BK virus to induce anti-DNA antibodies in rabbits, and the ability of these antibodies to bind natural eukaryotic DNA and synthetic polynucleotides have been analysed. The specificity of the binding was assayed by inhibition of anti-dsDNA and -ssDNA ELISA tests with dsDNA, ssDNA, and synthetic single-stranded as well as double-stranded polynucleotides. The anti-dsDNA activity of two rabbit antisera was effectively inhibited by dsDNA and ssDNA and poly(dAdT)-poly(dAdT). The other nucleotide antigens produced relatively less inhibition. The anti-ssDNA binding was most efficiently inhibited by the homologous antigen, whereas inhibition by dsDNA only reached approximately 70% of the maximum as defined by ssDNA as inhibitor. This indicates the existence of a selective anti-ssDNA antibody population and a population recognizing both ssDNA and dsDNA within the sera. Cross-reaction of the induced anti-DNA antibodies with phospholipid antigens, such as cardiolipin, phosphatidylic acid, and bacterial cell surface, could not be demonstrated. We conclude that antibodies resulting from inoculation with BK virus specifically bind to dsDNA and ssDNA and possess a high affinity for the synthetic duplex poly(dAdT). In this way, they have some similarities with anti-DNA antibodies encountered in SLE (systemic lupus erythematosus) in both man and mouse.

Animals↗

PubMed1975

Kinetics and effect of salts and polyamines on T4 polynucleotide ligase.

The kinetics of T4 polynucleotide ligase has been investigated at pH 8,20 degrees C and using the double-stranded DNA substrate (dA)n - [(dT)10]n/10. Double-reciprocal plots of initial rates vs substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to a ping-pong mechanism. The overall mechanism was found to be non-processive. The true Km for the DNA substrate was 0.6 muM and that of ATP 100 muM. Several attempts were made to reverse the T4 polynucleotide ligase joining reaction using 32-p-labelled (dA)n - [(DT)40]n/40 as substrate. No breakdown of this DNA could be detected. The joining reaction was inhibited by high concentrations, i.e. above approximately 70mM, of salts such as KCl, NaCl, NH4Cl and CsCl. At a concentration of 200 mM almost 100% inhibition was observed. Polyamines also caused inhibition of the enzyme, the most efficient inhibitor being spermine followed by spermidine. At a concentration of 1 mM spermine, virtually no joining took place. Addition of salts or polyamines resulted in a large increase in the apparent Km for the DNA substrate whereas the apparent Km for ATP remained unchanged. It is suggested that the affinity of the enzyme for the DNA substrate is decreased in the presence of inhibiting agents.

Adenosine Monophosphate↗

PubMed1976

Specificity and properties of the destabilization, induced by initiation factor IF-3, of ternary complexes of the 30-S ribosomal subunit, aminoacyl-tRNA and polynucleotides.

Initiation factor IF-3 causes the destabilization of preformed ternary complexes of 30-S ribosomal subunit, codons and aminoacyl-tRNAs or peptidyl-tRNA. This destabilization is dilution-dependent and affects all ternary complexes with the exception of those containing the initiator fMet-tRNA, which remain more resistant to IF-3-induced destabilization under the various conditions studied. Several possible reasons for this specificity have been examined. It was found that the basis for the specificity is not: (a) an intrinsic greater stability of the ternary complexes containing fMet-tRNA, (b) the amoung of aminoacyl-tRNA bound to the ribosome, (c) the conditions under which the ternary complex is made or (d) the formylation of the amino group. On the other hand, the nature of the polynucleotide in response to which the ternary complex is formed was found to influence the amount of aminoacyl-tRan bound to the ribosome, and to some extent the amount of aminoacyl-tRNA which can be relased. The ternary complex containing the mischarged initiator tRNA fVal-tRNAfMet displays greater resistance to the IF-3-induced destabilization than the complex containing fVal-tRNAVal. These results indicate that the specificity of the IF-3 activity is due to the special structural feature of the initiator tRNA molecule and to some extent to the nature of the polynucleotide. The IF-3-induced destabilization of ternary complexes was found to be little affected by variations in reaction conditons, so that this IF-3 activity can be used to measure the stoichiometric binding of IF-3 to the ribosome over a broad range of pH and K+ and Mg2+ concentrations. Several antibiotics have been tested for their capacity to interfere with this reaction; only high concentrations of tetracycline blocked this IF-3 activity.

Anti-Bacterial Agents↗