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PubMed1984

The binding of actinomycin D and Adriamycin to supercoiled DNA, single-stranded DNA and polynucleotides.

The effect of actinomycin D and adriamycin on synthetic polynucleotides, single-stranded viral DNA and supercoiled DNA has been studied employing the fluorescent probe, terbium. Marked displacement of the probe was observed when any deoxyribose-containing polynucleotide was pretreated with either drug. With supercoiled DNA, an unwinding of the supercoil was observed at very low drug concentrations (at approx. 1:500 molar ratio of drug:DNA) prior to the displacement of the terbium. This unwinding was visualized by agarose gel electrophoresis at molar ratios of approx. 1:200. The effect was more apparent and occurred at lower drug: DNA ratios with actinomycin D than with adriamycin. Unlike cis-dichlorodiammine platinum(II), actinomycin D did not protect pBR322 DNA from cleavage at its BamHI site. The hydrolysis of phi chi 174 DNA by a series of G-C-specific restriction nucleases (including HhaI, HpaII and HaeIII) was also not affected by prior treatment of the DNA with actinomycin D.

Animals↗
PubMed1976

Thermodynamic and kinetic parameters of ion condensation to polynucleotides. Outer sphere complex formed by Mg++ions.

The coupling of ion binding to the single strand helix-coil transition in poly (A) and poly (C) is used to obtain information about both processes by ion titration and field-jump relaxation methods. Characterisation of the field-jump relaxation in poly (C) at various concentrations of monovalent ions leads to the evaluation of a stability constant K = 71 M(-1) for the ion binding to the polymer. the rate constant of helix formation is found to be 1.3 X 10(7)s(-1), whereas the dissociation rate is 1.0 X 10(6)s(-1). Similar data are preseented for poly (A) and poly (dA). The interaction of Mg(++) and Ca(++) with poly (A) and poly (C) is measured by a titration method using the polymer absorbance for the indication of binding. The data can be represented by a model with independent binding "sites". The stability constants increase with decreasing salt concentration from 2.7 X 10(4) M(-1) at medium ionic strengths up to 2.7 X 10(7) M(-1) at low ionic strength. The number of ions bound per nucleotide residue is in the range 0.2 to 0.3. Relaxation time constants associated with Mg(++) binding are characterised over a broad range of Mg(++) concentrations form 5 muM to 500 muM. The observed concentration dependence supports the conclusion on the number of binding places inferred from equilibrium titrations. The rate of Mg(++) and Ca(++) association to the polymer is close to the limit of diffusion control kR =1 X 10(10) to 2 X 10(10) M(-1) s(-1)). This high rate demonstrates that Mg(++) and Ca(++) ions do not form inner-sphere complexes with the polynucleotides. Apparently the distance between two adjacent phosphates is too large for a simultaneous site binding of Mg(++) or Ca(++), and inner sphere complexation at a single phosphate seems to be too weak. The data support the view that the ions like Mg(++) and Ca(++) surround the polynucleotides in the form of a mobile ion cloud without site binding.

Ions↗
PubMed1985

Purification and kinetic properties of polynucleotide kinase from rat testes.

Polynucleotide kinase (EC 2.7.1.78) has been purified from rat testes, and an approximately 2000-fold purification was obtained. The purified enzyme had an Mr of 38000 +/- 3800. The enzyme phosphorylated micrococcal nuclease-treated calf thymus DNA and (dT)10 while 5'-HO-tRNA was a very poor substrate. A certain degree of specificity towards purine-containing 5'-HO-nucleotides was observed. The polynucleotide kinase had an absolute requirement for a divalent cation. Both Mg2+ and Mn2+ could be used, but 10 mM MgCl2 gave optimal activity. The monovalent cations Na+, K+ and NH4+ all stimulated enzyme activity, and the optimal concentration was 0.1 M. The enzyme was inhibited by inorganic phosphate, pyrophosphate and sulphate. A 50% inhibition was obtained with 20, 0.3 and 2 mM, respectively. At 2 mM MgCl2, 1 mM spermine enhanced the enzyme activity 3-times. The apparent KATP was estimated to be 36 microM and KHO-DNA was found to be 2 microM.

Adenosine Triphosphate↗
PubMed2003

Polysaccharide-polynucleotide complexes. Part 7. Hydrogen-ion and salt concentration dependence of complexation between schizophyllan and single-stranded homo RNAs.

Schizophyllan belongs to a beta-1,3-D-glucan family, which exists as a random coil in dimethyl sulfoxide (DMSO) and as a triple helix in water, respectively. The schizophyllan single chain forms a complex with single-stranded homo RNAs in water/DMSO mixed solvents. Using circular dichroism, we studied the complexation and its stability as a function of apparent pH (pH(*)) in a mixed solvent system and as a function of the salt concentration. The complex is formed in the pH(*) range 6.5-10, and dissociated in the pH(*) range 4-6. Both poly(A) and poly(C) adopt a double strand in the pH(*) range 4-6 and a single strand in the pH(*) range 6.5-10. Therefore, the conformational change of each polynucleotide is responsible for dissociation/association of the complex, i.e., the single strand of the polynucleotides can form complexes, whereas the double one cannot. This result indicates that hydrogen bonding and similarity of the helix parameters are essential for the complex formation. The melting temperature of the complex reaches the maximum around 0.05 M of NaCl and KCl, and the value of the maximum temperature depends on the cation species.

Circular Dichroism↗
PubMed2001

XRCC1 stimulates human polynucleotide kinase activity at damaged DNA termini and accelerates DNA single-strand break repair.

XRCC1 protein is required for DNA single-strand break repair and genetic stability but its biochemical role is unknown. Here, we report that XRCC1 interacts with human polynucleotide kinase in addition to its established interactions with DNA polymerase-beta and DNA ligase III. Moreover, these four proteins are coassociated in multiprotein complexes in human cell extract and together they repair single-strand breaks typical of those induced by reactive oxygen species and ionizing radiation. Strikingly, XRCC1 stimulates the DNA kinase and DNA phosphatase activities of polynucleotide kinase at damaged DNA termini and thereby accelerates the overall repair reaction. These data identify a novel pathway for mammalian single-strand break repair and demonstrate a concerted role for XRCC1 and PNK in the initial step of processing damaged DNA ends.

Animals↗
PubMedDate not supplied

The distance of manganese to phosphorus atoms of polynucleotides, and dynamics of binding.

The structure of manganese - polynucleotide complexes is studied by phosphorous NMR. An average value for the phosphorus-manganese distance is derived from the longitudinal relaxation rate. It is larger than the distance for direct coordination and decreases as a function of temperature. An inner-sphere/outer sphere equilibrium is proposed. The outer-sphere dominates in the doublestranded polynucleotides, whereas the inner-sphere contribution is important in single-stranded species. The kinetic parameters of the model are derived from the transverse relaxation time. The similar properties of DNA fragments and tRNA argue strongly against entrapment of of manganese in special sites of tRNA.

Chemical Phenomena↗
PubMed1998

Oxidative stress causes a general, calcium-dependent degradation of mitochondrial polynucleotides.

Oxidative stress has many effects on biological cells, including the modulation of gene expression. Reactive oxygen species are known to up-regulate and down-regulate RNA expression in prokaryotic and eukaryotic cells. We have previously reported that a preferential and calcium-dependent down-regulation of mitochondrial RNAs occurs when HA-1 hamster fibroblasts are exposed to hydrogen peroxide. Here we extend these studies to determine whether this down-regulation is specific to mitochondria RNA or involves general polynucleotide degradation. Degradation and associated decreases in the levels of 16S mitochondrial rRNA following exposure of cells to 400 microM hydrogen peroxide were found to be dependent on calcium at 2 and 5 h. Degradation of mitochondrial genomic DNA was also observed following peroxide exposure, and occurred at similar time points as for mitochondrial RNA degradation. As with mitochondrial RNA degradation, this mitochondrial genomic DNA degradation was dependent on calcium. These results indicate that there is a general, calcium-dependent degradation of mitochondrial polynucleotides following exposure of HA-1 fibroblasts to oxidative stress, and suggest that a dramatic shut-down in mitochondrial biosynthesis is an early-stage response to oxidative stress.

Animals↗
PubMed1995

Kinetic and thermodynamic characterization of the reaction catalyzed by a polynucleotide kinase ribozyme.

We have previously isolated a series of ribozymes with polynucleotide kinase activity [Lorsch, J.R., & Szostak, J.W. (1994) Nature 371, 31-36]. In order to learn how such newly evolved RNAs effect catalysis, we have determined a number of the kinetic and thermodynamic parameters for the reaction catalyzed by one of these ribozymes. This ribozyme, a class I polynucleotide kinase, catalyzes the transfer of the gamma-(thio)phosphate from ATP(-gamma S) to the 5'-hydroxyl of a 7-mer oligoribonucleotide. The kcat for the reaction with ATP-gamma S is 0.17 min-1 with a Km of approximately 3 mM. The Km for the oligoribonucleotide substrate 5'-HO-GGAACCU-3' is 2 microM, the same as the Kd for this substrate in the presence or absence of ATP-gamma S. Neither the binding of substrates nor the release of products is the rate-limiting step of the reaction. The binding of substrates and release of products appear to occur in a random fashion, with no synergy of binding between the ATP(-gamma S) and oligoribonucleotide substrates. The ribozyme binds the oligoribonucleotide substrate no more strongly than would be expected for the formation of a simple RNA-RNA duplex, suggesting that there are no tertiary contacts between the ribozyme and the RNA substrate. The oligoribonucleotide substrate binding site has been located, and the sequence specificity of the ribozyme could be altered by mutating this binding site. The ribozyme is specific for adenosine triphosphate substrates; GTP-gamma S reacts approximately 650-fold slower than ATP-gamma S. With ATP as the substrate, the Kms remain unchanged, but kcat decreases by a factor of 50, consistent with a rate-limiting chemical step occurring through a dissociative transition state. The pH independence (from pH 5.5 to 8.5) of kcat/Km and of the rate constant for the conversion of the ternary substrate complex into the ternary products complex is also consistent with a dissociative phosphoryl transfer mechanism. These results suggest that this newly evolved catalyst operates in a relatively simple manner, with independent substrate binding sites and without changing the mechanism of the underlying chemical reaction.

Adenosine Triphosphate↗
PubMed1982

Interaction between antibodies to Z-form deoxyribonucleic acid and double-stranded polynucleotides.

Antibodies to Z-DNA have been elicited in rabbits immunized with chloro(diethylenetriamine)platinum(II)chloride (Cl2dienPt) modified poly(dG-dC).poly(dG-dC). Under physiological conditions this polynucleotide in which 12% of the bases were substituted is in the Z form. The antibodies are mainly immunoglobulins G as shown by gel filtration of the antiserum and by ultracentrifugation of the antibodies purified on a Sepharose--poly(dG-dC).poly(dG-dC) column. From the quantitative precipitin curve, it is concluded that each binding site of the antibody covers four nucleotide residues. The Z-form DNA-antibody complex is stabilized by electrostatic interactions. There was no interaction between poly(dI-br5dC).poly(dI-br5dC) in the Z form and the antibodies, which strongly suggests that the exocyclic amino group of guanine residues in Z-poly(dG-dC).poly(dG-dC) interacts with the antibody binding site. The modification of poly(dG-dC).poly(dG-dC) by 2-(acetylamino)fluorene residues decreases but does not prevent the binding to the antibodies. It is also shown that the polynucleotide poly(dG-br5dC).poly(dG-br5dC) adopts the Z form over a large range of salt concentration [5 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.5, to 3 M NaCl].

Animals↗
PubMed1988

Oligonucleotide N-alkylphosphoramidates: synthesis and binding to polynucleotides.

A few different methods for the preparation of oligonucleotide N-alkylphosphoramidates were compared directly. One of these, involving the use of protected nucleoside phosphites as building blocks, provided the requisite N-alkylphosphoramidates via oxidation of the intermediate dinucleoside methyl phosphites with iodine in the presence of the appropriate alkylamine. This method was found to have several attractive features, including the use of building blocks identical with those employed for the synthesis of DNA and compatibility with procedures and instruments employed for the stepwise synthesis of oligonucleotides by solution and solid-phase methods. This procedure was used to make several di-, tri-, and tetranucleotide N-alkylphosphoramidates derived from deoxyadenosine and thymidine; alkyl substituents included N,N-dimethyl, N-butyl, N-octyl, N-dodecyl, and N-(5-aminopentyl). The aminoalkyl derivative of d(TpT) (24) was used to demonstrate the feasibility of introducing an intercalative agent to the alkylphosphoramidate moiety of such derivatives. The oligonucleotide N-alkylphosphoramidates were separated into their component diastereomers and characterized structurally by a number of techniques including circular dichroism, high-field 1H NMR spectroscopy, FAB mass spectrometry, and enzymatic digestion to authentic nucleosides and nucleotides. Physicochemical characterization of several di- and trinucleotide alkyl-phosphoramidates revealed that the adenine nucleotide analogues formed stable complexes with poly-(thymidylic acid). The stabilities of these complexes were found to increase with increasing chain length of the N-alkylphosphoramidate substituents. The finding that N-alkylphosphoramidate substituents can enhance the binding of certain oligonucleotides to their complementary polynucleotides suggests the existence of a novel source of polynucleotide affinity.

Chemical Phenomena↗
PubMed1989

Interactions of the dimethyldiazaperopyrenium dication with nucleic acids. 1. Binding to nucleic acid components and to single-stranded polynucleotides and photocleavage of single-stranded oligonucleotides.

The binding of dimethyldiazaperopyrenium dication (1) with nucleosides, nucleotides, and single-stranded polynucleotides has been studied by photophysical methods. It has been shown that 1 may be a potential selective fluorescent probe for A- and/or T-rich polynucleotides. 1 efficiently cleaves oligonucleotides at guanine sites, under illumination with visible light, and therefore may be used as a sequence-specific artificial photonuclease.

Base Sequence↗
PubMed1978

T4 polynucleotide ligase catalyzed joining on triple-stranded nucleic acids.

dT1O will form triple-stranded complexes with dAn and these complexes can serve as substrate for T4 polynucleotide ligase (EC 6.5.1.1). The rate of phosphodiester formation was found to be approximately the same as for the double-stranded complex and, furthermore, the rate appears to be similar on the two strands in the complex. Joining of dT1O also took place in the presence of the double-stranded complexes dAn.dTn and dAn.rUn. Polyamines increase the rate of joining catalyzed by T4 polynucleotide ligase under certain conditions.

Coliphages↗
PubMed1977

RNA phosphorylation: a polynucleotide kinase function in mouse L cell nuclei.

Analysis of [gamma-32P]ATP in vitro labeled nuclear RNA shows transfer of gamma-32P from ATP to form the 5'-terminal monophosphate for large RNA molecules. This finding is an actively transcribing nuclear system capable of guanylation and methylation reactions indicates that polynucleotide kinase activity in the eukaryotic nucleus may be functional in kinase reactions involving RNA. It further suggest a participation in the posttranscriptional modification reactions involved in RNA processing. All four nucleosides were found to act as acceptors at the 5' termini of RNA. It is also shown that both ATP and GTP can serve as donors in the nuclear polynucleotide kinase reaction.

Adenosine Triphosphate↗
PubMed1977

3'-Phosphatase activity in T4 polynucleotide kinase.

The purification of T4 polynucleotide kinase results in the copurification of an activity which will specifically remove the 3'-terminal phosphate from a variety of deoxyribonucleotides and ribonucleotides in the absence of ATP. This phosphatase activity requires magnesium, has a pH optiumum of 6.0, and is more active with deoxyribonucleotides than ribonucleotides. T4 polynucleotide kinase and the 3'-phosphatase activity copurify by gradient elution column chromatography on DEAE-cellulose, phosphocellulose, and hydroxylapatite. The two activities are included in and comigrate on Sephadex G-200. Polyacrylamide gel electrophoresis at PH 9.2 results in conigration of the two activities together with the major protein band. The two activities respond in parallel to heat inactivation at 35 degrees C and ATP, a substrate for the kinase only, protects both activities from heat inactivation. It is therefore suggested that the two activities are functions of the same protein molecule.

Coliphages↗
PubMed1975

Interactions of aromatic residues of proteins with nucleic acids. Fluorescence studies of the binding of oligopeptides containing tryptophan and tyrosine residues to polynucleotides.

The binding of oligopeptides of general structure Lys-X-Lys (where X is an aromatic residue) to several polynucleotides has been studied by fluorescence spectroscopy. Two types of complexes are formed, both involving electrostatic interactions between lysyl residues and phosphate groups as shown by the ionic strength and pH dependence of binding. The fluorescence quantum yield of the first complex is identical with that of the free peptide. The other complex involves a stacking of the nucleic acid bases with the aromatic amino acid whose fluorescence is quenched. Fluorescence data have been quantitatively analyzed according to a model involving these two types of complexes. Association constants and the size of binding sites have been determined. Stacking interactions are favored in single-stranded polynucleotides as compared to double-stranded ones. A short oligopeptide such as Lys-X-Lys is thus able to distinguish between single-stranded and double-stranded nucleic acids. Fluorescence results are compared to those obtained by proton magnetic resonance and circular dichroism.

Binding Sites↗
PubMed2003

Spectroscopic studies of DNA and ATP binding to human polynucleotide kinase: evidence for a ternary complex.

Human polynucleotide kinase (hPNK), which possesses both 5'-DNA kinase and 3'-DNA phosphatase activities, is a DNA repair enzyme required for processing and rejoining of single- and double-strand-break termini. Full-length hPNK was subjected to sedimentation and spectroscopic analyses in association with its ligands, a 20-mer oligonucleotide, ATP, and AMP-PNP (a nonhydrolyzable analogue of ATP). Sedimentation equilibrium measurements indicated that hPNK was a monomer in the presence and absence of the ligands. Circular dichroism measurements revealed that the ligands induced different conformational changes in hPNK, although AMP-PNP induced the same conformational changes as ATP. CD also indicated that the oligonucleotide could bind to the protein-AMP-PNP complex. Protein-ligand binding affinities and stoichiometries were determined by measuring changes in protein intrinsic fluorescence. Titrating hPNK with the oligonucleotide indicated tight binding with a K(d) value of 1.3 microM and with 1:1 stoichiometry. A 5'-phosphorylated oligonucleotide with the same sequence exhibited an almost 6-fold lower affinity (K(d) value, 7.2 microM). ATP and AMP-PNP bound with high affinity (K(d) values, respectively, of 1.4 and 1.6 microM), and the observed binding stoichiometries were 1:1. Furthermore, the nonphosphorylated oligonucleotide was able to bind to hPNK in the presence of AMP-PNP with a K(d) value of 2.5 microM, confirming the formation of a ternary complex. This study provides the first direct physical evidence for such a ternary complex involving a polynucleotide kinase, AMP-PNP, and an oligonucleotide, and supports a reaction mechanism in which ATP and DNA bind simultaneously to the enzyme.

Adenosine Triphosphate↗
PubMed1989

Magnesium ion catalyzed P-N bond hydrolysis in imidazolide-activated nucleotides. Relevance to template-directed synthesis of polynucleotides.

Magnesium, an ion necessary in enzymatic as well as in nonenzymatic template-directed polynucleotide-synthesizing reactions, has been found to catalyze the hydroxide ion attack on the P-N bond of selected 5'-monophosphate imidazolide derivatives of nucleotides, such as guanosine 5'-monophosphate 2-methylimidazolide (2-MeImpG), guanosine 5'-monophosphate imidazolide (ImpG), and adenosine 5-monophosphate 2-methylimidazolide (2-MeImpA). Calcium ion behaves similarly, but quantitatively the effects are smaller. Pseudo-first-order rate constants of 2-MeImpG and ImpG hydrolysis as a function of Mg2+ concentration have been obtained in the range 6 < or = pH < or = 10 at 37 degrees C. Mg2+ catalysis is particularly effective around pH 10 where a 0.02 M concentration leads to 15-fold acceleration and a 0.2 M concentration to a 115-fold acceleration of the rate. At other pH values Mg2+ catalysis is less dramatic, mainly because the noncatalyzed reaction is faster. Mg2+ catalysis is attributed to the reaction of the zwitterionic form of the substrate (SH+/-, imidazolide moiety protonated) with OH- rather than reaction of the anionic form (S-, imidazolide moiety deprotonated) with water. This conclusion is based on a study of the N-methylated substrates N-MeImpG and 1,2-diMeImpg, respectively, which were generated in situ by the equilibrium reaction of ImpG with N-methylimidazole and 2-MeImpG with 1,2-dimethylimidazole, respectively. In contrast, the absence of Mg2+ the reaction of S- with water competes with the reaction of SH+/- with OH-. The present study bears on the mechanism of the Mg2(+)-catalyzed template-directed synthesis of oligo-and polynucleotides derived from 2-MeImpG and on the competition between oligonucleotide synthesis and hydrolysis of 2-MeImpG.

Calcium↗
PubMed2002

Hypochlorite-induced damage to DNA, RNA, and polynucleotides: formation of chloramines and nitrogen-centered radicals.

Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is a key bactericidal agent, but can also damage host tissue. As there is a strong link between chronic inflammation and some cancers, we have investigated HOCl damage to DNA, RNA, and polynucleotides. Reaction of HOCl with these materials is shown to yield multiple semistable chloramines (RNHCl/RR'NCl), which are the major initial products, and account for 50-95% of the added HOCl. These chloramines decay by thermal and metal-ion catalyzed processes, to give nucleoside-derived, nitrogen-centered, radicals. The latter have been characterized by EPR spin trapping. The propensity for radical formation with polynucleotides is cytidine > adenosine = guanosine > uridine = thymidine. The rates of decay, and yield of radicals formed, are dependent on the nature of the nucleobase on which they are formed, with chloramines formed from ring heterocyclic amine groups being less stable than those formed on exocyclic amines (RNH2 groups). Evidence is presented for chlorine transfer from the former, kinetically favored, sites to the more thermodynamically favored exocyclic amines. EPR experiments have also provided evidence for the rapid addition of pyrimidine-derived nitrogen-centered radicals to other nucleobases to give dimers and the oxidation of DNA by radicals derived from preformed nucleoside chloramines. Direct reaction of HOCl with plasmid DNA gives rise to single- and double-strand breaks via chloramine-mediated reactions. Preformed nucleoside chloramines also induce plasmid cleavage, though this only occurs to a significant extent with unstable thymidine- and uridine-derived chloramines, where radical formation is rapid. Overall the data rationalize the preferential formation of chlorinated 2'-deoxycytidine and 2'-deoxyadenosine in DNA and suggest that DNA damage induced by HOCl, and preformed chloramines, occurs at sequence-specific sites.

Chloramines↗