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PubMed1986

Investigation of complexes formed between gene 32 protein from bacteriophage T4 and heavy-atom-modified single-stranded polynucleotides using optical detection of magnetic resonance.

Optical detection of triplet-state magnetic resonance (ODMR) is employed to study the complexes formed between gene 32 protein (GP32), a single-stranded DNA-binding protein from bacteriophage T4, and the heavy-atom-derivatized polynucleotides poly(5-HgU) and poly(5-BrU). The triplet-state properties of some of the tryptophan (Trp) residues in the complexes are dramatically different from those in the free protein, in that they are subject to an external heavy-atom effect. Direct evidence for the presence of a heavy-atom effect, and hence a close-range interaction between mercurated or brominated nucleotide bases and Trp residues in the complex, is provided by the observation of the zero-field (D) + (E) ODMR transition of Trp, which is not normally observed in the absence of a heavy-atom perturbation. The amplitude-modulated phosphorescence-microwave double-resonance (AM-PMDR) technique is employed to selectively capture the phosphorescence spectrum originating from the heavy-atom-perturbed Trp residue(s) in the GP32-poly(5-HgU) complex. Arguments based on our experimental results lead to the conclusion that the heavy-atom perturbation arises from aromatic stacking interactions between Trp and mercurated bases. Wavelength-selected ODMR measurements reveal the existence of two environmentally distinct and spectrally different types of Trp in GP32. One of these types is perturbed selectively by the heavy atom and hence undergoes stacking interactions with the heavy-atom-derivatized bases of the polynucleotide while the second type of Trp residue is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗

PubMed1986

Targeted cleavage of polynucleotides by complementary oligonucleotides covalently linked to iron-porphyrins.

Oligothymidylates covalently linked to iron-porphyrins were synthesized to target the nuclease activity of Fe-porphyrin to complementary polynucleotides. In the presence of oxygen and a reducing agent, oligo(dT)7 bearing the reactive group attached to the 3'-phosphate was shown to be active in the cleavage of poly(dA) and poly(rA) but not poly(dT). When poly(dA) was used as a matrix, the reaction yield was higher at low temperature where the complexes are stable; upon increasing temperature, the reaction yield decreased in agreement with the dissociation of the oligonucleotide-polynucleotide complex as measured by absorption spectroscopy. Thus, oligonucleotides covalently linked to iron-porphyrin derivatives can be used to cleave selectively the target sequence of the oligonucleotide.

Cobalt↗

PubMed1987

Interaction between adenovirus DNA-binding protein and single-stranded polynucleotides studied by circular dichroism and ultraviolet absorption.

The adenovirus DNA-binding protein (AdDBP) is a multifunctional protein required for viral DNA replication and control of transcription. We have studied the binding of AdDBP to single-stranded M13 DNA and to the homopolynucleotides poly(rA), poly(dA), and poly(dT) by means of circular dichroism (CD) and optical density (OD) measurements. The binding to all these polynucleotides was strong and nearly stoichiometric. Titration experiments showed that the size of the binding site is 9-11 nucleotides long for M13 DNA, poly(dA), and poly(rA). A higher value (15.0 +/- 0.8) was found for poly(dT). Pronounced changes in the circular dichroism and optical density spectra were observed upon binding of AdDBP. In general, both the positive peak around 260-270 nm and the negative peak around 240-250 nm in the CD spectra decreased in intensity, and a shift of the crossover point to longer wavelengths was found. The OD spectra observed upon binding of AdDBP are remarkably similar to those obtained with prokaryotic helix-destabilizing proteins like bacteriophage T4 gene 32 protein and fd gene 5 protein. The data can best be interpreted by assuming that the AdDBP-polynucleotide complex has a regular, rigid, and extended configuration that satifies two criteria: (1) a considerable tilt of the bases in combination with (2) a small rotation per base and/or a shift of the bases closer to the helix axis.

Adenoviridae↗

PubMed1981

Esterification of terminal phosphate groups in nucleic acids with sorbitol and its application to the isolation of terminal polynucleotide fragments.

The exposure of mono- and polynucleotides to 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and high concentrations of sorbitol results in the esterification of their monosubstituted phosphate groups. The presence of the sorbitol moiety permits these derivatives to bind strongly at pH 8.7 to columns of chromatographic supports containing the dihydroxyboryl group and to be subsequently released by elution with buffers at pH 5.5. The procedure constitutes a method for the isolation of polynucleotide fragments arising from the terminals of nucleic acids. A new method for the preparation of the chromatographic supports involves the synthesis of the 1,3-propanediol cyclic ester of m-[[3-(N-succinimidoxycarbonyl)propanoyl]amino]benzeneboronic acid and its condensation with aminoethylcellulose or amino-ethylpolyacrylamide. The reagent is readily prepared by reaction of N-[m-(dihydroxyboryl)phenyl)]succinamic acid with 1,3-propanediol to protect the boronate moiety followed by esterification with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide.

Animals↗

PubMed1981

Equilibrium studies of ethidium--polynucleotide interactions.

We report equilibrium dialysis studies of the binding of ethidium to a variety of double-helical synthetic polynucleotides containing A.U (or T) and I.C base pairs. The results are interpreted in terms of the neighbor exclusion model of drug binding, with allowance both for cooperativity of binding and for a structural switch of the helix to a different form which binds the drug more effectively. Both DNA and the alternating copolymers examined [poly[d(A-T)] and poly[d(I-C)]] showed high affinity (10(4)--10(5) M-1) in 1 M salt. Homopolynucleotides showed a more complicated pattern of affinities: poly(rA).poly(rU), poly(rA).poly(dT), and poly(dA).poly(rU) showed high affinity, whereas poly(dA).poly(dT), poly(rI).poly(rC), and poly(dI).poly(dC) showed low affinity (less than or equal to 10(3) M-1). The neighbor exclusion range was inferred to be two base pairs for DNA or B family helices and three for RNA or A family helices. Generally, polynucleotides showed some cooperativity in their ethidium binding. The data reveal a switch of poly[d(I-C)] to a form able to bind ethidium more effectively.

Animals↗

PubMed1981

Interactions of poly (N epsilon , N epsilon , N epsilon ,-trimethyllysine) and poly(lysine) with polynucleotides: circular dichroism and A-T sequence selectivity.

Complexes of (Lys) and [Lys(Me3)]n with natural and synthetic DNAs have been studied by CD as a function of ionic strength. In dilute EDTA, (Lys)n and [Lys(Me3)]n produce the same distortions to the CD spectrum of calf thymus DNA at r (peptide residue/nucleotide residue) values less than 0.6. At higher r values, the distortions are somewhat different. [Lys(me3)]n alters the conformation of some polynucleotides differently from (Lys)n under non- psi conditions. Therefore, methylation of histones may serve to alter the structure of chromatin. At low ionic strength, [Lys(Me3)]n and (Lys)n alter the viscosity of DNA to the same extent between r values of 0.0 and 1.0. In contrast to (Lys)n-DNA, at high ionic strengths, [lys(Me3)]n-DNA does not show psi - type CD spectra. (Lys)n forms psi - structures with (dA-dT)n and (dG-dC)n. [Lys(Me3)]n forms psi + structures with (dA-dT)n. Between 0.05 and 0.3 M NaCl, [Lys(Me3)]n forms psi+ structures with (dG-dC)n, while between 0.35 and 0.45 M NaCl, it forms a psi - structure with (dG-dC)n. Neither (Lys)n nor [Lys(Me3)]n forms psi structures with (dA)n.(dT)n or (dG)n.(dC)n. These results, in conjunction with the work of others on reconstitution of nucleosome-like particles from synthetic polynucleotides, suggest that the ability of DNA and histones to form nucleosomes is related to the formation of psi structures. (Lys)n binds preferentially to (dA)n.(dT)n over (dA-dT)n. [Lys(Me3)]n binds to (dA)n.(dT)n and (dA-dT)n with equal affinity.

Base Sequence↗

PubMed1982

On the mechanism of de novo polymerization by form I polynucleotide phosphorylase of Micrococcus luteus.

The diastereomers of adenosine 5'-O-(1-thiodiphosphate) (ADP alpha S) have been tested as substrates for the polymerization reaction of primer-independent polynucleotide phosphorylase from Micrococcus luteus. The preferred substrate is ADP alpha S(Sp), which has a similar Km and a greatly reduced Vmax when compared to the natural substrate ADP. The other diastereomer, ADP alpha S(Rp), is preferentially cleaved by a polyphosphate kinase activity (present with the phosphorylase) that may be responsible for the removal of the 5'-beta-phosphate during de novo polymerization, leading to the observed 5'-phospho-poly(A). Inhibitor studies suggest that the kinase and de novo polymerization sites are not coincident. During de novo polymerization of the diastereomeric mixture, ADP alpha S(Rp) is selectively used to form 5' termini, whereas ADP alpha S(Sp) serves to support the chain elongation. Thus there are two stereochemically distinct subsites for initiating polymerization. ADP beta S functions as a substrate for polynucleotide phosphorylase with kinetic properties similar to those of ADP, indicating that removal of the beta-phosphate (a thiophosphate) is not a kinetically important step and probably occurs after polymerization is complete. The average chain length of the polymeric product is considerably smaller for ADP alpha S vs. ADP beta S or ADP, suggesting that the degree of processivity of the polymerization is determined by competition between the rate of polymerization and the rate of dissociation of the growing chain.

Adenosine Diphosphate↗

PubMed1979

Stereochemistry of internucleotide bond formation by polynucleotide phosphorylase from Micrococcus luteus.

Polynucleotide phosphorylase catalyzes the formation of polynucleotides from the Sp diastereomer of adenosine 5'-O-(l-thiodiphosphate) ADPalphaS), whereas the Rp diastereomer is a competitive inhibitor. The absolute configuration of the phosphorothioate diester bond in the polymer was determined by copolymerizing ADPalpha S, Sp isomer with UDP and degrading the resulting copolymer with R Nase A and spleen phosphodiesterase to give, inter alia, uridine 2',-3'-cyclic phosphorothioate. The latter product was shown to be the endo isomer by high-performance liquid chromatography. No evidence for the presence of the exo isomer was obtained. It can thus be concluded that the Sp diastereomer of ADPalphaS polymerizes with inversion of configuration at phosphorus without racemization to give a phosphorothioate diester bond with the Rp configuration.

Kinetics↗

PubMed1977

Preparation of Escherichia coli tRNAs terminating of modified nucleosides by the use of CTP(ATP):tRNA nucleotidyltransferase and polynucleotide phosphorylase.

Two procedures were investigated for the modification of tRNAs at the 3'-terminal nucleoside. The first involved the incubation of an enzymatically abreviated tRNA (tRNA-C-COH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from Escherichia coli and yeast. The E. coli enzyme did not utilize 2'- or 3'-deoxyadenosine 5'-triphosphate as substrates, but affected incorporation of the 2'- and 3'-O-methyladenosine triphosphates onto tRNA-C-Cou to the extent of 30 and 37%, respectively. Although incorporation of the deoxynucleotides could not be effected using the E. coli enzyme, yeast CTP(ATP:tRNA nucleotidyltransferase produced the desired tRNAs in yields of 45-65%. The second modification procedure involved incubation of tRNA-C-COH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase. This procedure afforded the tRNAs terminating in 2'- and 3'-deoxyadenosine in yields of 4% (and the yield of the former was increased to 36% when the incubation was carried out in the presence of 20% methanol). The yields of tRNAs terminating in 2'- and 3'-O-methyladenosing produced by this procedure were 55 and 17%, respectively. Because only single isomers of most of the tRNAs terminating in 2'- and 3'-deoxy- and O-methyladenosine are aminoacylated, attempts were made to obtain the other isomericaminoacyl-tRNA by enzymatic introduction of chemically preaminoacylated nucleotides onto tRNA-C-COH. Although incubation of tRNA-C-COH with three aminoacylated nucleoside 5'-triphosphates and E. coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNAs to a detectable extent, incubation with 2'-deoxy-3'-O-L-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase afforded E. coli tRNA terminating with the corresponding aminoacylated deoxynucleoside.

Adenosine Diphosphate↗

PubMed1968

A study by polyacrylamide-gel electrophoresis of the effect of proteolysis on Micrococcus lysodeikticus polynucleotide phosphorylase.

1. Trypsin digestion of Micrococcus lysodeikticus polynucleotide phosphorylase (nucleoside diphosphate-polynucleotide nucleotidyltransferase) causes a progressive increase in electrophoretic mobility in polyacrylamide gels of the single active degradation product. 2. A marked increase in primer requirement for CDP polymerization occurs before a more mobile product is formed. 3. alpha-Chymotrypsin digestion yields a product that separates into several active species on polyacrylamide-gel electrophoretograms. 4. No separation of ADP-and CDP-polymerization activities occurs during electrophoresis after either trypsin or alpha-chymotrypsin treatment.

Adenine Nucleotides↗

PubMed1969

The preferential loss of the polylysine- or polyornithine-stimulated activity of Clostridium perfringens polynucleotide phosphorylase during proteolysis.

1. An improved method for the purification of Clostridium perfringens polynucleotide phosphorylase (nucleoside diphosphate-polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is described. The product was stable and was highly stimulated by polylysine or polyornithine. 2. It migrated as a single enzyme during sucrose-density-gradient centrifugation, and no separation of polymerization and phosphorolytic activities was observed. 3. Trypsin digestion caused a rapid, preferential loss of the polylysine- or polyornithine-stimulated activity, which was prevented by low concentrations of polyornithine. 4. The protection by polyornithine was not specific. 5. It is concluded that charge effects on the clostridial polynucleotide phosphorylase itself are primarily responsible for the stimulation of this enzyme by polylysine or polyornithine.

Centrifugation, Density Gradient↗

PubMed1971

Nucleic acid enzymology of extremely halophilic bacteria. Halobacterium cutirubrum polynucleotide phosphorylase.

1. Polynucleotide phosphorylase was purified 200-fold from Halobacterium cutirubrum. 2. It is membrane-associated and can be solubilized by sonication. 3. The purified enzyme requires a high ionic strength for both stability and activity. 4. It is Mn(2+)-dependent, has all three typical polynucleotide phosphorylase activities and is specific for nucleoside diphosphates. 5. The enzyme is of low molecular weight.

Carbon Isotopes↗

PubMed1972

A study of the localization of polynucleotide phosphorylase within rat liver cells and of its distribution among rat tissues and diverse animal species.

1. Rat liver polynucleotide phosphorylase was localized in the mitochondrion, but may also occur in the nucleus. 2. The mitochondrial enzyme was found in rat heart, kidney, liver, muscle and spleen. 3. Mitochondrial polynucleotide phosphorylase is also present in calf, chicken, guinea-pig and rabbit liver and in goldfish muscle. 4. A possible physiological role for the enzyme in the control of the intramitochondrial ADP concentration is suggested.

Adenosine Diphosphate↗

PubMed2006

Monitoring the direct and indirect damage of DNA bases and polynucleotides by using time-resolved infrared spectroscopy.

The nucleotide 5'-dGMP and polynucleotide poly(dGdC).poly(dGdC) have been irradiated by using a 200-fs, 200-nm laser pulses and spectrally characterized by using time-resolved infrared spectroscopy. Under the experimental conditions, 200-nm excitation generates both electronic excited states and radical cations through photoionization; the former decay rapidly to vibrationally hot ground state. By using infrared signatures we have been able to follow these processes, and at time scales of >1 ns we observe an infrared marker band at 1,702 cm(-1) within both 5'-dGMP and the polynucleotide assigned to a photoionized product of guanine. This transient has also been reproduced through indirect chemistry through the reaction with photogenerated carbonate radical with 5'-dGMP. The ability to use time-resolved infrared spectroscopy in this way paves the way for developing solution-phase studies to investigate both direct and indirect radiation chemistry of DNA.

DNA↗

PubMed1976

Platinum binds selectively to phosphorothioate groups in mono- and polynucleotides: a general method for heavy metal staining of specific nucleotides.

Platinum binding to nucleoside phosphorothioates has been examined to determine their suitability as heavy metal labeling sites for the potential electron microscopic sequencing of nucleic acids. The complex platinum terpyridine nitrate forms a 1:1 adduct with either adenosine or uridine monophosphorothioate. Spectroscopic evidence strongly indicates the presence of platinum-sulfur bonds. Both platinum terpyridine nitrate and chloroterpyridineplatinum(II) bind to poly(sA-U), a polymer prepared from adenosine 5'-O-(1-thiotriphosphate) and UTP. Binding to the sulfur atoms of the phosphorothioate groups is quantitative, as shown by double label experiments using [35S]poly(sA-U) and [3H]chloroterpyridine-platinum(II). Similar experiments with [14C]poly(A-U) indicated no platinum binding. No evidence of nicking or loss of sulfur from poly(sA-U) could be detected after platinum binding. The phosphorothioate group is a strong, highly selective binding site for platinum in polynucleotides. Previous studies have demonstrated quantitative enzymatic incorporation of phosphorothioate groups into a polynucleotide adjacent to a specific base [Matzura, H. & Eckstein, F. (1968) Eur. J. Biochem. 3, 448-452]. The use of heavy metal-labeled phosphorothioate groups for the sequencing of nucleic acids by electron microscopy therefore appears feasible.

Adenosine Triphosphate↗

PubMed1977

Spatial configuration of ordered polynucleotide chains: a novel double helix.

Structural details are reported for a novel right-handed polynucleotide double helix stabilized by vertical base stacking and hydrogen bonding. The primary difference between this duplex and the familiar Watson-Crick horizontally stabilized polynucleotide complex arises in the glycosyl rotation of the heterocyclic bases with respect to the sugar-phate backbone. In the vertical double helix the bases adopt a fairly unusual (although not sterically impossible) high-anti conformation, while in the horizontal models deduced from x-ray analyses the bases occur in the favored anti arrangement. The base pairing scheme in both duplexes is the standard Watson-Crick type. The vertical double helix is demonstrated to be a plausible model for the unusual complex formation observed between complementary synthetic polycyclonucleotides (the bases of which are fixed by a chemical linkage in the high-anti orientation) and also a potential alternative ordered structure available to naturally occurring nucleic acid systems that can adopt both anti and high-anti glycosyl arrangements.

Computers↗

PubMed1977

Binding of mouse interferon to polynucleotides.

The polynucleotides poly(I) and poly(U), and to a lesser extent poly(G), are capable of desorbing mouse interferon from blue dextran-Sepharose columns, whereas poly(A) andpoly(C) are without effect. When covalently bound to agarose, poly(I), poly(U), and also poly(A) act as potent ligands for purification of interferon by affinity chromatography. Furthermore, poly(I) and poly(U) confer a significant degree of protection to interferon against thermal denaturation. Taken together, these observations point to a direct interaction of interferon with these polymers and suggest that interferon molecules have a polynucleotide attachment site. Possible implications for the concept of interferon induction are discussed.

Cell Line↗

PubMed1981

Effects of methylation on a synthetic polynucleotide: the B--Z transition in poly(dG-m5dC).poly(dG-m5dC).

We have compared the behavior in solution of the synthetic polynucleotide poly(dG-m5dC).poly(dG-m5dC) with that of the unmethylated polynucleotide poly(dG-dC).poly(dG-dC). In solutions containing high concentrations of salt, poly(dG-dC).poly(dG-dC) is known to exhibit altered circular dichroic and absorption spectra correlated with formation of a left-handed Z DNA structure. Poly(dG-m5dC).poly(dG-m5dC) behaves similarly, but the spectral transition from the B to the Z form occurs at much lower salt concentrations, close to usual physiological conditions. Divalent and polyvalent ions are particularly effective: The B--Z transition of poly(dG-m5dC).(dG-m5dC) can be induced at a Mg2+ concentration three orders of magnitude lower than that required for the unmethylated polymer. We have also studied mixed copolymers containing both dC and m5dC. Our results suggest that the sequence m5dC-dG, which occurs in eukaryotic DNA, can have a disproportionately large effect on the B--Z transition.

Cations↗