The strand-separation transition of T2 bacteriophage DNA

Strand separation of T2 DNA has been investigated in a helix-destabilizing solvent. Temperature-shift experiments in which the conformation of the DNA is monitored by its viscosity, sedimentation behavior, and kinetics of helix formation show that a well-defined strand-separation transition follows the helix–coil transition usually observed by changes in absorbance. For T2 DNA, this strand-separation transition is 70% as broad as the helix–coil transition, and is characterized by extremely slow kinetics of conformational change in the population. Strand separation requires the expansion of the two-stranded coil observed at the end of the helix–coil transition. This expansion is apparently coupled with the disurption of the last remaining base pairs in the molecule. The expansion process increases the viscosity, and can be readily followed as a function of time and/or temperature. Subsequent separation of the expanded form into complementary strands results in a viscosity decrease, the net result of a reduction in hydrodynamic volume and the halving of the molecular weight. Only under conditions where the driving force for strand separation is large are these events at all synchronous in the population. When the kinetics of conformational change are complete in the strand-separation transition, a mixture of expanded forms and separate strands is observed; the breadth of the transition reflects differences in stability with respect to strand separation among the molecules in the population. The transition exhibits hysteresis and is not a reversible equilibrium between double-stranded and single-stranded forms. It appears that renucleation is kinetically forbidden within the strand-separation region.     

Studies on conformational changes in the DNA structure induced by protonation: reversible and irreversible acid titrations and sedimentation measurments

Reversible and irreversible conformational changes in the acid-induced denaturation of DNA were studied by spectrophotometric titration, sedimentation, and melting measurements. A GC-rich DNA (72 mole-%) shows complete or partial reversibility of the titration profiles within the pH region of transition from helix to coil, while AT-rich DNA (29 mole-%) is irreversible in its titration behavior at each acid pH below the onset of the transition. The results for GC-rich DNA further indicate distinct differences in the titration behavior, which can be attributed to differences in the frequency of GC clusters along the DNA molecule. Plots of the sedimentation coefficient and the parameter a_s^app against pH lead to the conclusion that conformational changes occur before the onset of the acid-induced helix–coil transition. These alterations are more pronounced upon protonation of larger GC-rich domains than of smaller ones, as concluded from very marked differences observed in the sedimentation–pH behavior of two GC-rich DNA's. An acid denaturation scheme for a GC-rich DNA segment is suggested. Reversibility of the acid denaturation is explained by the existence of stable, protonated, single GC base pairs in nonprotonated stacked single-stranded domains formed in the acid-induced transition region.     

Intermediates in the strand-separation transition of T7 DNA.

Sedimentation velocity runs as a function of temperature in the region of the alkaline helix-coil transition have enabled us to demonstrate the existence of stable two-stranded intermediates in the strand-separation process for T7 DNA. The strand-separation transition under these conditions has an intrinsic breadth of ～1°C, and within this temperature range (T_m + 2°C < T < T_m + 3°C) the intermediate forms are progressively converted (as a function of temperature) to single-stranded DNA. Parallel characterizations of the strand-separation transition by viscosity and absorbance–renaturation studies in the alkaline solvent are entirely consistent with the sedimentation experiments. Comparison of the experimental mean sedimentation coefficient of the intermediate forms with theoretical predictions for branched structures suggests that in these molecules the two strands are connected at a single point, not centrally located with respect to the ends of the molecule.     

Kinetics of the helix-coil transition in DNA

The kinetics of the helix-coil transition have been investigated for T2 and T7 phage DNA in a formamide-water-salt mixed solvent using a slow temperature perturbation technique (applicable to kinetic processes with rate constants ? 3 min^?1). In this solvent degradation of the DNA is effectively suppressed. Complex kinetic curves are observed by absorbance and viscosity measurements for the response to denaturing perturbations in the transition region. Analysis of the decay curves indicates that the denaturation reaction in this time range can be treated as a first-order reaction with a variable first-order rate parameter, k, the derivative of the logarithm of the absorbance or viscosity change with respect to time. In the approach to denaturation equilibrium in the transition region, the rate parameter is determined only by the instantaneous extent of denaturation of the molecules. Near equilibrium, the rate parameter assumes a constant value characteristic of the equilibrium state. In this region, where the denaturation reaction proceeds as a simple first-order process, both the decay of absorbance (reflected local conformational change) and the decay of solution viscosity (reflecting macromolecular conformational change) are characterized by the same constant value of k. In 83% formamide, 0.3M Na⁺, the rate parameter k for T2 DNA decreases from an extrapolated value of 2.0 min^?1 at 0% denaturation to 0.11 min^?1 at 90% denaturation. Rate parameters determined for T7 DNA at the same counterion concentration and fraction of denaturation are approximately five times as large as those cited for T2 DNA, indicating an inverse proportionality of rate constant to molecular length. On the other hand, simple first-order kinetic responses with constant k are obtained for renaturing perturbations within the transition, indicating that the mechanism of rewinding differs, in most cases, from that of unwinding. Only in the limit of very small perturbations about a given equilibrium position are the rate constants k obtained from denaturing and renaturing perturbations equal. For perturbations of finite size, it appears possible that an intramolecular initiation or nucleation event may precede rewinding and limit the rate of this reaction. The rate parameters again are approximately inversely proportional to molecular weight. The one exception to the first-power dependence on molecular weight appears when temperature jumps are made upward into the post-transition region. Here the molecular-weight dependence is second power, but complications arising from the different strand-separation properties of T2 and T7 DNA's make interpretation difficult. The previously used model of friction-limited unwinding appears to fit all the observations except for the molecular-weight dependence.     

Sedimentation and intrinsic viscosity behavior of PM2 bacteriophage DNA in alkaline solution

The sedimentation coefficient and intrinsic viscosity of nicked and closed circular PM2 bacteriophage DNA have been measured as a function of pH in the alkaline region. A gradual increase in the sidimentation coefficient, and a corresponding decrease in the intrinsic viscosity, are observed for the superhelical (closed) circle in the pH region from 10.5 to about 10.9. This has been tentatively interpreted in terms of the known dependence of sedimentation coefficient upon the number of superhelical turns. At slightly higher pH values, the curve passes through the minimum (sedimentation coefficient) and maximum (intrinsic viscosity) expected when the superhelical turns present at neutral pH are unwound by partial alkaline denaturation. Sedimentation studies of the relaxed (nicked) circular species have revealed the existence of DNA forms in the pH region from 11.27 to 11.37 which sediment considerably faster than the closed circle in the same pH region. These have been identified as partially denatured nicked circles, in which varying fractions of the duplex structure have undergone alkaline denaturation, but strand separation has not yet occurred. Varying fractions of a slower species, either undenatured or completely denatured nicked circles, are also observed in some of these experiments. A corresponding result is observed in the intrinsic viscosity vs. pH curve. When nicked circular PM2 DNA is exposed to various alkaline pH's, rapidly neutralized, and sedimented at neutral pH, the expected sharp transition from native to denatured (strand-separated) molecules is seen. However, a very narrow pH range is noted in which native and denatured forms coexist in a single experiment. The above experiments carried out upon the closed form also reveal a narrow pH range in which the bulk of the transition from native closed circles to the collapsed cyclic coil takes place, in acccord with an earlier study on a different DNA. This transition is shown never to be completely effected, however, as there is a fraction (7–8%)of the closed circles which renature to the native form, regardless of the alkaline pH employed. This same phenomenon was not observed in the case of artificially closed λb₂b₅c DNA circles. Possible explanations for some of the above results are discussed.     

Thermal transition of DNA measured by NMR spin-echo technique

The thermal helix–coil transition of DNA can be studied by means of the spin-echo technique. The longitudinal spin–lattice relaxation time T1 and the transvense spin–spin relaxation time T2 of the DNA sample show a similar phase transition as observed spec-trophotometrically with increasing and decreasing temperatures. Four slopes on the T1 and T2 temperature relationship curves were found and interpreted as functions of nonrelational hydration of the DNA molecule. The T1 and T2 values differ depending on the native or denatured state of the DNA molecule. The importance of the dynamic equilibrium between water molecules in the hydration lattice and steps in the denaturation of the DNA molecule are discussed. This phenomenon may be directly related to the nonrotational hydration.     

Volume changes accompanying thermal denaturation of deoxyribonucleic acid. II. Denaturation at alkaline pH

The change in apparent molal volume ? of DNA on thermal denaturation in carbonate buffer at pH 11.0 has been determined by the dilatometric method. It was found that ? increases sigmoidally during the helix–coil transition. Several methods, including a colorimetric technique that closely simulates the conditions used in the dilatometric experiments, were employed to estimate the protons lost by the DNA during the transition. These measurements indicated that the extent of the proton loss depends on the counterion present, increasing in the order Li⁺ < Na⁺ < K⁺ < Cs⁺. The major part of the volume changes observed during the denaturation is due to the volume changes expected to accompany the transfer of protons from the bases guanine and thym ne to carbonate ions. As has been previously reported for the denaturation of DNA at neutral pH, the volume change directly due to the change in shape of the polymer molecules is so small as to be experimentally undetectable.     

Pressure dependence of the helix-coil transition temperature for polynucleic acid helices

Thermal denaturation of DNA's and the corresponding helix–coil transformation of artificial polyribonucleic and polydeoxyribonucleic acids have been studied extensively both theoretically^1–13 and experimentally. ^14–30 Much less work has been carried out on the properties of these polynucleic acids at high pressure, and in particular, on the presure dependence of the helix–coil transition temperature.^31–33 Light-scattering techniques have been used in this study to measure the pressure dependence of the helix–coil transition temperature of the two- and three-stranded helices of polyriboadenylic and polyribouridilic acids and of calf thymus DNA. From the slopes of the transition temperature vs. pressure curves and heats of transition obtained from the literature,^20,34 the following volume changes from these helix–coil transitions have been obtained: (a) ?0.96 cc/mole of nucleotide base pairs for the poly (A + U) transition, (b) +0.35 cc/mole of nucleotide base trios for the poly (A + 2U) transition, and (c) +2.7 cc/mole of nucleotide base pairs for the DNA transition. The relative magnitudes and signs of these volume changes which show that poly (A + U) is destabilized by increased pressure, whereas poly (A + 2U) and calf thymus DNA are stabilized by increased pressure, indicates that further development of the helix–coil transition theory for polynucleotides is needed.     

Alpha-particle-induced changes in the stability and size of DNA

The effect of alpha-particle radiation on the thermal stability and size of calf thymus DNA molecules in deoxygenated aqueous solutions was investigated by thermal transition spectrophotometry, pulsed-field gel electrophoresis, and standard agarose gel electrophoresis. The thermal transition of DNA from helix to coil was studied through analysis of the UV A(260) absorbance. The results obtained for alpha particles of mean LET of 128 keV microm(-1) reveal a dual dose response: a tendency for thermal stability of the DNA helix at "low" doses, followed by an increasing instability at higher doses. The same phenomenon was observed for the mean molecular weight of DNA molecules exposed to alpha particles. The results reported here for alpha particles in the low-dose region of 0-16 Gy are consistent with our previous hypothesis of inter- and intramolecular interactions of a covalent character in gamma-irradiated DNA molecules in the dose region of 0-4 Gy.     

Bifilar enzyme-sensitive sites in ultraviolet-irradiated DNA are indicative of closely opposed cyclobutyl pyrimidine dimers.

Incubation of UV-irradiated DNA with pyrimidine dimer-DNA glycosylase in cell-free lysates prepared from Micrococcus luteus results in the appearance of double-strand breaks. It has previously been assumed that such double-strand breaks result from cleavage at closely opposed dimers. We have used hybrid molecules of bacteriophage T7 DNA comprised of two unirradiated strands, two UV-irradiated strands, or one unirradiated and one UV-irradiated strand to test this hypothesis. Bifilar cleavage was observed only with molecules consisting of two irradiated strands and no bifilar cleavage was observed after the monomerization of pyrimidine dimers by enzymatic photoreactivation. Our results indicate that at least 80% of the double-strand breaks result from cleavage at closely opposed dimers and that the induction of dimers in one strand does not influence the induction of dimers at closely opposed positions in the complementary strand of a DNA double helix.     

Kinetics of denaturation of DNA

The kinetics of denaturation of DNA have been studied by relaxation techniques. Examination of the terminal relaxation times for a variety of DNA's under a variety of conditions has shown that DNA denaturation is principally a hydrodynamically limited process. Measurements within the helix–coil transition have demonstrated that the experimentally measured terminal relaxation times are a function of the following: (1) position in the helix–coil transition; (2) ionic strength of the solvent; (3) solvent viscosity; (4) DNA concentration; (5) molecular weight; (6) number and position of single-strand breaks. The dependence of the terminal relaxation time on the above mentioned factors can be attributed to hydrodynamic effects. Thus a hydrodynamic model for DNA unwinding is required. The model which best fits the data involves the assumption of a rotational frictional coefficient independent of molecular weight. This assumption is suggested by the fact that the relaxation time is proportional to the first power of the molecular weight.     

Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA

The vaccinia virus genome is a single, linear, duplex DNA molecule whose complementary strands are naturally cross-linked. The molecular weight has been determined by contour length measurements from electron micrographs to be 122 ± 2.2 × 10⁶. Denaturation mapping techniques indicate that the nucleotide sequence arrangement of the DNA is unique. Two forms of cross-linked vaccinia DNA were observed in alkaline sucrose gradients. The relative S-values of the two cross-linked species were appropriate for a single-stranded circle and a linear single strand, each with a molecular weight twice that expected for an intact, linear, complementary strand of vaccinia DNA. The fraction of sheared vaccinia DNA able to “snap back” after denaturation suggested a minimum of two crosslinks per molecule. Full-length single-stranded circles were observed in the electron microscope after denaturation of vaccinia DNA. Partial denaturation produced single-stranded loops at the ends of all full-length molecules. Exposure of native vaccinia DNA to a single strand-specific endonuclease isolated from vaccinia virions caused disruption of the cross-links, as assayed by alkaline sedimentation, and produced free single-strand ends when partially denatured DNA was observed in the electron microscope. We conclude that vaccinia DNA contains two cross-links, one at or near (within 50 nucleotides) each end in a region of single-stranded DNA. Two models for the cross-links are presented.     

On the double peak of NMR in the helix—coil transition region

To attempt to resolve the controversy over “fast” and “slow” helix–coil transition rates in polypeptides, nuclear magnetic resonance spectra were measured for monodisperse poly-γ-benzyl-L -glutamate (PBLG). These results were compared with simulated line spectra which were computed by taking the molecular-weight distribution into consideration. Broad but single peaks have been observed in 220 mHz nmr for the α-CH and NH proton resonance spectra in the transition region. The shape of the line changes with the extent of polydispersity. Assuming a fast conversion rate, a molecular model of the helix–coil transition simulates these results. Consequently, the double peak which has been observed in the nmr of polypeptides at the helix–coil transition region is shown to result from the polydispersity in molecular weight.     

Study of helix-coil transition of DNA by dielectric constant measurement

The thermal helix–coil transition of DNA was studied by means of dielectric constant measurements. The dielectric dispersion of native helical DNA is characterized by a large dielectric increment and a large relaxation time, whereas that of denatured coil DNA is characterized by a small dielectric increment and a small relaxation time. The dielectric dispersion of partially denatured DNA is of particular interest. At the intermediate stage of the helix–coil transition, dispersion curves which are different from either that of helix DNA or that of coil DNA appear. This is particularly pronounced for large DNA. This indicates the presence of an intermediate form of DNA. Flow birefringence measurements were carried out simultaneously. The negative birefringence of helical DNA diminishes as the helix–coil transition proceeds. However, the extinction angle remains constant, as long as it can be measured. These results indicate the absence of intermediate forms during the helix–coil transition. The discrepancy between dielectric and birefringence measurements can be resolved by assuming that the intermediate forms are not birefringent. The size distribution of native DNA and of the indicated intermediate form of DNA was studied. It is found that a logarithmic normal distribution function explains the distribution of size of DNA reasonably well.     

NMR characterization of residual structure in the denatured state of protein L

Triple-resonance NMR experiments were used to assign the (13)C(alpha), (13)C(beta), (15)N and NH resonances for all the residues in the denatured state of a destabilized protein L variant in 2 M guanidine. The chemical shifts of most resonances were very close to their random coil values. Significant deviations were observed for G22, L38 and K39; increasing the denaturant concentration shifted the chemical shifts of these residues towards theory random coil values. Medium-range nuclear Overhauser enhancements were detected in segments corresponding to the turn between the first two strands, the end of the second strand through the turn between the second strand and the helix, and the turn between the helix and the third strand in 3D H(1), N(15)-HSQC-NOESY-HSQC experiments on perdeuterated samples. Longer-range interactions were probed by measuring the paramagnetic relaxation enhancement produced by nitroxide spin labels introduced via cysteine residues at five sites around the molecule. Damped oscillations in the magnitude of the paramagnetic relaxation enhancement as a function of distance along the sequence suggested native-like chain reversals in the same three turn regions. The more extensive interactions within the region corresponding to the first beta-turn than in the region corresponding to the second beta-turn suggests that the asymmetry in the folding reaction evident in previous studies of the protein L folding transition state is already established in the denatured state.     

The rate of strand separation in alkali of DNA of irradiated Mammalian cells

When mammalian cells are treated with alkali of pH at about 12, the cells are lysed and the released DNA starts to uncoil. This process of DNA strand separation is accelerated if the cells have been exposed to ionizing radiation, and the effect is clearly detectable in the dose range 10-100 rads. The rate of strand separation is also influenced by temperature and ionic strength of the alkaline solution. The kinetics of DNA strand separation in alkali is studied for three conditions in terms of ionic strength and temperature, chosen in such a way that the effect of irradiation may conveniently be studied in the dose range 10 rads to 20 krads. The accelerating effect of ionizing radiation on DNA strand separation is probably due to DNA strand breakage and the technique described is thus a sensitive method of studying such damage to DNA. A model for the strand-separation process, based on the assumption that strand breakage causes the accelerating effect, is proposed and found to describe the experimental data adequately.     

Helix–coil transitions of compositionally heterogeneous DNA

Fragmented and mitomycin C cross-linked E. coli DNA was fractionated according to base composition by means of hydroxylapatite chromatography and density-gradient centrifugation in order to determine the effect of compositional heterogeneity on the breadth of the helix–coil transition. The transitions of some of the fractions are broader than that of the unfractionated DNA, due, presumably, to nonrandom sequences in molecules of 5 × 10⁵ daltons. Analysis of the transition breadths in terms of the known heterogeneity leads to reconsideration of current DNA helix–coil transition theory. We propose that partially denatured states include those for which the chains do not remain in strict register. Denaturation profiles are comprehensible only if this multitude of entropically favorable, degenerate states is included in the theory.