Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H.

A model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.     

Hydrogen exchange study of some polynucleotides and transfer RNA

The apparent disagreement between published transfer RNA hydrogen exchange results and the tRNA cloverleaf model, prompted a re-investigation of the relationship between hydrogen exchange data and nucleic acid structure. Hydrogen-tritium exchange experiments were carried out with samples of pure and mixed tRNA and with the synthetic polynucleotide bihelices: poly(rA) · poly(rU), poly(rI) · poly(rC), poly(rG) · poly(rC) and poly(dG) · poly (dC).     

Induced circular dichroism in nucleic acid-acridine derivative complexes

Visible absorption and circular dichroism (CD) spectra have been measured for complexes formed between nucleic acids (calf thymus DNA, poly(rA).poly(rU) and poly(rI).poly(rC)) and 9-aminoacridines (quinacrine, acranil and 9-amino-6-chloro-2-methoxy acridine). With poly(rA).poly(rU), a new absorption band was observed at longer wavelengths. The nucleic acid-drug complexes showed considerable different induced CD spectra. Analysis of these CD spectra suggests that the cationic side chains of quinacrine and acranil play an important role on the binding properties to DNA and poly(rA).poly(rU).     

A spin label study of purple membranes from Halobacterium halobium.

Mammary tumors induced in Sprague-Dawley Rats by the carcinogen 7,12-dimethylbenz(a)anthracene contain a DNA polymerase similar to that found in RNA tumor viruses. It has a molecular weight of 105,000 daltons and is active on the synthetic templates poly(rA):oligo(dT) and poly(rC):-oligo(dG) but is inactive on poly(dA):oligo(dT). This polymerase may be purified more than 300 fold with a 25% yield by ammonium sulfate precipitation, phosphocellulose chromatography and hydroxyapatite chromatography. A similar polymerase is also found in lactating normal rat mammary tissues.     

Effect of isonicotinic acid hydrazide-copper complex on Rous sarcoma virus and its genome RNA.

The copper complex of the antituberculous drug, insonicotinic acid hydrazide (INH), inhibits the RNA-dependent DNA polymerase of Rous sarcoma virus and inactivates its ability to malignantly transform chick embryo cells. The INH-copper complex binds to the 70S genome RNA of Rous sarcoma virus (RSV), which may account for its ability to inhibit the RNA-dependent DNA polymerase. The complex binds RNA more effectively than DNA in contrast to M-IBT-copper complexes, which bind both types of nucleic acids equally. The homopolymers, poly rA and poly rU, are bound by the INH-copper complex to a greater extent than poly rC. Isonicotinic acid hydrazide alone and CuSO4 alone bind neither DNA, RNA, poly (rA), poly (rU), nor poly (rC). However, CuSO4 alone binds poly (rI); INH alone does not. In addition to viral DNA synthesis, chick-embryo cell DNA synthesis is inhibited by the INH-copper complex. The extent of inhibition of cellular DNA synthesis is greater than that of cellular RNA and protein synthesis. No selective inhibition of transformation in cells previously infected with Rous sarcoma virus is observed.     

RNA-dependent DNA polymerase from a cell line derived from the bone marrow of a patient with polycythemia vera.

A RNA-dependent DNA polymerase was isolated from a human cell line derived from the bone marrow of a patient with polycythemia vera. The purification procedure included chromatography on phosphocellulose and oligo(dT)-cellulose, and glycerol gradient centrifugation. The enzyme could be distinguished from polymerase A by salt elution from phosphocellulose, utilization of poly(rC) - oligo(dG) and its molecular size of about 70000, as determined by centrifugation. Throughout the purification procedure ribonuclease H activity was co-purified. Upon dodecylsulfate-polyacrylamide electrophoresis on microgradient gels two main bands with molecular weights of 68000 and 66000 and three minor bands were detected. The enzyme preferentially used poly(rA) - oligo(dT) as template-primer compared with poly(dA) - oligo(dT). It incorporated dGMP into polymer on poly(rC) - oligo(dG).     

Characterization of an alpha-like DNA polymerase from Bombyx mori silkglands.

A soluble DNA polymerase has been purified near to homogeneity from Bombyx mori silkglands. The following characteristics were observed: high molecular weight (about 150 000 - 220 00); optimum pH about 8; inhibition by high salt concentrations, sulfhydryl-group blocking agents and polyamines; absence of nuclease activity; preference for magnesium as required divalent cation with all the efficient template-primers tested; and clear template-primer specificity, the purified enzyme being able to copy primed - polydeoxyribonucleotide templates [activated DNA, poly(dA).oligo(dT), poly(dA).oligo(rU)] but not polyribonucleotide chains [poly(rA).oligo(dT), poly(rA).oligo(rU)] in the presence of either Mg++ or MN++. Believed to represent the bulk of silkgland DNA polymerase activity, the purified soluble enzyme most resembles vertebrate DNA polymerases alpha when it is compared to other eukaryotic DNA polymerases as yet characterized.     

Purification and characterization of the RNA-directed DNA polymerase of a primate type-D retrovirus: Mason-Pfizer virus

The RNA-directed DNA polymerase of the primate type-D retrovirus Mason-Pfizer virus was purified using ion-exchange and affinity chromatography, and molecular sieving. The enzyme was shown to have a molecular weight of approx. 80 000 as determined by sedimentation analysis, molecular sieving and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified RNA-directed DNA polymerase retained its ability to use a heteropolymeric RNA as a template. The Mason-Pfizer virus RNA-directed DNA polymerase was also characterized as to its divalent cation preference for several synthetic primertemplates and for heteropolymeric RNA. Mg2+ was preferred as its divalent cation for all primer-templates except oligo(dG).poly(rC)m for which it prefers Mn2+. The Mason-Pfizer virus enzyme was also shown to have a pH optimum of 8-8.5 and a temperature optimum of 37-40 degrees C. The stability of the Mason-Pfizer virus RNA-directed DNA polymerase was shown to differ when measured using different primer-templates.     

Human cytomegalovirus. III. Virus-induced DNA polymerase.

Infection of WI-38 human fibroblasts with human cytomegalovirus (CMV) led to the stimulation of host cell DNA polymerase synthesis and induction of a novel virus-specific DNA polymerase. This cytomegalovirus-induced DNA polymerase was purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. It can be distinguished from host cell enzymes by chromatographic behavior, template primer specificity, sedimentation property, and the requirement of salt for maximal activity. This virus-induced enzyme has a sedimentation coefficient of 9.2S and is found in both the nuclei and cytoplasm of virus-infected cells, but not in uninfected cells. This enzyme could efficiently use activated calf-thymus DNA, oly(dA)-oligo(dT)12-18, and poly(dC)-oligo(dG)12-18 as template primers, especially poly(dA)-oligo(dT)12-18, but it could not use poly(rA)-oligo(dT)12-18, poly(rC)-oligo(dG)12-18, or oligo(dT)12-18. The enzyme requires Mg2+ for maximal activity, is sensitive to p-hydroxymercuribenzoate, and is not a zinc metalloenzyme. In addition, the cytomegalovirus-induced DNA polymerase activity can be enhanced by adding 0.06 to 0.12 M NaCl or 0.03 to 0.06 M (NH4)2SO4 to the reaction mixture.     

Interaction of sanguinarine with double stranded RNA structures

Interaction of sanguinarine with A-form RNA structures of poly(rI)poly(rC) and poly(rA).poly(rU) has been studied by spectrophotometric, spectrofluorimetric, UV melting profiles, circular dichroism and viscometric analysis. The binding of sanguinarine to A-form duplex RNA structures is characterised by the typical bathochromic and hypochromic effects in the absorption spectrum, increasing steady state fluorescence intensity, an increase in fluorescence quantum yield of sanguinarine, an increase in fluorescence polarization anisotropy, an increase of thermal transition temperature, an increase in the contour length of sonicated rod-like RNA structure and perturbation in circular dichroic spectrum. Scatchard analysis indicates that sanguinarine binds to each polymer in a non-cooperative manner. Comparative binding parameters determined from absorbance titration by Scatchard analysis, employing the excluded site model, indicate a higher binding affinity of sanguinarine to poly(rI).poly(rC) structure than to poly(rA).poly(rU) structure. On the basis of these observations, it is concluded that the alkaloid binds to both the RNA structures by a mechanism of intercalation.     

Destabilization of the secondary structure of RNA by ribosomal protein S1 from escherichia coli

S1 is an acidic protein associated with the 3′ end of 16S RNA; it is indispensable for ribosomal binding of natural mRNA. We find that S1 unfolds single stranded stacked or helical polynucleotides (poly rA, poly rC, poly rU). It prevents the formation of poly (rA + rU) and poly (rI + rC) duplexes at 10–25 mM NaCl but not at 50–100 mM NaCl. Partial, salt reversible denaturation is also seen with coliphage MS2 RNA, $\text{E. coli}$ rRNA and tRNA. Generally, only duplex structures with a Tm greater than about 55° are formed in the presence of S1. The protein unfolds single stranded DNA but not poly d(A·T).     

Nucleic acid vibrational circular dichroism, absorption, and linear dichroism spectra. II. A DeVoe theory approach.

The DeVoe polarizability theory is used to calculate vibrational circular dichroism (VCD) and infrared (IR) absorption spectra of four polyribonucleotides: poly(rA) x poly(rU), poly(rU) x poly(rA) x poly(rU), poly(rG) x poly(rC), and poly(rC+) x poly(rI) x poly(rC). This is the first report on the use of the DeVoe theory to calculate VCD, oriented VCD, IR absorption, and IR linear dichroism (LD) spectra of double- and triple-stranded polyribonucleotides. Results are reported for DeVoe theory calculations--within the base-stretching 1750-1550 cm(-1) spectral region--on several proposed multistranded polyribonucleotide geometries. The calculated spectra obtained from these proposed geometries are compared with previously reported measured and calculated VCD and IR spectral results. Base-base hydrogen-bonding effects on the frequencies and magnitudes of the base carbonyl stretching modes are explicitly considered. The good agreements found between calculated and measured spectra are proposed to be further evidence of the usefulness of the DeVoe theory in drawing three-dimensional structural conclusions from measured polyribonucleotide VCD and IR spectra.     

Topography of nucleic acid helices in solutions. The interaction specificities of optically active amino acid derivatives

The synthesis and interactions of the d- and l-enantiomers of the amino acid amide derivatives [Formula: see text] (I) and lysyl dipeptides [Formula: see text] (II) with poly rI.poly rC, poly rA.poly rU and calf thymus DNA is reported. The following results were found. (1) The degree of stabilization of the helices as measured by the T(m) (;melting' temperature) of the helix-coil transition was dependent on the nature of the amino acid. (2) For the poly rI.poly rC helix, the l-enantiomers of salts (I) and (II) stabilized more than the d-enantiomers. The same was true for calf thymus DNA in the presence of salts (II) and for poly rA.poly rU in the presence of salts (II) and the proline derivatives of salts (I). (3) As R increased in size and became more apolar, the amount of stabilization of the poly rI.poly rC helix in the presence of salts (I) decreased. On the other hand, the amount of stabilization increased with more polar substituents. An attempt was then made to determine whether the difference in stabilization of the double-stranded helices at the T(m) in the presence of the l- and d-enantiomers of salts (I) is due to the interaction with the helix, the random coil or both. A new method was developed for determining the binding of small ions to polyions that involves a competition between an insoluble polystyrene ion-exchange resin and the soluble polyion for the counterion. Dissociation constants are obtained for the complexes of single- and double-stranded helices with the salts (I). The results are illuminating and indicate that with certain helices, i.e. poly rA.poly rU, the interactions of salts (I) with the single strands may not be ignored. It is concluded that the high optical specificity found in Nature, i.e. d-ribose in nucleic acids and l-amino acids in proteins, cannot be attributed solely to monomer-polymer interactions described by Gabbay (1968).