Changes in Raman vibrational bands of calf thymus DNA during the B-to-A transition

The B -to-A conformational transition of calf thymus DNA fibers was followed employing Raman spectroscopy. The transition was induced by soaking DNA fibers in water/ethanol mixtures increasing from 60 to 85% ethanol (v/v). Intensity changes of 17 Raman vibrational bands were quantified in the region from 400 to 860 cm^?1. Two bands at 500 and 784 cm^?1 were employed as internal standards. These bands do not appear to change in intensity with ethanol concentration. Large intensity changes relative to these two bands are observed between 70 and 74% ethanol for backbone vibrations at 708, 808, and 835 cm^?1, and base vibrations at 682, 730, and 750 cm^?1. These results indicate that a highly cooperative conformational change takes place between different portions of DNA in the B -to-A transition. Relative intensity changes preceding the onset of the major transition are observed in only two bands; at 835 cm^?1, assigned to a ribose–phosphate vibration, and at 750 cm^?1, assigned to thymine. The implications of these pretransition changes are discussed.     

Phosphorus NMR spectra of natural DNA fragments in the course of the B-to-A conformational transition

Changes in the ³¹P-nmr spectra of sonicated natural DNA fragments were investigated in ethanol solutions where the fragments underwent, as checked by CD, the B-to-A conformational transition. The study produced the following conclusions: (1) The high DNA concentrations used for the ³¹P-nmr measurements promote the transition compared to dilute solutions that are commonly used for CD measurements. (2) The B-to-A transition was reflected in a cooperative downfield shift of the DNA ³¹P-nmr resonance, consistent with unwinding of the double helix. (3) Prior to the transition, the changes in chemical shift of double-and single-stranded DNAs were almost identical. It thus appears that the effect of ethanol on the geometry and hydration of phosphodiester linkages does not depend heavily on DNA base–base interactions. (4) The A-form resonances were 30–40% narrower than the B-form resonances, which is attributed to marked sequence-dependent variations in the latter conformation and to their reduction in the former. (5) The B-form DNA aggregated in the concentrated ³¹P-nmr samples in the presence of ethanol, judged from a milky opalescence of the solution and a substantial broadening of its ³¹P-nmr resonance. The broadening abruptly disappeared as soon as DNA adopted the A-form so that DNA, in dependence on the secondary structure, showed different tendencies to condense in the presence of ethanol. The condensation increased cooperativity of the B-to-A interconversion.     

Infrared and X-ray diffraction study of the effect of protonation of DNA on its B-to-A transition

The influence of H+ on the secondary structure of DNA and on its B-to-A transition has been studied by employing X-ray diffraction and infrared spectroscopy. Helical parameters for DNA molecules with different degrees of protonation were determined. It was shown that H+ binding stabilizes the B-form of DNA in fibers over a wide range of water and inorganic salt content. Only 0.03 H+ bound per nucleotide is sufficient to prevent the B-to-A transition caused by decreasing relative humidity in DNA fibers containing 4% NaCl. The effectiveness of B-form stabilization by H+ is explained by changes in DNA-solvent molecule interactions, especially in the major groove of double helices.     

A reduced set of coordinates for modeling DNA structures: (I). A B-to-A transition pathway driven by pseudorotational angle.

The A-DNA and the B-DNA are two well characterized polymorphous forms of DNA duplex. By using Metropolis Monte Carlo Simulations in a reduced coordinate space, we have shown that the B in equilibrium with A transitions can be induced by forcing pseudorotational angle (W) to change between C3'-endo and C2'-endo puckerings. The energy barrier for the transition pathway is less than 10 Kcal.mol-1. Base-pair parameters x-displacement (Dx) and roll (rho), which have the largest differences between the two forms of structures, cannot drive the transition. Our results support the view that the bistable states of the DNA duplex are due to the bistable structures of the sugar ring.     

Partial B-to-A DNA transition upon minor groove binding of protein Sac7d monitored by Raman spectroscopy

Members of the Sso7d/Sac7d protein family and other related proteins are believed to play an important role in DNA packaging and maintenance in archeons. Sso7d/Sac7d are small, abundant, basic, and nonspecific DNA-binding proteins of the hyperthermophilic archeon Sulfolobus. Structures of several complexes of Sso7d/Sac7d with DNA octamers are known. These structures are characterized by sequence unspecific minor groove binding of the proteins and sharp kinking of the double helix. Corresponding Raman vibrational signatures have been identified in this study. A Raman spectroscopic analysis of Sac7d binding to the oligonucleotide decamer d(GAGGCGCCTC)(2) reveals large conformational perturbations in the DNA structure upon complex formation. Perturbed Raman bands are associated with the vibrational modes of the sugar phosphate backbone and frequency shifts of bands assigned to nucleoside vibrations. Large changes in the DNA backbone and partial B- to A-form DNA transitions are indicated that are closely associated with C2'-endo/anti to C3'-endo/anti conversion of the deoxyadenosyl moiety upon Sac7d binding. The major spectral feature of Sac7d binding is kinking of the DNA. Raman markers of minor groove binding do not largely contribute to spectral differences; however, clear indications for minor groove binding come from G-N2 and G-N3 signals that are supported by Trp24 features. Trp24 is the only tryptophan present in Sac7d and binds to guanine N3, as has been demonstrated clearly in X-ray structures of Sac7d-DNA complexes. No changes of the Sac7d secondary structure have been detected upon DNA binding.     

A study of the reversibility of helix-coil transition in DNA.

The reversibility of DNA melting has been thoroughly investigated at different ionic strengths. We concentrated on those stages of the process that do not involve a complete separation of the strands of the double helix. The differential melting curves of pBR 322 DNA and a fragment of T7 phage DNA in a buffer containing 0.02M Na+ have been shown to differ substantially from the differential curves of renaturation. Electron-microscopic mapping of pBR 322 DNA at different degrees of unwinding (by a previously elaborated technique) has shown that the irreversibility of melting under real experimental conditions is connected with the stage of forming new helical regions during renaturation. In a buffer containing 0.2M Na+ the melting curves of the DNAs used (pBR322, a fragment of T7 phage DNA, a fragment of phage Lambda DNA, a fragment of phiX174 phage DNA) coincide with the renaturation curves, i.e. the process is equilibrium. We have carried out a semi-quantitative analysis of the emergence of irreversibility in the melting of a double helix. The problem of comparing theoretical and experimental melting curves is discussed.     

A study of the B-A transition in DNA by gel electrophoresis

A procedure is developed for studying the B-A transition in DNA using gel electrophoresis. The starting point has been the idea that the junction between the A and B sections, which appear within the transition interval would increase the mobility of the DNA molecules. Indeed, the mobility of DNA in a gel is shown to increase in the middle of the B-A transition due to the formation of the largest possible number of boundaries between the B and A forms. The middle of the B-A transition in supercoiled DNA appears to be shifted against the middle of the transition in open circular (as well as linear) DNA by about 1.3% towards lower ethanol concentrations under the influence of the superhelical stress.     

A pH-dependent structural transition in the homopurine-homopyrimidine tract in superhelical DNA

We have inserted the 509-bp-long fragment of sea urchin P. miliaris histone gene spacer region into plasmid pUC19. The fragment contains the 60-bp-long homopurine-homopyrimidine tract that is known to be hypersensitive to the S1 endonuclease. Using two-dimensional gel electrophoresis we have observed a sharp structural transition in the insert with increasing DNA superhelicity. As in the cases of cruciform and Z form formation, the observed transition partly relaxes the superhelical stress. In contrast with the other two well documented transitions, the observed transition strongly depends on pH. At pH7 and above the transition occurs at negative superhelicities exceeding the physiological range (- sigma greater than 0.08). For pH6 the transition occurs at -sigma = 0.055, whereas for pH4.3 it takes place at -sigma = 0.001. A comprehensive analysis of the obtained data has made it possible to define the nature of the observed transition. We conclude that under superhelical stress or/and at low pH homopurinehomopyrimidine tracts adopt a novel spatial structure called the H form.     

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.     

The energetics of the B-Z transition in DNA

The paper deals with the energetics of the transition to left-handed Z form in DNA with an arbitrary base sequence. There is a brief outline of the statistical-mechanical model of the B-Z transition allowing for three possible states of each base pair. The parameters of the model can be determined by comparing the theory with experimental data for the B-Z transition in inserts with given sequences in circular DNA. The model contains six energy parameters, most of which have been determined before. In order to find the remaining parameters of the model and test its adequacy, a number of oligonucleotide sequences were synthesized and inserted into the pUC 19 plasmid. Two-dimensional gel electrophoresis was used to determine the superhelical density at which the inserts adopt the Z form. A statistical-mechanical treatment of these data yielded a complete set of six energy parameters for the B-Z transition. The theoretical assumption that the free energy of Z-form pairs does not depend on the type of adjacent pairs proved to be in agreement with the experimental data.     

The origin of the A to B transition in DNA fibers and films

We have studied the hydration of Na-DNA and Li-DNA fibers and films, measuring water contents, x-ray fiber diffraction patterns, low-frequency Raman spectra (below 100 cm^?1), high-frequency Raman spectra (600–1000 cm^?1), and swelling, as a function of relative humidity. Most samples gain weight equilibrium (though not conformational equilibrium) in one day. The volume occupied by a base pair as the DNA is hydrated (obtained from the x-ray and swelling data) shows anomalies for the case of Na-DNA in the region where the A-form occurs. Our Raman and x-ray data reproduce the well-known features of the established conformational transitions, but we find evidence in the Raman spectra and optical properties of a transition to what may be a disordered B-like conformation in Na-DNA below 40% relative humidity. We have studied the effects of crystallinity on the A to B transition. We find that the transition to the B-form is impeded in highly crystalline samples. In most samples, the transition occurs in three days (after putting the sample at 92% relative humidity) but in highly crystalline samples, the transition may take months. By comparing the high-frequency Raman spectra of highly ordered and disordered films, we show that the extent of crystallinity controls the amount of A-DNA formed when ethanol is used to dehydrate the films. We show that rapid dehydration (by laser heating) does not result in a B to A transition. A fiber that gives A-type x-ray reflections probably contains B-like material in noncrystalline regions. The low-frequency Raman spectrum is dominated by a band at about 25 cm^?1 in both Na- and Li-DNA. Another band is seen near 35 cm^?1 in Na-DNA at humidities where the sample is in the A-form. In contrast to earlier reports, we find that the Raman intensity does not depend on fiber orientation relative to the scattering vector. The “35-cm^?1” band is largely depolarized (i.e. vertical polarization incident and horizontal polarization scattered, VH, or vice versa, HV) while the “25-cm^?1” band appears in both VV, VH and HV polarizations. These bands are all weaker in HH polarization. The “25-cm^?1” band may be due to a shearing motion of the phosphates and their associated counterions, while the “35-cm^?1” band may be characteristic of A-DNA crystallites. We consider mass-loading, relaxational coupling to the hydration shell, and softening of interatomic potentials as possible explanations of the observed softening of the low-frequency Raman bands on hydration. Relaxation data suggest that the added water binds tightly (on these time scales) and a mass-loading model accounts for the observed softening rather well. We conclude that the A to B transition is not driven by softening of the “25-cm^?1” band. Rather, it is most probably a consequence of crystal-packing forces, with the more regular A-form favored in crystals when these forces are strong.     

Insight into the denaturation transition of DNA

DNA undergoes a helix-to-coil transition (also called denaturation transition) upon heating. This transition can also be facilitated by using solvent mixtures (for example water–alcohol). An increase in the hydrophobic tail of the second solvent molecule first decreases then increases the melting temperature appreciably. Measurement on 4% DNA in a series of water–alcohol mixtures shows that the helix-to-coil melting transition is driven by the solvent ability to cross the hydrophobic sugar-rich region. DNA is behaving like a cylindrical micelle.     

S-N transition of the saccharide ring in B-form DNA

A conformational transition of a single deoxyribose was analyzed in B-form trimers dA3:dT3 and dG3:dC3, both in the purine and pyrimidine chains. The main results were obtained for the duplexes with frozen ends, which could be extended by regular double helixes. The geometry of the central sugar ring in the duplexes may strongly deviate from the regular conformation. When deoxyribose changed its conformation in the central pyrimidine, the energy increase was proved to be less significant in comparison with that for purine. In the case of Thy, a decrease in pseudorotation angle P from 140 to 80 degrees causes the energy increase of 0.5 kcal/mol only, the barrier being 1.2 kcal/mol. The energy profile for Cyt has several local minima. The results of calculations were compared with numerous experimental data, they help to explain some NMR data. A perturbation of the duplex AAA:TTT structure caused by the thymine sugar ring transition, produces 5 degrees bend of the DNA axis directed toward adenines. We also investigated the influence of such conformational disturbance on the neighbouring base pairs, in particular the transition in the trimers with unfrozen ends.     

Fine structure in the B-A transition of native DNA

The B-A transition caused by high ethanol concentrations has been studied by the multi-dimensional spectrophotometer equipped with the computer-controlled microbullet. When ethanol concentration is increased, the CD signal at 270 nm of linearized ColEl DNA exhibits a biphasic transition; the first broad one and the second sharp one. The B-A transition of the ColEl DNA is much broader than that of alternative copolymers with shorter lengths. In addition, each PvuII restriction fragment of ColEl DNA has a different transition curve. Therefore the stability of the B-A transition varies along a long DNA molecule. The second transition is speculated to be caused by aggregation. When ethanol concentration is decreased, on the other hand, only a single transition shifted to lower ethanol concentration is observed. Thus the B-A transition curve has a hysteresis. A slow dissociation rate of the aggregation seems to cause the hysteresis.     

CC/GG contacts facilitate the B to A transition of DNA in solution

Self-complementary decadeoxynucleotides, CCGATATCGG, CCAGATCTGG, CCCTGCAGGG, GGGGGCCCCC, were designed and synthesized to estimate the A-philic free energy of CC/GG contacts. First, regions of temperature-stability of the double-stranded conformation were determined for each 10-mer. Then, circular dichroism spectra were recorded for the B-family forms at different temperatures, counter-ion concentrations and trifluoroethanol contents. A cooperative change typical of the B-A transition is observed in the CD spectra at a trifluoroethanol content specific for each duplex. The positions of half-transition points were functions not only of the nucleotide sequence but of the duplex length as well: the B to A transitions were hindered in these 10-mers in comparison with a lengthy DNA. The B-phility value was estimated to be 3 kcal/mol of 10-mer. The B-A transition point was shown to drop with an increase in the number of CC/GG contacts in a duplex. The designed 10-mers made it possible to estimate quantitatively the A-phility of CC/GG contact as compared with an average DNA: (FA-FB)CC = 0.2 Kcal/mol, (FA-FB)DNA = 0.7 Kcal/mol.