Single-stranded Polydeoxyribonucleotide Research Articles

Purification and properties of a DNA-dependent ATPase induced by bacteriophage T4.

A DNA-dependent ATPase formed after T4 phage infection is purified to apparent homogeneity. The molecular weight of the purified enzyme is 50 000 when determined by glycerol gradient centrifugation and by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The enzyme at an earlier stage in purification (prior to DEAE-cellulose chromatography) exists as a complex with a molecular weight of 100000. However, molecular weight determinations by Sephadex gel chromatography give considerably decreased molecular weights for the complex and for the enzyme after DEAE-cellulose chromatography. The enzyme is stimulated to varying degrees by a variety of single-stranded polydeoxyribonucleotides or by single-stranded DNA, but no chemical change in the polynucleotide has been detected as a result of the enzyme action.

Proton magnetic resonance studies of ultraviolet-irradiated apurinic acid.

In apurinic acid, a single-stranded polydeoxyribonucleotide easily obtained upon depurination of DNA, the proton resonances arising from thymine and cytosine are readily observable in aqueous solution of 25 degrees C. Two methyl thymine resonances, centered at 1.88 ppm and separated by 0.045 ppm, are observed. We attribute the downfield methyl resonance to thymines with no pyrimidine nearest neighbors and the upfield methyl resonance to thymines having pyrimidine neighbors in the 3' and/or 5' positions. Upon ultraviolet irradiation, the upfield methyl and thymine H-6 resonances decrease in amplitude and two methyl resoances appear at 1.63 and 1.52 ppm, corresponding, respectively, to cytosine-thymine and thymine-thymine cyclobutane dimers. Photoreversal eliminates these two minor methyl resonances from the pmr spectrum. We conclude that apurinic acid provides a suitable model system for pmr studies of chemically modified pyrimidine bases in DNA.

Synthesis of N-acetylated deoxyribonucleoside 5'-triphosphates and their utilization in enzymatic formation of single-stranded polydeoxyribonucleotides.

The N‐acetylated deoxyadenosine and deoxyguanosine 5′‐triphosphates (dAAcTP and dGAcTP) have been synthesized and found competent as substrates for enzymatic chain lengthening of polydeoxyribonucleotides without concomitant loss of the acetyl group. The enzyme used was terminal deoxyribonucleotidyltransferase. The addition of dGAc units proceeds without aggregation that self‐limits addition of nonacetylated dG units. The resulting polymer 3′‐ended with dGAc units is an efficient initiator for subsequent chain lengthening. Hydrogen bonding is not destroyed by N‐acetylation of the dA‐dT Watson‐Crick pair, although it is considerably weakened. As a practical result of this, a polymer 3′‐ended with dAAc units is a much more efficient initiator for enzymatic chain lengthening by dT units than is a corresponding polymer with dA units. Venom phosphodiesterase does not hydrolyze a polymer 3′‐ended with dAAc units.

Studies on polynucleotides: LXXI. Sedimentation and buoyant density studies of some DNA-like polymers with repeating nucleotide sequences

The properties of the following DNA-like polymers with repeating sequences were studied by ultraeentrifugation: poly dA:dT, poly dTG:dCA, poly dTC:dGA, poly dTTC:dGAA, poly dTTG:dCAA, poly dTAC:dGTA, poly dATC:dGAT, poly dTATC:dGATA and poly dTTAC:dGTAA. Sedimentation velocity experiments indicated the following molecular weight values for the polymers: poly dA:dT, at least 4 × 10 6; the two repeating dinucleotide polymers (poly dTC:dGA and poly dTG:dCA), approximately 5 × 10 5; the four repeating trinucleotide polymers, approximately 2·5 × 10 5; and the two repeating tetranucleotide polymers, approximately 1 × 10 5. The buoyant densities of the DNA-like polymers were studied by cesium chloride density-gradient centrifugation, The polymers, except for the two repeating tetranucleotide polymers, gave single symmetrical bands by this technique. The two repeating tetranucleotide polymers failed to band in either cesium chloride or cesium sulfate density-gradients. The density values of the DNA-like polymers were not as would be predicted from previously established relations correlating the base composition of naturally occurring DNA's and bouyant density values. Also, heat treatment of the polymers did not cause a uniform increase in the densities, as had been previously found for naturally occurring DNA's; but instead, no density change was found for four polymers, a density decrease was found for poly dTC:dGA and density increases were found for poly dATC:dGAT and poly dTTG:dCAA. The restricted base composition of the DNA-like polymers permitted separation of the individual strands in some cases by centrifugation in alkaline cesium chloride solution. The four single-stranded polydeoxyribonucleotides poly dA, poly dT, poly dTG and poly dAC were isolated by this technique on a preparative scale and their spectral properties are recorded.