Relationship between the molecular structure of aqueous solutions of polyethylene glycol and the compactness of double-stranded DNA molecules

The dependence of viscosity of the water solutions of poly(ethylene glycol) (PEG) on the molecular weight has been studied. It has been shown that there is a "transitional" region in PEG properties which accounts for the formation of fluctuation polymer network of the PEG molecules. It has been shown that the "transitional" region in properties of PEG which appears at a certain concentration of PEG (CtrPEG) is characteristic of the PEG preparations with molecular weights exceeding 600 and dependence of the value of CtrPEG on the molecular weight of PEG was obtained. Compactization of double-stranded DNA molecules in PEG-containing water-salt solutions has been studied and the dependence of the value of CcrPEG, . i.e. the concentration of PEG at which the compact particles of DNA appear in the solution, on the molecular weight of PEG was obtained. The correlation between these two dependences reflecting quite different physico-chemical processes shows that the double-stranded DNA molecules are constrained within the polymer network of the PEG molecules. The influence of ionic strength and ionic composition of the solution on the formation of a compact form was investigated. The transition of the DNA molecules from a linear to a compact state may occur only at a definite value of ionic strength of the solution. This transition may occur at the change of K+ for Na+ cations (at a constant value of CPEG). The extent of compactization of the DNA molecules in PEG-containing water-salt solutions is monitored by the molecular structure and by the ionic strength of the solvent. It is supposed that the peculiarities of compactization of the DNA molecules in PEG-containing water-salt solutions reflect some characteristics of conformational transitions of the DNA molecules which occur in vivo.     

Electric birefringence of restriction enzyme fragments of DNA: optical factor and electric polarizability as a function of molecular weight

The electric birefringence of restriction enzyme fragments of DNA has been investigated as a function of DNA concentration, buffer concentration, and molecular weight, covering a molecular weight range from 80 to 4364 base pairs (bp) (6 × 10⁴–3 × 10⁶ daltons). The specific birefringence of the DNA fragments is independent of DNA concentration below 20 μg DNA/ml, but decreases with increasing buffer concentration, or conductivity, of the solvent. At sufficiently low field strengths, the Kerr law is obeyed for all fragments. The electric field at which the Kerr law ends is inversely proportional to molecular weight. In the Kerr law region the rise of the birefringence is accurately symmetrical with the decay for fragments ≤ 389 bp, indicating an induced dipole orientation mechanism. The optical factor calculated from a 1/E extrapolation of the high field birefringence data is ?0.028, independent of molecular weight; if a 1/E² extrapolation is used, the optical factor is ?0.023. The induced polarizability, calculated from the Kerr constant and the optical factor, is proportional to the square of the length of the DNA fragments, and inversely proportional to temperature. Saturation curves for DNA fragments ≤ 161 bp can be described by theoretical saturation curves for induced dipole orientation. The saturation curves of larger fragments are broadened, because of a polarization term which is approximately linear in E, possibly related to the saturation of the induced dipole in high electric fields. This “saturated induced dipole” is found to be 6400 D, independent of molecular weight. The melting temperature of a 216-bp sample is decreased 6°C in an electric field of 8 kV/cm, because the lower charge density of the coil form of DNA makes it more stable in an electric field than the helix form.     

The double-stranded DNA stability in presence of a flexible polymer

The mixture of the short segments of double-stranded DNA and a flexible polymer are addressed. It is shown that in the condensed phase, rigid DNA molecules exhibit transition between isotropic and orientationally ordered phases. It is shown that orientational ordering stabilizes the secondary structure of double-stranded DNA that could be relevant for the regulation of the gene expression at the condensed state of DNA.     

Dissimilar kinetic behavior of electrically manipulated single- and double-stranded DNA tethered to a gold surface

We report on the electrical manipulation of single- and double-stranded oligodeoxynucleotides that are end tethered to gold surfaces in electrolyte solution. The response to alternating repulsive and attractive electric surface fields is studied by time-resolved fluorescence measurements, revealing markedly distinct dynamics for the flexible single-stranded and stiff double-stranded DNA, respectively. Hydrodynamic simulations rationalize this finding and disclose two different kinetic mechanisms: stiff polymers undergo rotation around the anchoring pivot point; flexible polymers, on the other hand, are pulled onto the attracting surface segment by segment.     

Nonlinear electrophoresis and focusing of macromolecules

Effects of nonlinear dependence drift velocity of (double-stranded) DNA vs. electric field strength were investigated. In comparatively weak fields, the molecular drift velocity is proportional to the external electric field, while in strong fields there is additional nonlinear component. This effect offers possibilities to manipulate the total drift velocity at will-the macromolecules of different size can be made to move in opposite directions in pulsed field gel electrophoresis.A new approach for focusing DNA molecules based on nonlinear electrophoresis and geometric trapping in electric fields is proposed. The focusing is carried out in an alternating nonuniform electric field, created by using a wedge gel with hyperbolic boundaries. It is shown that the fractions separated in such wedge retain their rectilinear shape.Gel electrophoresis experiments supported the possibility of a pronounced nonlinear focusing of DNA molecules. This nonlinear separation technique presents encouraging prospects for micromanipulating systems and also for preparative isolation of long DNA fragments and development of new separation methods for bacterial fingerprinting.     

A DNA glycosylase from Escherichia coli that releases free urea from a polydeoxyribonucleotide containing fragments of base residues.

A poly (dA, [2-14C]dT) copolymer has been synthesized using terminal deoxynucleotidyltransferase. Treatment of the polydeoxyribonucleotide with potassium permanganate converts the thymine residues to urea and N-substituted urea derivatives, while the adenine residues are resistant to oxidation. This damaged polymer has been annealed with an equimolar amount of poly (dT) to generate a double-stranded polydeoxyribonucleotide containing scattered fragmented base residues, which are radioactively labeled selectively. On incubation of the latter with crude cell extracts from E. coli, free urea is released by a DNA glycosylase activity. The enzyme has been partly purified, and appears to be different from previously studied DNA glycosylase. It shows a strong preference for a double-stranded substrate, exhibits no cofactor requirement, and has a molecular weight of 20000 - 25000. Since fragmentation of pyrimidine residues is a major type of base lesion introduced in DNA by exposure to ionizing radiation, it seems likely this DNA glycosylase is active in repair of X-ray-induced lesions.     

Electro-optical studies on synthetic polyelectrolytes: IV. Electric polarizability and conformation of poly-N-methyl-2-vinylpyridinium bromide in aqueous solution

Measurements of the relaxation time on aqueous solutions of the title polyelectrolyte as a function of the concentration and of the molecular weight show that its conformation at very high dilution can be better accounted for by a weakly bending rod or worndike chain model, with persistence length ranging from 200 to 400 Å. The analysis of the field strength dependence of the electric birefringence yields electric polarizability values which increase sharply with the dilution and are not significantly dependent upon the molecular weight. This has been tentatively interpreted on the basis of the theories derived by Oosawa and by Mendel and Van der Touw. The partially flexible model proposed by the latter authors allow to identify the electric polarizability calculated from electro-optical data to the specific dielectric increment measured in the high frequency range; both parameters appear to be molecular weight independent. The electric polarizability obtained from the Kerr effect would be originated in the induced dipoles caused by the delocalization of the bound counterions along rigid subunits whose length seems however to differ from the persistence length.     

Enzymes from Micrococcus luteus involved in the initial steps of excision repair of spontaneous DNA lesions: uracil-DNA-glycosidase and apurinic-endonucleases.

Uracil-DNA-glycosidase that releases free uracil from single-stranded or double-stranded deaminated DNA and poly d(A-U) has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 16,000 and can be separated from uracil-endonuclease and endonucleases (AP-endonucleases) specific for apurinic and apyrimidinic sites. Uracil-DNA-glycosidase does not act on guanine residues opposite uracil in double-stranded DNA and on xanthine in deaminated DNA. The glycosidase generates apyrimidinic sites which can serve as substrate sites for different AP-endonucleases from M. luteus.     

Morphology and Physical Properties of Staphylococcus Bacteriophage P11-M15

The Group B Staphylococcus phage P11-M15 is shown to be 51% protein and 49% deoxyribonucleic acid (DNA). The intact virion has a molecular weight of 66.7 x 10(6) daltons. The purified viral DNA has a molecular weight of 32.7 x 10(6) daltons. The intact virion is shown to be composed of a polyhedral head which is attached at one of its vertices to a flexible tail having helical symmetry. The tail structure is terminated by a complex baseplate which has sixfold symmetry. The virion contains a single molecule of double-stranded DNA which has no apparent single-strand nicks or single-stranded terminal redundancies.     

Isolation and purification of poly(ADP-ribose) glycohydrolase from pig thymus

Poly(ADP-ribose) glycohydrolase has been purified about 12 300-fold from pig thymus with a recovery of 8.5%. The specific activity of the purified enzyme is 13.8 mumol min -1 mg protein -1. The molecular weight was estimated to be 59 000 by gel filtration through Sephadex G-100 in a non-denaturing solvent. Analysis of the final preparation by sodium dodecyl sulphate gel electrophoresis reveals two protein bands of molecular weight, 61 500 and 67 500. The Km value for poly(ADP-ribose) is estimated to be 1.8 microM monomer units. The enzyme preparation is free from phosphodiesterase, NADase and ADP-ribosyltransferase activities. The purified enzyme is inhibited by cyclic AMP, ADP-ribose, naphthylamine, histones H1, H2A, H2B, H3, polylysine, polyarginine, polyornithine and protamine. The inhibition by histone is relieved by an equal mass of DNA. Single-stranded DNA, poly(A), poly(I) and polyvinyl sulphate were inhibitory, but double-stranded DNA was not inhibitory.     

The dichroism of DNA in electric fields

We have studied the dichroism of various samples of calf thymus DNA (of molecular weight from 3 × 10⁵ to 7 × 10⁶) in pulsed electric fields. The results may be summarized as follows:

• 1 We find that calf thymus DNA behaves in electrical orientation as if it possessed a large permanent dipole moment. This apparent moment is sensitive to such effects as Mg⁺⁺ binding which lower the net charge on DNA.
• 2 The limiting dichroism at infinite field corresponds to an angle of at least 80% between the transition moments at 265 nm and the helix axis, and could be consistant with a number of known forms of DNA. This result is independent of DNA molecular weight. There is evidence that the conformation may be different in 80% ethanol.
• 3 The dichroism relaxation curves contain a component with a relaxation time of about 8 μsec, which is nearly independent of molecular weight, and a longest component which behaves either according to the Broersma theory for low-molecular-weight samples, or the Zimm-Rouse theory at high molecular weights.

     

Solution conformation of DNA

Optical observations on linear B-form DNA by the method of electric linear dichroism show that the value of the limiting reduced dichroism is molecular weight-dependent, increasing with molecular weight to a limit of about ?1.41 ± 0.02 in aqueous solution. These data and the rotational relaxation times obtained from the decay of the dichroism when the orienting field is instantaneously removed, imply the existence of a non-linear tertiary equilibrium structure for DNA. The data indicate that the essential B-form parameters of the double-stranded DNA are retained in this tertiary structure, and are not consistent with a DNA structure in which the base-pairs have a 34 ° propeller-like twist (Hogan et al., 1978). The interpretation of the dichroism data is supported by a clear demonstration that the magnitude of dichroism change in ethanol corresponds to that expected for the B to A-form structural transition as determined from X-ray diffraction. We propose that the tertiary structure of B-DNA is a helical coil and suggest the limits of the structural parameters of the coil consistent with the observations.     

The role of the ionic environment in the orientation of nucleic acids in electric fields

The relationship between polyelectrolyte theories based on linear charge density models and the electric-field induced orientation of the polyelectrolytes, poly(A), poly(C) and DNA is examined by varying their ionic environment with respect to ionic strength and acidity. The degree of counterion condensation on the polyelectrolytes predicted by the theories of Manning and Record is shown to be related linearly to the orientation as measured by their dichroism in the field. Micro-structural differences between poly(A) and poly(C) account for the differences in their dependence on the pH of the medium which affects the counterion condensation and thus the polarization in the orienting electric fields. The results consequently support recent treatments of linear polyelectrolytes having a high charge density which model them as smoothly charged linear polyions, but indicate that these models are insufficient to account for some of the effects of microstructural variations.     

Adjustable ‘cross-linking’ of neighboring DNA molecules in liquid-crystalline dispersions through (daunomycin-copper) polymeric chelate complexes

The interaction of daunomycin molecules with double-stranded DNA in the liquid-crystalline state was investigated. It was shown that at a certain extent of daunomycin binding a change of the mechanism of anthracycline orientation with reference to the DNA chain occurs. This is testified by the alteration of the sense of spatial packing of the DNA molecules in liquid-crystalline dispersions formed as a result of phase separation in poly(ethyleneglycol)-containing solutions, as well as by the onset of the reaction of daunomycin with divalent copper ions. Using this reaction, polymeric (daunomycin-copper) chelate cross-links between the DNA molecules fixed in the liquid-crystalline dispersions were formed. The length of such cross-links is adjusted by the distance between the DNA molecules, which, in turn, depends on the concentration of poly(ethyleneglycol) used for phase separation. The above molecular building mechanism may lead to new interesting applications.     

Transient orientation of linear DNA molecules during pulsed-field gel electrophoresis.

The transient orientation of lambda DNA and lambda-DNA oligomers has been measured during pulsed field gel electrophoresis. The DNA becomes substantially aligned parallel to the electric field E. In response to a single rectangular pulse, orientation shows an overshoot with a peak at 1 second, then a small undershoot, and finally a plateau. When the field is turned off, the orientation dissipates in two distinct exponential phases. Field inversion leads to periods of orientation with intervening periods of reduced orientation as the chains reverse direction. Field inversion pulses applied to linear oligomers of lambda-DNA show that orientation responses slow down but increase in amplitude as molecular weight increases, for a given field. Because DNA stretching and alignment parallel to E are expected to correlate with DNA velocity, the velocity in response to a pulsed field is also expected to exhibit an overshoot.     

Effect of pulsed and reversing electric fields on the orientation of linear and supercoiled DNA molecules in agarose gels

When linear or supercoiled DNA molecules are imbedded in agarose gels and subjected to electric fields, they become oriented in the gel matrix and give rise to an electric birefringence signal. The sign of the birefringence is negative, indicating that the DNA molecules are oriented parallel to the electric field lines. If the DNA molecules are larger than about 1.5 kilobase pairs, a delay is observed before the birefringence signal appears. This time lag, which is roughly independent of DNA molecular weight, decreases with increasing electric field strength. The field-free decay of the birefringence is much slower for the DNA molecules imbedded in agarose gels than observed in free solution, indicating that orientation in the gel is accompanied by stretching. Both linear and supercoiled molecules become stretched, although the apparent change in conformation is much less pronounced for supercoiled molecules. When the electric field is rapidly reversed in polarity, very little change in the birefringence signal is observed for linear or supercoiled DNAs if the equilibrium orientation (i.e., birefringence) had been reached before field reversal. Apparently, completely stretched, oriented DNA molecules are able to reverse their direction of migration with little or no loss of orientation. If the steady-state birefringence had not been reached before the field reversal, complicated orientation patterns are observed after field reversal. Very large, partially stretched DNA molecules exhibit a rapid decrease in orientation at field reversal. The rate of decrease of the birefringence signal in the reversing field is faster than the field-free decay of the birefringence and is approximately equal to the rate of orientation in the field (after the lag period).(ABSTRACT TRUNCATED AT 250 WORDS)     

The flexibility of alternating dA-dT sequences

The flexibility of alternating poly (dA-dT) has been investigated by the technique of transient electric dichroism. Rotational relaxation times, which are very sensitive to changes in the end-to-end length of flexible polymers, are determined from the field free dichroism decay curves of four, well defined fragments of poly (dA-dT) ranging in size from 136 to 270 base pairs. Persistence lengths, calculated from the results of Hagerman and Zimm (Biopolymers (1981) 29, 1481-1502), are in the range 200-250 A. This makes alternating dA-dT sequences about twice as flexible as naturally occurring, "random" sequence DNA. Considering a bend around a nucleosome, for example, this difference in persistence length translates to an energy difference between poly (dA-dT) and random sequence DNA of 0.17 kT/base pair or 1 kcal per 10 base pair stretch. This energy difference is sufficiently large to suggest that dA-dT sequences could serve as markers in DNA packaging, for example, at sites where DNA must tightly bend to accommodate structures.     

Light scattering during the hysteresis effect in poly(A)·2poly(U)

Light-scattering studies on buffered aqueous solutions of the triple-stranded polyribonucleic acid poly(A)·2poly(U) were carried out at neutral pH and during titration. At pH 7.1 and 22°C, a sample of commercially available polymer in 0.005M phosphate buffer gave a Zimm plot which yielded values for the weight-average molecular weight, M _w, of 874,000 ± 1800 g/mol, a root-mean-square radius, ρ of 930 ± 22 Å, and a second viral coefficient of 0.51 ± 0.05 × 10 ^?3 cm³g^?1 mol. The light-scattering data were also analyzed by serval linear and nonlinear least-squares programs which were devised to determine the model (e.g., rod, coil, or zigzag) which could best describe the shape of the molecule. It was found that a rodlike model, perhaps with a few bends, was in best overall agreement with the data. The assumption that the molecule is a thin rod leads to a value for the linear density of 206 g mol^?1 Å^?1 and a translation of 3.3 Å per residue. These values are also in close agreement with those expected for a triple-stranded, thin, base-stacked molecule. During titration from neutral pH with 0.1M HCl, the observed apparent molecular weight slowly increased until at about pH 3.5 a sudden, large increase (about 30-fold) occurred. The root-mean-square radius, on the other hand, after an initial small decrease (of about 25%), also exhibited a large increase (about 4-fold). Upon back titration with 0.1M NaOH, the molecular parameters did not retrace the original path, but instead exhibited hysteresis—the M _w and ρ _z are both larger on the basic branch than on the acid branch at a corresponding pH. A plot of long ρ _z against log(M _w) during the interval in which the high-moelcular-weight form was present (below pH 3.5 on the acid branch, and on the basic branch) gave a straight line with a slope of ?. This suggests that the aggregates were composed of some tens of rather open radom coils, presumably of poly(A)·poly(A), and that the hysteresis may be caused under conditions by the metastability of the entangled coils.     

A conformation change of single stranded polyriboadenylate induced by an electric field

Absorbance measurements performed with high molecular weight poly A at pH 8 show that the degree of single strand stacking present at high ionic strength is reduced at low ionic strengths. The salt dependence of the poly A conformation is assigned to an electrostatic repulsion between subsequent turns of the single strand “helix” structure. - Electric fields of 5 to 80 kV/cm induce an increase in the poly A absorbance consistent with a decrease in the ion concentration in the environment of the polymer. The increase of the absorbance is a linear function of the field strength suggesting that the conformation change is caused by a dissociation field effect. At increasing ionic strength, threshold values of the electric field strength have to be exceeded in order to induce measurable absorbance changes. - The time required for the conformation change decreases from about 2 μsec at 10⁻⁴ M ionic strength to about 0.3 μsec at high ionic strengths. At low ionic strengths the ion equilibration may influence the rate limiting step, whereas the arrangement of the nucleotide residues into the ordered structure is rate limiting at high ionic strengths.     

Bovine thymus poly(adenosine diphosphate ribose) polymerase. Physical properties and binding to DNA

Purified bovine thymus poly(adenosine diphosphate ribose) polymerase is a monomeric protein with a single polypeptide chain having a molecular weight of approximately 130,000, determined by sodium dodecyl sulfate-gel electrophoresis, analytical ultracentrifugation, and gel filtration. A high frictional ratio (1.81) indicated that the molecule has an elongated shape, or a high solvation, or both. The enzyme is a basic protein (pI 9.8), and amino acid analysis showed a relatively high lysine content. The enzyme activity is dependent on double-stranded DNA and is solely correlated with single- or double-stranded breaks on the DNA. Filter binding assay technique showed that the enzyme-activating efficiency of DNA correlated sufficiently with its enzyme-binding efficiency. Thus, a very high enzyme-activating efficiency of a DNA fraction (active DNA) which was separated from the crude enzyme fraction is mainly due to its high enzyme-binding efficiency. It was also shown that single-stranded DNA and heparin had a strong inhibitory effect on the binding of the enzyme to double-stranded DNA, whereas competitive inhibitors did not affect the binding, We interpret these results to indicate that the binding of the enzyme to double-stranded DNA is a prerequisite step to its catalytic activity and has a dual function: (a) to position the enzyme on specific binding sites such as single- or double-stranded breaks on the DNA, and (b) to induce an active conformation of the enzyme.