Robert D. Wells

Physicochemical studies on polydeoxyribonucleotides containing defined repeating nucleotide sequences

Absorbance-temperature transition, analytical buoyant density, and circular dichroism studies are reported on a variety of high molecular weight doublestranded DNA polymers containing defined repeating nucleotide sequences. These studies provide further evidence that the structure of a DNA is dictated by its primary nucleotide sequence. The DNAs studied include three homopolymer pairs, five repeating dinucleotide DNAs and four repeating trinucleotide DNAs. n nEquilibrium absorbance-temperature studies over a 100-fold salt concentration range reveal the following: (1) DNAs which have the same base composition but different nucleotide sequences (sequence isomers) do not show identical helixcoil transitions. Sequence isomeric DNAs† have Tm values differing by as much as 9 deg. C. (2) For sequence isomeric polymers, the DNA containing both purines and pyrimidines on each of the complementary strands is more stable than the isomer containing only purines on one strand and only pyrimidines on the complementary strand. This generalization is valid for 80% of the cases examined. (3) All polymers undergo a single helix-random coil transition over a narrow temperature range at all salt concentrations studied. (4) The transitions are virtually completely reversible, particularly at salt concentrations above 0.1m-Na+. (5) Poly d(I-C)·poly d(I-C)‡ shows a marked increase in the breadth of the melting transitions as the salt concentration is raised. A similar behavior for poly d(A-T)·poly d(A-T) has been reported. n nDensity gradient analyses were performed on the DNA polymers. The ordering of polymer densities in Cs2SO4 solution is similar to that previously reported for CsCl gradients. In comparing sequence isomeric DNAs (for example poly dA · poly dT and poly d(A-T)·poly d(A-T)), the DNA which is more thermostable is always less dense in a CsCl gradient analysis. n nCircular dichroism studies on eleven different DNA polymers show that nucleotide sequence strongly affects spectra. Semi-empirical nearest-neighbor calculations are used to attempt to predict some spectra. Only moderate agreement between calculated and observed spectra is found, thus providing further evidence that the configuration of all polymers is not the same. However, the inclusion of certain approximations facilitates the prediction of the circular dichroism spectrum of naturally occurring DNA from the experimentally obtained polymer spectra. The calculated spectrum for salmon sperm DNA is in excellent agreement with its observed spectrum. n nThe synthesis and characterization of a new DNA polymer, poly d(G-C)·poly d(G-C), is described. n nThe possible biological implications of the influence of nucleotide sequence on DNA physicochemical properties are discussed.

Specificity of the three-stranded complex formation between double-stranded DNA and single-stranded RNA containing repeating nucleotide sequences

Abstract Three-stranded nucleic acid complexes have been shown to be formed from double-stranded DNA and single-stranded RNA polymers with restricted base sequences. Complex formation takes place only when the DNA contains all purine bases in one strand and all pyrimidine bases in the other strand and the RNA must be a polypyrimidine polymer. Hence, poly (dA)·(dT) forms a three-stranded complex with poly U, and poly d(T-C)·d(G-A) † forms a three-stranded complex with poly (U-C) in acidic solutions. No other RNAs including the random co-polymer poly (U,C) interact with poly d(T-C)·dG-A) to give a stoichiometric three-stranded structure. Under no condition is a complex detected between poly d(T-G)·d(C-A) or poly d(A-T)·d(A-T) and any polyribonucleotide. The three-stranded poly d(T-C)·d(G-A)·(U-CH + ) was characterized by cesium sulfate buoyant density studies, by continuous variations study, by ultraviolet spectral properties and by optical density-temperature profiles. Addition of poly (U-C) to poly d(T-C)·d(G-A) and addition of poly U to poly (dA)·(dT), prior to the addition of Escherichia coli RNA polymerase, effectively reduced the transcription rate of both strands of these DNAs. Other combinations of RNAs with DNAs showed essentially no inhibition; all of these RNA-DNA mixtures were also found to be inert as regards triplex formation. Possible biological roles of the triplexes are discussed.

Studies on the binding of actinomycin D to DNA and DNA model polymers

Abstract The ability of 17 different DNAs to bind actinomycin D was studied by a variety of techniques including equilibrium dialysis, in vitro transcription and analytical buoyant density centrifugation. The major conclusions are as follows: (1) The presence of deoxyguanylic acid in a DNA is not necessary for complex formation. Poly dI binds approximately one-quarter as much AM as DNAs which contain 50% G + C; the equilibrium constant for the poly dI-AM complex is as large as that observed for 50% G + C DNAs. (2) The presence of deoxyguanylic acid in a DNA is not sufficient for complex formation. Poly d(A-T-C) · poly d(G-A-T), which contains 33% G + C, binds little or no AM as judged by five different techniques. (3) A marked nucleotide sequence preference exists for the binding reaction. When comparing sequence isomeric DNAs, the poly d(pur n -pyr m ) · poly d(pur m -pyr n ) isomer binds more AM and binds AM more tightly than does the poly d pur · poly d pyr isomer. (4) The guanine-containing DNAs tested, with the exception of poly d(A-T-C) · poly d(G-A-T), bind AM and are: Micrococcus luteus DNA, salmon sperm DNA, poly d(G-C) · poly d(G-C), poly dG · poly dC, poly d(T-G) · poly d(C-A), poly d(T-C) · poly d(G-A), poly d(T-T-G) · poly d(C-A-A), poly d(T-T-C) · poly d(G-A-A) and poly d(T-A-C) · poly d(G-T-A). (5) The DNAs which are devoid of deoxyguanine, with the exception of poly dI, do not bind AM and are: poly d(A-T) · poly d(A-T), poly dA · poly dT, poly dI · poly dC and poly d(I-C) · poly d(I-C). The results are discussed in relation to two models for the AM-DNA complex, the hydrogen-bonded, “outside-binding” model of Hamilton, Fuller & Reich (1963) and the intercalation model of Muller & Crothers (1968) . The data are not consistent with the hydrogen-bonded model.

A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis

Abstract A simple and rapid microscale technique is described for the isolation of plasmid DNA which involves cell lysis with phenol, centrifugation, phenol extraction, ethanol precipitation, and RNase digestion. The plasmid DNA is of suitable purity and quantity for multiple restriction endonuclease digestions and bacterial transformations. This “miniprep” procedure is applicable for a variety of types of plasmids ranging in size from 2900 to 18,400 base pairs (bp) and for a number of Escherichia coli strains. The plasmids are rapidly cleaved by all restriction enzymes (total of 14) tested to date. Recombinant clones have been screened for insertions as small as 10 bp and as large as 5000 bp. The procedure takes ~3 h and has been routinely used to simultaneously analyze 24 candidate clones. This procedure is reliable and useful for rapid screening of recombinant DNA candidates where analysis by restriction endonuclease digestion is necessary.

DNA Structure and Gene Regulation

Publisher Summary The chapter discusses the role of deoxyribonucleic acid (DNA) structure in gene regulation. It also considers the properties and conformations of various DNAs and review static structure as well as dynamic transitions. Some of the goals of current research in this area are the following: (1) determination of the properties of regions of DNA along the high molecular-weight chromosomes; (2) identification of the interactions between the neighboring regions of DNA; (3) determination of how the properties of DNA influence the specificity or affinity of regulatory proteins that interact with specific regions of DNA; (4) identification of how the interaction of regulatory proteins, with DNA, modifies the properties of the DNA target site; (5) investigation of the presumed correlation between the physical properties of a region of DNA and its genetic function. A complete knowledge of the kinetic and equilibrium properties of the interaction of specific DNA target sites, with important regulatory proteins, is fundamental for the eventual comprehension of gene regulation. Cellular differentiation is the orderly and programmed expression of a family of genes. Alternatively, the malignant transformation of a cell will eventually be recognized as a faulty interaction between one or more key proteins and their DNA receptor sites.

Orientation relaxation of DNA restriction fragments and the internal mobility of the double helix.

The orientation relaxation of 15 DNA restriction fragments (43-4361 base-pairs) is characterized by measurements of linear dichroism using high electric field pulses. The off-field relaxation of fragments of 84 base-pairs or less can be described by single exponentials, which are related to the transverse rotational diffusion of the helix. Fragments of 95 base-pairs or greater exhibit an additional fast component with time constants around 100 ns for fragments of approx. 100 base-pairs, increasing with chain length to about 700 ns for a fragment with 258 base-pairs. The amplitude of this process increases from virtually zero at low fields (approximately equal to 10 kV) to a substantial limit contribution at high fields. According to these results, we suggest that electric fields induce stretching of the DNA fragments from a weakly bent to a more straight form and that the fast component reflects the internal mobility of the DNA chain. The slow off-field components of the orientation are discussed in terms of different models. The data up to helix lengths of about 400 base-pairs can be described by the weakly bending rod model from Hearst using 3.4 A rise per base-pair and 13 A axial radius of the helix. Both the weakly bending rod according to Hearst and the wormlike chain according to Hagerman and Zimm provide a persistence length of 500 A. The on-field relaxation is slower than the corresponding off-field process at low field strengths, but the on-field process is accelerated substantially at high electric fields. These observations are compared with model calculations of Schwarz.

Electric properties and structure of DNA restriction fragments from measurements of the electric dichroism

The electric dichroism of 17 homogeneous DNA fragments, ranging in size from 43 to 4362 base-pairs, has been analyzed in high electric fields. The orientation of the small fragments can be described in terms of an induced dipole moment, whereas the large fragments are oriented according to a constant dipole mechanism. In the intermediate size range, DNA orients according to an induced dipole mechanism at low field strengths and according to a constant dipole mechanism at high field strengths. From these observations we propose an orientation mechanism with a saturating induced dipole. The induced dipole observed at low field strengths is saturated at a field strength Eo within a transition range Em to give a constant dipole moment at high field strengths. These parameters together with the polarizability and the limit reduced dichroism are evaluated by a least-squares analysis of the experimental data. Eo and Em are found to decrease with increasing chain length from Eo approximately 40 kV/cm (Em approximately 14 kV/cm) at 65 base-pairs to 10 kV/cm (6 kV/cm) at 194 base-pairs. The polarizability is found to increase with the square of the chain length, whereas the saturated dipole increases with chain length N at low N and goes to a limit value at high N. The temperature dependence of the orientation parameters is found to be very small. The values obtained for the limit dichroism are between -1.0 and -1.3 for chain lengths between 60 and 1000 base-pairs, whereas values around -1.4 are observed at chain lengths greater than 1000 base-pairs. These data indicate that electric fields extend the contour of DNA strands at high chain lengths from a weakly bent to a more linear form. The variations of the limit dichroism observed for short fragments suggest sequence-dependent differences in the secondary structure of the helix. The experimental results are compared with numerical calculations based on simple polyelectrolyte models. For short fragments the magnitude of several electrochemical parameters can be adequately explained by a polarization of the ion cloud around the DNA molecules. However, these polyelectrolyte models do not adequately describe the observed chain length dependence of the orientation phenomena.

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

Abstract 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 × 106; the two repeating dinucleotide polymers (poly dTC:dGA and poly dTG:dCA), approximately 5 × 105; the four repeating trinucleotide polymers, approximately 2·5 × 105; and the two repeating tetranucleotide polymers, approximately 1 × 105. 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 DNAs 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 DNAs; 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.

The facile generation of covalently closed, circular DNAs with defined negative superhelical densities.

Abstract A rapid and simple method for generating samples of circular DNAs with defined superhelical densities is presented. A wide range of negative superhelical densities can be produced; namely, from 0 to 0.165 under the conditions employed. This method should facilitate the study of the detailed relationship between superhelical density and biochemical processes such as replication, transcription, and recombination.

Studies on polynucleotides: LXX. Synthetic deoxyribopolynucleotides as templates for the DNA polymerase of Escherichia coli: DNA-like polymers containing repeating tetranucleotide sequences☆☆☆

Abstract Short-chain deoxyribopolynucleotides containing repeating trinucleotide sequences serve as templates for the DNA-polymerase-catalyzed polymerization of deoxyribonucleoside triphosphates. The products are double-stranded DNA-like polymers containing, in both of the complementary strands, repeating trinucleotide sequences which were present in the short-chain templates. Four DNA-like polymers were thus prepared: poly dTTC:dGAA, poly dTTG:dCAA, poly dTAC:dGTA and poly dATC:dGAT. In every case, it was necessary to use as the templates, short segments of chain length 9 to 15 nucleotide units corresponding to both of the complementary strands of the desired DNA-like polymer. All of the polymers were shown to contain repeating trinucleotide sequences by comprehensive nearest-neighbor frequency analyses. Three of the four DNA-like polymers effectively served as templates for the synthesis of more of the same polymer in the presence of DNA-polymerase. Characterization of the DNA-like polymers with respect to molecular weight and buoyant density in a cesium chloride density-gradient is described in an accompanying paper.