Formation of triple-helical nucleic acids studied by using antibodies specific for poly(A).poly(U).poly(U).

The formation of the triple helix of poly(A).poly(U).poly(U) was studied by using antibodies specific to poly(A).poly(U).poly(U). the 10-11 base chain length for oligo(A) and the 20-30 base chain length for oligo(U) may be the minimum sizes required to maintain a stable triple helix. Double-stranded poly(A).poly(U) which was the core of triple-stranded poly(A).poly(U).poly(U) could bind poly(U) and produce an analogue of poly(A).poly(U).poly(U) reactive with the antibodies even if the poly(A) or poly(U) was brominated or acetylated to the extent of 35-55%. However, brominated or acetylated poly(U) did not produce a stable triple helix with double-stranded poly(A).poly(U).

Investigation of the role of individual tryptophan residues in the binding of Escherichia coli single-stranded DNA binding protein to single-stranded polynucleotides. A study by optical detection of magnetic resonance and site-selected mutagenesis.

Fluorescence and optical detection of triplet state magnetic resonance (ODMR) spectroscopy have been employed to study the complexes formed between single-stranded polynucleotides and Escherichia coli ssb gene products (SSB) in which tryptophans 40, 54, and 88 are selectively, one residue at a time, replaced by phenylalanine using site-specific oligonucleotide mutagenesis. Fluorescence titrations and ODMR results indicate that tryptophans 40 and 54 are the only tryptophan residues in E. coli single-stranded DNA binding protein that are involved in stabilizing the protein-nucleic acid complexes via stacking interactions. Wavelength-selected ODMR measurements on E. coli SSB reveal the presence of two spectrally distinct tryptophan sites (Khamis, M. I., Casas-Finet, J. R., and Maki, A. H. (1987) J. Biol. Chem. 262, 1725-1733). Our present results indicate that tryptophan 54 belongs to the blue-shifted site, while tryptophan 40 belongs to the red-shifted site of the protein.

Optically detected magnetic resonance of tryptophan residues in complexes formed between a bacterial single-stranded DNA binding protein and heavy atom modified poly(uridylic acid).

Optically detected magnetic resonance (ODMR) methods were employed to study three single-stranded DNA binding (SSB) proteins encoded by plasmids of enteric bacteria: pIP71a, R64, and F. Equilibrium binding isotherms obtained by fluorescence titrations reveal that the complexes of the plasmid SSB proteins with heavy atom modified polynucleotides are readily disrupted by salt. Since all the plasmid SSB proteins show limited solubility at low ionic strength (pIP71a greater than R64 greater than F), we were able to bind only the pIP71a protein to mercurated poly(uridylic acid) [poly(5-HgU)] and brominated poly(uridylic acid) [poly(5-BrU)]. ODMR results reveal the existence of at least one heavy atom perturbed, red-shifted, stacked Trp residue in these complexes. Amplitude-modulated phosphorescence microwave double resonance spectra display selectively the phosphorescence associated with Hg-perturbed Trp residue(s) in the pIP71a SSB protein-poly(5-HgU) complex, which has a broad, red-shifted 0,0-band. Our results suggest that Trp-135 in Escherichia coli SSB, which is absent in the plasmid-encoded SSB proteins, is located in a polar environment and is not involved in stacking interactions with the nucleotide bases. Phosphorescence spectra and lifetime measurements of the pIP71a SSB protein-poly (5-BrU) complex show that at least one Trp residue in the complex does not undergo stacking. This sets a higher limit of two stacking interactions of Trp residues with nucleotide bases in complexes of pIP71a SSB with single-stranded polynucleotides.

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)

Interactions of 2-methyladenines and poly(m2A) with poly(br5U).

Mixing curve experiments and melting curve analyses have shown that poly(m2A) forms complexes with poly(br5U) with stoichiometries of either 1:1 or 1:2 in high ionic strengths. CD spectra of poly(m2A).poly(br5U) and poly(m2A).2 poly(br5U) both resemble quite well to those of poly(A). poly(br5U) and poly(A).2poly(br5U), respectively. This suggests that the corresponding complexes are closely related in the structural details. Significant similarities of the CD spectra were observed for poly(m2A).2poly(br5U) and complexes between 2,9-dimethyladenine or 2-methyladenosine and poly(br5U) in the presence of spermine, indicating also the 1:2 stoichiometry. Thus, a methyl group at the position 2 of adenine ring is not necessarily hindering a formation of the Watson-Crick type base pairings.

Affinity labelling of rat liver ribosomal protein S26 by heptauridylate containing a 5'-terminal alkylating group.

Heptauridylate bearing a radioactive alkylating [14C]-4-(N-2-chloroethyl-N-methylamino)benzylamine attached to the 5'-phosphate via amide bond, was bound to ribosomes and small ribosomal subunits from rat liver which thereby were coded to bind N-acylated Phe tRNA. After completion of the alkylating reaction and subsequent hydrolysis of the phosphamide bond ribosomal proteins were isolated. Radioactivity was found covalently associated preferentially with protein S26 and, to a very small extent, with proteins S3 and S3a. The affinity labelling reaction could be abolished by (pU)14 and poly(U). From the results it is concluded that ribosomal protein S26 is located at the mRNA binding site of rat liver ribosomes.

Interaction of N6-substituted-9-methyladenines to poly-5-bromouridylic acid.

Interaction of N6-methyl-9-methyladenine (m6m9A) to poly-5-bromouridylic acid (poly(BU] was investigated. The stoichiometry of interaction was determined to be 1 m6A:2 BU by equilibrium dialysis. Structural information was obtained from circular dichroism and absorption spectra and a similarity of the structure to the corresponding polymer complex was suggested. Thermodynamic parameters of the interaction was calculated from dependence of TmS on the monomer concentration and compared to those from calorimetric measurements. N6-(delta 2-Isopentenyl) - and N6-allyl-9-methyladenine were also examined of the binding to poly(BU).

Structure and stability of complexes between poly-5-bromouridylic acid and 2,9-dimethyladenine or 2-methyladenosine.

The complexes formed between poly-5-bromouridylic acid (poly(BU)) and 2,9-dimethyladenine (m2m9A) or 2-methyladenosine (m2Ado) were investigated. The stoichiometry of the m2m9A-poly(BU) complex was found to be 1:1 as determined by equilibrium dialysis measurements. The CD spectra of the complexes showed the formation of helically ordered structure. This is also confirmed by the sharp melting profiles. A comparison of the CD spectra with that of poly(m2A) . poly(BU) complex revealed a close relationship of the helical structures for these three types of complexes. It was observed that the m2m9A was able to form a thermally more stable complex with the poly(BU) than the m2Ado did.

Poly(4-thiouridylic acid) as messenger RNA and its application for photoaffinity labelling of the ribosomal mRNA binding site.

Poly(4-thiouridylic acid) [poly(s4U)] synthesized by polymerization of 4-thiouridine 5'-diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein-synthesizing system from E. coli. It stimulates binding of Phe-tRNA to ribosomes both in the presence of EF-Tu-Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration. It codes for the synthesis of polyphenylalanine. Poly(s4U) competes with poly(U) for binding to E. coli ribosomes. Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site. Upon irradiation of 70-S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16-S RNA. Ribosomes pre-incubated with R17 RNA are protected against the photoaffinity reaction. The labelling of 16-S RNA can be reduced by treatment of ribosomes with colicin E3.

Action of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver.

The effects of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver were investigated. The enzyme was isolated from the nuclear fraction essentially according to the method of Baril et al.; it was characterized as the alpha polymerase on the basis of its response to synthetic templates and its inhibition with N-ethylmaleimide. Although polycytidylic acid had no effect on the DNA polymerase alpha either as a template or as an inhibitor, partially thiolated polycytidylic acid (MPC) was found to be a potent inhibitor, its activity being directly related to its extent of thiolation (percentage of 5-mercaptocytidylate units in the polymer). In comparison, the DNA polymerase beta which was purified from normal rat liver nuclear fraction, was much less sensitive to inhibition by MPC. Analysis of the inhibition of the alpha polymerase by the method of Lineweaver and Burk showed that the inhibitory action of MPC was competitively reversible with the DNA template, but the binding of the 7.2%-thiolated MPC to the enzyme was much stronger than that of the template (Ki/Km less than 0.03). Polyuridylic acid as such showed some inhibitory activity which increased on partial thiolation, but the 8.4%-thiolated polyuridylic acid was less active than the 7.2% MPC. When MPC was annealed with polyinosinic acid, it lost 80% of its inhibitory activity in the double-stranded configuration. However, 1 to 2%-thiolated DNA isolates were significantly more potent inhibitors than were comparable (1.2%-thiolated) MPC and showed competitive reversibility with the unmodified (but "activated") DNA template. These results indicate that the inhibitory activities of partially thiolated polynucleotides depend not only on the percentage of 5-mercapto groups but also on the configuration, base composition, and other specific structural properties.

Modified nucleotide polymers as inhibitors of DNA polymerases.

We have compared the relative inhibitory activity of poly (A) with its analogues poly N6-isopentenyl adenylic acid (poly(i6 A)) and poly N6-benzyl adenylic acid (poly(bzl6A)), and of poly (U) with its analogue poly 2'-fluoro-2'-deoxyuridylic acid (poly(dUfl)), against DNA polymerase, alpha, beta and gamma and terminal deoxynucleotidyl transferase from human cells and two oncorna virus DNA polymerases. Although poly (A) and its analogues were equally inhibitory against endogenous RNA-directed DNA polymerases of murine and feline leukemia viruses, the analogues in contrast to poly (A) were strongly inhibitory against all four cellular enzymes. Poly (dUfl), on the other hand, was up to 100-fold more potent than poly (U) against both viral and cellular enzymes. Since poly (U) at 100 mug/ml and poly (dUfl) at 1 mug/ml had no effect on terminal deoxynucleotidyl transferase while inhibiting other enzymes by 80--100 per cent these polymers could be useful in the characterization and assay of terminal deoxynucleotidyl transferase. In addition, the polymers such as poly (igA) and poly (bzl5A) which were strongly inhibitory to all cellular enzymes, could be useful in cancer chemotherapy if taken up preferentially by the malignant calls due to their high pinocytic activity. The results also demonstrate potential for large variation in inhibitory activity of polyribonucleotides as related to their chemical composition.

Inhibition of vesicular stomatitis virus replication by poly(2'-fluoro-2'-deoxyuridylic acid).

Poly(2'-fluoro-2'-deoxyuridylic acid) is known to be an effective inhibitor of the deoxyribonucleic acid polymerase found within the oncornaviruses. This synthetic polynucleotide was found to inhibit the replication of vesicular stomatitis virus in mouse L cells. The polymer was shown to be capable of inhibiting the viral ribonucleic acid (RNA)-dependent RNA polymerase, and it is proposed that this is the mechanism of antiviral activity. The following observations support this viewpoint: (i) the polymer is most active when added after virus adsorption; (ii) the antiviral activity is not species specific; and (iii) the polynucleotide is nontoxic to the host cell. Conventional methodologies designed to increase nucleic acid uptake by cultured cells do not show an increase in antiviral potency.

Physical and coding properties of poly(5-aminouridylic acid) and of 5-aminouridine-containing trinucleotides.

This report concerns the synthesis of poly(5-aminouridylic acid) and of 5-aminouridine-containing trinucleotides. Starting from 5-aminouridine the nucleoside 5'-phosphate was prepared enzymatically with carrot phosphotransferase whereas the nucleoside 5'-diphosphate was prepared chemically and polymerised with polynucleotide phosphorylase. The aminouridine-containing trinucleotides were prepared by known enzymatic procedures. Besides an increase of stability in the secondary structure poly(nh25U) forms a triple-stranded complex with poly(A) and stimulates the poly(Phe) synthesis like poly(U). In contrast to U-nh25U-U, the triplet containing the 3'-terminal aminouridine does not stimulate the binding of Phe-tRNA to 70-S ribosomes. This behavior is discussed with respect to the influence of a modification on the stacking geometry of a codon and the base pairing scheme between the 5'-nucleotide of the anticodon and the 3'-nucleotide of the condon.

Role of Mn2+ in the reaction of polynucleotide phosphorylase with 2'-O-methylated substrates.

Polymerization of 2'-O-methylcytidine-5'-diphosphate (CmDP) with polynucleotide phosphorylase in the presence of Mn2+ proceeds with 65% yield after 72 h, and in the presence of Mg2+ the yield does not exceed 10%. Phosphorolysis of poly 2'-O-methylcytidylic acid and poly 2'-O-methyluridylic acid, as well as exchange of the beta-phosphate group of CmDP in the presence of Mn2+ and Mg2+, proceed with a yield of only a few percent. A possible mechanism of Mn2+ action on CmDP polymerization is discussed.

Inhibition of oncornavirus functions by 2'-azido polynucleotides.

The 2'-azido analogs of poly(U) and poly(C), poly(dUz) [poly(2'-azido-2'-deoxyuridylic acid)], and poly-(dCz [poly(2'-azido-2'-deoxycytidylic acid)], were found to inhibit the RNA-directed DNA polymerase (reverse transcriptase) activity of murine leukemia (Moloney, Rauscher) and sarcoma (Moloney) virus, and feline leukemia (Theilen) and sarcoma (Gardner) virus, while under the same conditions the unsubstituted parent compounds failed to do so. In addition, poly(dUz) and poly(dCz) inhibited the replication of exogenous murine sarcoma virus (Moloney) in nontransformed cells (as assessed by an infectious center assay), but poly(dUz) failed to suppress the formation of endogenous sarcoma and leukemia viruses in transformed cell lines (MO-P, JLSV5). In these same cells, poly(dUz) failed to inhibit the multiplication of vesicular stomatitis virus. These data add further strength to the contention that reverse transcriptase is necessary for the productive infection and transformation of normal cells by oncornaviruses but is not essential maintenance of this transformed state and the continuous production of new viruses particles by these transformed cells.