Evidence for two forms of RNA-dependent DNA polymerase in Visna virus.

The visna viral RNA-dependent DNA polymerase has been resolved into two forms by affinity chromatography. Glycerine gradient centrifugation of the two forms showed that one form sedimented at 6.9 S corresponding to an apparent molecular weight of 135 000 and the other at 6.3 S corresponding to 118 000. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the two forms indicated that the 6.9 S enzyme is composed of 2 molecules of 68 000 mol. wt. chain and the 6.3 S is a single chain enzyme. The latter form has been identified as a glycoprotein. The 6.9 S form can be completely inactivated in 20 min at 45 degrees C, prefers poly(rC) over poly(rA) as template and has high efficiency in utilizing visna 70 S RNA as template. The 6.3 S form is stable at 45 degrees C, active with 70 S viral RNA as template, prefers poly(rA) over poly(rC), and requires higher concentration of Mn2+ (0.4 mM) for maximum activity than the 6.9 S form does (0.1 mM) with synthetic homopolymers as templates. However, both 6.9 S and 6.3 S forms prefer Mg2+ over Mn2+ regardless of the nature of the templates.     

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).     

Polynucleotide conformation from flow dichroism studies

We demonstrate that the isotropic absorption and linear dichroism in an unknown flow field can be used to determine base tilt in polynucleotides if three transitions are measured and the directions of the corresponding dipoles are known. The method is applied here to reach conclusions about the base tilt in poly(rA), poly(rA)⁺·poly(rA), and poly(rC). The respective values are: 28° tilt about the axis + 50° toward C8 from the C1′ → N9, and 25° tilt about the axis + 118° toward C5 from C1′ → N1. The results for poly(rA)⁺·poly(rA) are consistent with the accepted model. Spectra were measured for poly(rC)⁺·poly(rC), but definite conclusions must await reliable directions for transition dipoles. The dipole direction for the 218-nm transition in rC is found to be +13° or +43° toward C5 from C1′ → N1. The CD spectra to about 168 nm are presented and discussed.     

High molecular weight deoxyribonucleic acid polymerase from crown gall tumor cells of periwinkle (Vinca rosea).

A high molecular weight (6 S) plant DNA polymerase from axenic Vinca rosea tissue culture cells has been purified 2200-fold and characterized. The enzyme has a molecular weight of 105 000 (+/-5000). Sodium dodecyl sulfate-acrylamide gel electrophoresis of the purified enzyme yields polypeptide subunits having molecular weights of 70 000 and 34 000. The purified enzyme has a pH optimum of 7.5; a cation requirement optimum of 6 mM Mg2+ or 0.5 mM Mn2+; an apparent requirement for Zn2+; a Km of 1 muM for dTTP; and a 3.5-fold stimulation by 50 mM KCl. The enzyme is sensitive to N-ethylmaleimide (1 mM), heparin (0.1 muM), ethanol (5%), pyrophosphate (0.05 muM), and o-phenanthroline (0.1 mM) but is insensitive to rifamycin. Denatured DNA is found to be the best natural template, and only negligible activity can be demonstrated with the ribopolymer templates poly(dT)n-poly(rA)n and p(dT)10-poly(rA)n. In addition to the polymerization reaction, the enzyme catalyzes a pyrophosphate exchange reaction. Antibody to calf thymus 6-8S DNA polymerase does not inhibit DNA polymerase from Vinca rosea, suggesting no antigenic relationships between the mammalian and plant enzymes.     

RNA-dependent ATPase from Saccharomyces cerevisiae

A new RNA-dependent ATPase has been isolated from yeast chromatin extracts and partially characterized. The protein has a sedimentation coefficient of about 7 S. The enzyme hydrolyzes specifically ATP (or dATP) to ADP (or dADP) and Pi in the presence of Mg2+ or Mn2+ ions and requires a single-stranded polynucleotide as cofactor. The order of efficiency of synthetic polymers is poly(rU) > poly(rI) greater than or equal to poly(dU) > poly(rA) greater than or equal to poly(rC). Among natural polymers, single-stranded DNA and poly(rA)-containing mRNA from yeast are also active but less so than poly(rU). The enzyme exhibits a pH optimum of 8 and is fully inhibited by 0.25 M NaCl. The Km for ATP is0.2 mM. The resemblance between this ATPase and DNA-dependent ATPases from other sources, as well as the termination factor rho, is discussed.     

Poly(rA) binds poly(rG).poly(rC) to form a triple helix.

Poly(rA) binds poly(rG).poly(rC) to form a triple helix. Evidence for this structure includes ultraviolet absorbance mixing curves and melting curves, and circular dichroism spectroscopy. The formation of the triple helix depends on the length of the poly(rC) strand. Triple helix forms when the average length is around 100 nucleotides but does not form when the average length is about 500 nucleotides.     

Inhibition of RNA-dependent DNA polymerase activity of oncornaviruses by caffeine.

Caffeine was found to inhibit RNA-dependent DNA polymerase activity of Rauscher leukemia virus when endogenous viral RNA and poly(rA)·(dT)_12–18 were used as templates. Similar results were also obtained with purified RNA-dependent DNA polymerase (deoxynucleoside triphosphate; DNA nucleotidyl transferase; EC 2.7.7.7) from avian myeloblastosis virus (AMV) utilizing 70S and 35S RNA of AMV, poly(rA)·(dT)_12–18, globin mRNA and activated calf thymus DNA as templates. The “caffeine effect” was evident only when it was present during the initiation of polymerization reaction. Increasing the template concentration in the reaction mixture partly reversed the effect of caffeine. Of the analogs of caffeine tested, only theophylline inhibited AMV DNA polymerase, whereas aminophylline showed no effect.     

A DNA polymerase from Ustilago maydis. 1. Purification and properties of the polymerase activity.

A DNA polymerase from Ustilago maydis has been purified to apparent homogeneity. The native enzyme possesses a subunit structure consisting of 50000 and 55000-dalton monomers. The apparent sedimentation coefficient of the polymerase activity in the absence of salt is 8.4 S (Mr=180000-200000), that in its presence (0.6 M NaCl or 0.12 M KCl) being 6.3 S (Mr=80000-100000). Low concentrations of EDTA also converted the 8.4-S to a 6.3-S form, whereas magnesium ions catalysed the reverse association. The enzyme has an absolute requirement for both a DNA or RNA template and a DNA primer. For homopolymer templates the primer requirement was satisified by a short complementary oligodeoxynucleotide, but oligoribonucleotides were extremely inefficient primers. With the template-primer poly(dA) X (dT)12, the enzyme added an average of 50 dTMP nucleotides on to each primer molecule, whereas with poly(rA) X (dT)12, this figure was 300. The enzyme also possesses an associated deoxyribonuclease activity. No other DNA polymerase activity was detected in cell-free extracts of U. maydis.     

Nucleic acid binding properties of Escherichia coli ribosomal protein S1. II. Co-operativity and specificity of binding site II.

The following properties characterize the interaction of nucleic acid binding site II of Escherichia coli ribosomal protein S1 with oligo- and polyribonucleotides; all have been determined with site I complexed with oligo- or polydeoxyribonucleotides. (1) The intrinsic binding constant (K) of site II to single-stranded polyribonucleotides is fairly independent of base composition, though cytidinecontaining polymers bind with approximately threefold higher intrinsic affinities than do the comparable adenine-containing species. (2) Poly(rC) is bound to site II co-operatively; the co-operativity parameter (ω) ? 31. Poly(rA) shows no binding co-operativity. The site size (n) for both polyribonucleotides binding at site II is about ten nucleotide residues. (3) The K value for site II is ? 4 × 10⁵m^?1 for poly(rA), and ? 1 × 10⁶m^?1 for poly(rC), in 0.12 m-Na⁺. Unlike site I, the binding affinity of site II increases somewhat with increasing salt concentration, suggesting that phosphate—basic protein residue contacts are not involved. (4) Varying Mg^{2 +} concentration has no effect on K, and changes in the concentration of either Mg²⁺ or Na⁺ do not affect the magnitude of site II co-operativity. (5) Reaction of the exocyclic amino groups of poly (rC) with formaldehyde drastically reduces the affinity of site II for this polynucleotide, while the affinity of poly (rC) for site I is not altered by this treatment. (6) No major sequence specificity of K for site II is found with either homogeneous polynucleotides or the 3′ terminal dodecanucleotide of 16 S ribosomal RNA; we conclude that selectivity of S1 binding via site II depends largely on the presence or absence of base compositiondependent binding co-operativity.The binding properties of site II probably account for the ability of S1 to inhibit translation at high S1 to ribosome ratios (“factor i” activity). Possible mechanisms for the role of S1 protein as a part of the phage Qβ replicase complex and in protein synthesis are discussed in relation to the binding properties of site I and site II.     

Presence of poly(rA):poly(dT)-dependent DNA polymerase in mouse myeloma cells

An RNA-dependent DNA polymerase analogous to that of normal cells has been found in mouse myeloma cells. This enzyme, which is activated by Mn²⁺ ion, specifically copies the poly A strand of poly (rA): poly (dT) hybrid to synthesize dTMP homopolymer.     

Distribution of Mn2+ ions around poly(rI).poly(rC)

The distribution of bound Mn²⁺ ions about poly(rI)·poly(rC) has been studied by measuring the effect of this paramagnetic metal ion on the relaxation behavior of poly(rI)·poly(rC) protons. By combining selective spin – lattice and spin – spin relaxation rates for various protons, some of the principle regions of ion association can be identified. The relaxation data on the CH6 proton are consistent with a < 10% occupancy of phosphate inner-sphere binding sites. The broadening of the imino proton resonance requires a substantial occupancy of sites located in the major groove, possibly near IN7. This would also be consistent with the observation that IH8 resonance is the proton most susceptible to relaxation by Mn²⁺. The relaxation data for the IH2 proton indicate a relatively low occupancy of minor-groove binding sites (e.g., IN3).