The B-A transition in superhelical DNA.

Relaxation of a DNA superhelical stress due to the B to A transition induced by trifluoroethanol has been studied by assessing the change of DNA orientation in a flow gradient. Using DNAs of different superhelical densities, a decrease in the winding angle during the B----A shift of DNA was found to be 1.5 degrees per base pair in solution. Accepting the winding angle for B-DNA in solution to be 34.1 degrees, that for A-DNA must have a value of 32.6 degrees which agrees with the X-ray data for A-DNA in the condensed state. The date obtained within the B-A transition interval make it possible to conclude that there is an increase in winding at each B/A junction, which is about 5 degrees per one junction.     

Mapping the B-A conformational transition along plasmid DNA

A simple method is presented to monitor conformational isomerizations along genomic DNA. We illustrate properties of the method with the B-A conformational transition induced by ethanol in linearized pUC19 plasmid DNA. At various ethanol concentrations, the DNA was irradiated with ultraviolet light, transferred to a restriction endonuclease buffer and the irradiated DNA was cleaved by 17 restriction endonucleases. The irradiation damaged DNA and the damage blocked the restrictase cleavage. The amount of uncleaved, i.e. damaged, DNA depended on the concentration of ethanol in a characteristic S-shape way typical of the cooperative B-A transition. The transition beginning and midpoint were determined for each restriction endonuclease. These data map the B-A transition along the whole polylinker of pUC19 DNA and six evenly distributed recognition sequences within the rest of the plasmid. The transition midpoints fell within the B-A transition region of the plasmid simultaneously determined by CD spectroscopy. The present method complements the previous methods used to study the B-A transition. It can be employed to analyze multikilobase regions of genomic DNA whose restriction endonuclease cleavage fragments can be separated and quantified on agarose gels.     

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.     

Theory of DNA melting in the interval of B-A transition

Theory of DNA melting within the B--A transition range is presented. The phase diagram in coordinates alcohol--temperature is plotted. The temperature shift of DNA helix--coil transition in the B--A transition point is predicted to be delta T = 3 degrees. The temperature rise of DNA melting in the range of B--A transition is caused by the presence of junctions between regions in B and A forms in helical sections.     

Sequence dependence of the B-A conformational transition of DNA

We have studied, by conformational analysis, the sequence dependence of DNA conformational transition between B- and A-forms. We have considered intramolecular interactions between base pairs, without backbone, to examine their role in the conformational transition between B- and A-forms, and found that base pairs themselves usually have intrinsic conformational preferences for the B- or A-form. Calculation of all ten possible base steps shows that the base combinations, CC (or GG), GC, AT, and TA, have tendencies to assume the A-conformation. Results show that it is particularly easy to slide along the long axis of the base pair for these steps, with AT and CC showing especially flat energies. These calculations show that a preference for the B- or A-conformation depends on the electrostatic energy parameters, in particular, on dielectric and shielding constants; the A-conformation is preferred for low dielectric constant or low shielding. Both the A- and B-conformations are mainly stabilized by electrostatic interactions between favorably juxtaposed atomic charges on base pairs; however, the B-conformation generally has more favorable van der Waals interactions than the A-form. These sequence-dependent conformational preference and environmental effects agree roughly with experimental observations, suggesting that the origin of the conformational polymorphism is attributable to the intrinsic conformational preference of base pairs.     

Dynamics of the B-A transition of DNA double helices

Although the transition from the B-DNA double helix to the A-form is essential for biological function, as shown by the existence of the A-form in many protein–DNA complexes, the dynamics of this transition has not been resolved yet. According to molecular dynamics simulations the transition is expected in the time range of a few nanoseconds. The B–A transition induced by mixing of DNA samples with ethanol in stopped flow experiments is complete within the deadtime, showing that the reaction is faster than ~0.2 ms. The reaction was resolved by an electric field jump technique with induction of the transition by a dipole stretching force driving the A- to the B-form. Poly[d(A-T)] was established as a favourable model system, because of a particularly high cooperativity of the transition and because of a spectral signature allowing separation of potential side reactions. The time constants observed in the case of poly[d(A-T)] with ~1600 bp are in the range around 10 µs. An additional process with time constants of ~100 µs is probably due to nucleation. The same time constants (within experimental accuracy ±10%) were observed for a poly[d(A-T)] sample with ~70 bp. Under low salt conditions commonly used for studies of the B–A transition, the time constants are almost independent of the ionic strength. The experimental data show that a significant activation barrier exists in the B–A transition and that the helical states are clearly separated from each other, in contrast to predictions by molecular dynamics simulations.     

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.     

Determination of enthalpy of B-A transition of DNA in aqueous-ethanol solutions

The enthalpies of the guanidinium chloride (Gu.HCl) with sodium DNA salt in the solutions in B- and A-conformations in the mixtures of ethanol-water at 298.15 K and the enthalpies of solution of guanidinium chloride in the mixtures of ethanol-water at 298.15 K in a whole range of the compositions of mixed solvents were measured calorimetrically. It was established that in a field of B-A-transition of DNA the values of interaction enthalpies of Gu.HCl with DNA practically do not depend on the composition of the solvent. The concentrations of Na-ions in water-ethanol solutions of DNA containing Gu.HCl were determined by the potentiometric method. It was revealed that the interaction of the equimolar quantities of Gu.HCl and DNA leads to the complete replacement of Na-cations, which are naturally linked with DNA, into solution. From the results obtained the enthalpy of B-A-conformation transition DNA at 298.15 K was determined (-2.50 +/- 0.10 kJ/mole).     

Condensation prevails over B-A transition in the structure of DNA at low humidity

B-A transition and DNA condensation are processes regulated by base sequence and water activity. The constraints imposed by interhelical interactions in condensation compromise the observation of the mechanism by which B and A base-stacking modes influence the global state of the molecule. We used a single-molecule approach to prevent aggregation and mechanical force to control the intramolecular chain association involved in condensation. Force-extension experiments with optical tweezers revealed that DNA stretches as B-DNA under ethanol and spermine concentrations that favor the A-form. Moreover, we found no contour-length change compatible with a cooperative transition between the A and B forms within the intrinsic-force regime. Experiments performed at constant force in the entropic-force regime with magnetic tweezers similarly did not show a bistable contraction of the molecules that could be attributed to the B-A transition when the physiological buffer was replaced by a water-ethanol mixture. A total, stepwise collapse was found instead, which is characteristic of DNA condensation. Therefore, a low-humidity-induced change from the B- to the A-form base-stacking alone does not lead to a contour-length shortening. These results support a mechanism for the B-A transition in which low-humidity conditions locally change the base-stacking arrangement and globally induce DNA condensation, an effect that may eventually stabilize a molecular contour-length reduction.     

The effect of transition metal ions on the resistance of bacterial spores to hydrogen peroxide and to heat.

The presence of 10 microM-Cu2+ increased the lethal effect of hydrogen peroxide on spores of Clostridium bifermentans but not on those of Clostridium sporogenes PA 3679, Clostridium perfringens, Bacillus cereus or Bacillus subtilis var. niger. Cu2+ at 100 muM also increased the lethal effect of heat on spores of C. bifermentans but not on those of B. sutilis var. niger. The rate and extent of Cu2+ uptake by spores of C. bifermentans and B. subtilis var. niger were similar, but examination of unstained sections of spores by electron microscopy suggested that Cu2+ is bound by the protoplasts of spores of C. bifermentans but not of B. subtilis var. niger.     

Modulation of the B-A transition of DNA by potential antitumor antibiotics. Influence of the base composition of DNA

The B-A transition of DNA in oriented films of DNA-drug complexes is more or less restricted as a consequence of drug binding as revealed by infrared linear dichroism. A fraction of DNA is irreversibly locked into the B form. This behavior is described by the number of DNA base pairs "frozen" in the B form by one drug molecule. This quantity is dependent on the DNA sequence the drug is attached to. In this paper, drug complexes of oriented films of NaDNA with a GC content of 42% from calf thymus and a GC-rich DNA from Micrococcus lysodeikticus were compared. The restriction of the B-A transition of DNA complexes with two intercalating antibiotics, aclacinomycin A and violamycin BI, is not severely influenced by the base composition of DNA. By contrast, the strong groove binding oligopeptide antibiotics netropsin and distamycin A are much less effective to restrict the B-A transition of GC-rich DNA than of AT-rich DNA. This finding is in agreement with previous results by other methods which support a model based upon a strong preference of AT clusters by these two non-intercalating drugs.     

Carbon nanotubes selective destabilization of duplex and triplex DNA and inducing B-A transition in solution

Single-walled carbon nanotubes (SWNTs) have been considered as the leading candidate for nanodevice applications ranging from gene therapy and novel drug delivery to membrane separations. The miniaturization of DNA-nanotube devices for biological applications requires fully understanding DNA-nanotube interaction mechanism. We report here, for the first time, that DNA destabilization and conformational transition induced by SWNTs are sequence-dependent. Contrasting changes for SWNTs binding to poly[dGdC]:poly[dGdC] and poly[dAdT]:poly[dAdT] were observed. For GC homopolymer, DNA melting temperature was decreased 40°C by SWNTs but no change for AT-DNA. SWNTs can induce B–A transition for GC-DNA but AT-DNA resisted the transition. Our circular dichroism, competitive binding assay and triplex destabilization studies provide direct evidence that SWNTs induce DNA B–A transition in solution and they bind to the DNA major groove with GC preference.     

Dependence of B-A Conformational Change in DNA on Base Composition

IT is generally accepted that all native DNAs, irrespective of their origin, give essentially identical X-ray diffraction patterns characteristic of the A form at high humidities and of the A form at lower humidities^{1, 2}. The purpose of the work described here was an accurate characterization of B-A conformational changes in a range of DNA by infrared dichroism, which seems to be particularly appropriate for the structural characterization of nucleic acids^3–6 as a complement of X-ray diffraction. In previous infrared studies the A form and B-A conformational changes were not observed⁷.     

Noncompetitive immunochemical determination of ribonuclease using transition metal ions and the effect of catalytic hydrogen release

A noncompetitive variant of immunochemical ribonuclease (RNase) determination has been developed, involving the use of Co(II) as a label. A variety of approaches to labeling the immunological reagent with the metal have been assessed. In the variant proposed, catalytic hydrogen release was used as a means of detecting the label, the amount of which was proportional to RNase concentration. Conditions making it possible to record catalytic hydrogen release fluxes were determined. In the presence of RNase, the electrocatalytic effect was maximum at a concentration of Co(II) in the ammoniac buffer, equal to 2 x 10(-4) M (pH 10.0). The dependence was linear in the range 4-2000 ng/ml RNase concentrations (threshold concentration, 2 ng/ml).     

A Study of the B-A Transition in DNA by Gel Electrophoresis

Abstract

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.