The Flexibility of A-Form DNA

Abstract

We have determined the rise per base pair and persistence length of A-form DNA in trifluoroethanol solutions for fragments 350–900 base pairs in length that best describe rotational diffusion coefficients determined by transient electric birefringence. The 2.6 A spacing between base pairs found in crystal and fiber A-form structures is preserved in solution. The persistence length is about 1500 A, or about three times longer than for B-form DNA. There is no apparent electrostatic contribution to the persistence length in the salt concentration range 0.2–2.0 mM Na cacodylate. This suggests an even closer association between DNA and its neutralizing counterions than predicted by condensation theory, perhaps due to a sheath of trifluoroethanol excluded water surrounding the A-form helix.     

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.     

Sedimentation of homogeneous double-strand DNA molecules.

Sedimentation velocity studies have been carried out with isolated double-strand DNA fragments prepared by digestion of PM2 phage with the restriction endonuclease Hae III. The results show that DNA molecules shorter than about 200 base pairs behave almost exactly as rigid rods with a diameter of 27 A. The behavior of the larger fragments (up to 1735 base pairs) can be described very well by either the theory of Yamakawa and Fujii (Yamakawa, H., and Fujii, M. (1973), Macromolecules 6. 407) using the same diameter and a persistence length of 575 A, or the theory of Hearst and Stockmayer (Hearst, J.E., and Stockmayer, W.H. (1962), J. Chem. Phys. 37, 1425) using a diameter of 20 A and a persistence length of 525 A.     

The subunit structure of chromatin from Physarum polycephalum.

Nucleosome DNA repeat lengths in Physarum chromatin, determined by nuclease digestion experiments, are shorter than those observed in most mammalian chromatin and longer than those reported for chromatin of certain other lower eukaryotes. After digestion with staphylococcal nuclease for short periods of time an average repeat length of 190 base pairs is measured. After more extensive digestion an average repeat length of 172 base pairs is measured. Upon prolonged digestion DNA is degraded to an average monomer subunit length of 160 base pairs, with only a small amount of DNA found in lengths of 130 base pairs or smaller. Mathematical analysis of the data suggests that the Physarum nucleosome DNA repeat comprises a protected DNA segment of about 159 base pairs with a nuclease-accessible interconnecting segment which ranges from 13 to 31 base pairs. The spacing data are compatible with measurements from electron micrographs of Physarum chromatin.     

The Intramolecular Impact to the Sequence Specificity of B→A Transition: Low Energy Conformational Variations in AA/TT and GG/CC Steps

Abstract It is well known, that local B→A transformation in DNA is involved in several biological processes. In vitro B?A transition is sequence-specific. The physical basis of this specificity is not known yet. Here we analyze the effect of intramolecular interactions on the structural behavior of the GG/CC and AA/TT steps. These steps exemplify sequence specific bias to the B- or A-form structure. Optimization of potential energy of the molecular systems composed of an octanucle-otide, neutralized by Na(+) and solvated with TIP3P water molecules in rectangular box with periodic boundary conditions gives the statistically representative sets of low energy structures for GG/CC and AA/TT steps in the middle of the diverse flanking sequences. Permissible 3D variations of GG/CC and AA/TT, and correlation of the relative motion of base pairs in these steps were analyzed. AA/TT step permits high variability for low energy conformers in the B-form DNA and small variability for low energy conformers in the A-form DNA. In contrast GG/CC step permits high variability for low energy conformers in the A-form DNA and small variability for low energy conformers in the B-form DNA. The relative motion of base pairs in GG/CC step is high correlated, while in AA/TT step this correlation is notably less. Atom-atom interactions inside-the-step always favors the B-form and their component - stacking interactions (atomatom interactions between nucleic bases) is crucial for the duplex stabilization. Formation of the A-form for both steps is a result of interactions with the flanking sequences and water-cation environment in the box. The average energy difference between conformations presenting B-form and A-form for the GG/CC step is high, while for the AA/TT step it is rather low. Thus, intramolecular interactions in GG/CC and AA/TT steps affect the possible conformational diversity ("conformational entropy") of the A- and B- type structures of DNA step. This determines the known bias of the A-form DNA depending on the enrichment of sequences with GG/CC. If structural tuning during the process of protein-DNA complex formation lead to the local B→A transformation of DNA, it is largely directed by high conformational diversity of GG/CC step in the A-form. In such a case the presence in the target site of both kinds of examined steps ensures the reversible character of ligand binding.     

Flexibility of duplex DNA on the submicrosecond timescale

Using a site-specific, Electron Paramagnetic Resonance (EPR)-active spin probe that is more rigidly locked to the DNA than any previously reported, the internal dynamics of duplex DNAs in solution were studied. EPR spectra of linear duplex DNAs containing 14-100 base pairs were acquired and simulated by the stochastic Liouville equation for anisotropic rotational diffusion using the diffusion tensor for a right circular cylinder. Internal motions have previously been assumed to be on a rapid enough time scale that they caused an averaging of the spin interactions. This assumption, however, was found to be inconsistent with the experimental data. The weakly bending rod model is modified to take into account the finite relaxation times of the internal modes and applied to analyze the EPR spectra. With this modification, the dependence of the oscillation amplitude of the probe on position along the DNA was in good agreement with the predictions of the weakly bending rod theory. From the length and position dependence of the internal flexibility of the DNA, a submicrosecond dynamic bending persistence length of around 1500 to 1700 A was found. Schellman and Harvey (Biophys. Chem. 55:95-114, 1995) have estimated that, out of the total persistence length of duplex DNA, believed to be about 500 A, approximately 1500 A is accounted for by static bends and 750 A by fluctuating bends. A measured dynamic persistence length of around 1500 A leads to the suggestion that there are additional conformations of the DNA that relax on a longer time scale than that accessible by linear CW-EPR. These measurements are the first direct determination of the dynamic flexibility of duplex DNA in 0.1 M salt.     

Peculiarities of the B to A Transition of the λ Phage Regulatory Site OR3 and of Its Fragment

Abstract

Conformations of the synthetic deoxyoligonucleotide 17 base pairs long, which is an OR3 operator of λ phage, and of its 9-b.p. fragment were studied by the circular dichroism method (CD). The regions of stability of the double-stranded state were determined for these duplexes. A comparison of the CD spectra for these oligonucleotides with the CD for a lengthy DNA showed the conformation of these short DNA pieces to belong to the B-family.

A cooperative change in the CD spectra is observed in trifluoroethanol (TFE) solutions at a TFE concentration specific for each oligonucleotide, which is supposed to stem from a B to A transition. The length of the fragment was found to affect the ability for the B-A transition. The transition into the A form is hindered by 13% TFE for the short 9-nucleotide in comparison with the 17-nucleotide. We suggest that this is due to the B form stabilization by terminal base pairs (B-phility of the ends).     

Conformation and circular dichroism of DNA.

CD spectra of calf thymus, C. perfringens, E. coli, and M. luteus DNA have been measured in the vacuum-uv region to about 168 nm for the A-, B-, and C-forms. The positive band at about 187 nm and the negative band at about 170 nm found for each type and form of DNA are sensitive to the source of the DNA and the base–base interactions of the double-stranded helix. The A-form spectra confirm that these bands are indeed sensitive to secondary structure. In the near-uv, the CD of B-form DNA is well analyzed as a linear combination of 27% A-form and 78% C-form. However, an analysis of the extended spectrum demonstrates that the near-uv analysis is not correct. The extended analysis shows that the base–base interactions are similar for B- and C-forms in solution, which implies that these two forms have nearly the same number of base pairs per turn. Various types of CD difference spectra are also discussed.     

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.     

Base tilt of DNA in various conformations from flow linear dichroism

We have measured the isotropic absorption (Aiso) and linear dichroism (LD) of Escherichia coli DNA in 0.01 M Na+ (10.4 base pairs per turn of B form), 5.5 M NH4F (10.2 base pairs per turn of B form), and 80% trifluoroethanol (A form) into the vacuum UV spectral region. The reduced dichroism spectrum (LD divided by Aiso) of DNA in the A conformation differed from those of the B conformations, demonstrating that LD is a sensitive method for distinguishing DNA conformation. The reduced dichroism spectra of the B conformations were similar, indicating little change in the orientation of the bases for DNA in high salt. The wavelength dependence of the reduced dichroism indicates that the angle between the base planes and the helix axis is less than 76 degrees for all three conformations of DNA.     

A note on crystal packing and global helix structure in short A-DNA duplexes

A simple relation exists between the packing density in crystals of short A-DNA duplexes and their global double-helical structure. The volume per nucleotide pair shows a linear inverse correlation with the mean displacement of base pairs from the best straight helix axis. The mean displacement is a measure of major groove depth and varies between -3.3 A and -4.9 A in A-form oligonucleotides analysed in the crystalline state. Since the mean displacement of base pairs from the helix axis determines other helical parameters such as base-pair longitudinal slide, its correlation with crystal packing is of considerable interest. The displacement-packing correlation is very clear for octamer duplexes which crystallize in three different lattices. Longer A-helical fragments sometimes deviate from the rule. It may be speculated whether A-form duplexes not completing a full helical turn are especially prone to distortions due to packing in crystals or arising from intermolecular contacts in solution.     

The Flexibility of Alternating dA—dT Sequences

Abstract

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.     

Breathing and bending fluctuations in DNA modeled by an open-base-pair kink coupled to axial compression

We develop a model designed to show that flexibility in the DNA molecule can arise from relatively improbable transient opening of base pairs. The axial direction changes at the site of an open base pair. The region between open base pairs is a double helix of hydrogen-bonded base pairs with a slightly decreased rise per residue and a slightly increased helical winding angle. An analysis of the model yields several testable predictions. For example, we predict probability 0.026 for a base pair to be open at 25°C, a value close to that measured by hydrogen-exchange experiments. Other predictions involve matters like the variation of persistence length with ionic strength and temperature, the variation of helical winding angle with temperature, and the kinetics of heat denaturation. An additional result of the analysis is an explanation of the high degree of local stiffness of the DNA molecule. Strong resistance to bending fluctuations is provided from two sources: increased polyelectrolyte repulsion among phosphate groups in the axially compressed stacks between open base pairs and the tendency of stacking forces to oppose opening of a base pair. Stacking forces, however, also support compression of the stacks between open base pairs, so that the net effect of stacking forces on elastic bending of DNA is small relative to the polyelectrolyte effect. If the ionic charges on the phosphate groups were absent, DNA would spontaneously fold, driven by the entropy gained when about 1% of its base pairs open.     

The stiffness of dsRNA: hydrodynamic studies on fluorescence-labelled RNA segments of bovine rotavirus.

The sedimentation coefficients of dsRNA segments of bovine rotavirus were determined in the analytical ultracentrifuge. The eleven segments were separated by preparative gel electrophoresis, and isolated by elution from gel pieces. The RNA was labelled by the intercalating fluorescent dye ethidium bromide at a ratio bound dye per base pair between 0.003 to 0.018. The analytical ultracentrifuge was equipped with a fluorescence recording optics. Sedimentation coefficients could be determined with amounts of RNA as little as 8 ng. All sedimentation coefficients were extrapolated to zero-concentration, zero-dye binding, and zero-impurities from the preparative gel electrophoresis. The hydrodynamic model of flexible cylinders was applied for the interpretation of the sedimentation coefficients. All dsRNA segments of rotavirus (663-3409 base pairs) and the dsRNA5 of cucumber mosaic virus (335 base pairs) fit the model of a "worm-like" or flexible cylinder with a persistence length of 1125 A and a hydrated diameter of 30 A. The results are compared with data from the literature on the persistence lengths of the B- and Z-forms of dsDNA and of viroids.     

Structures of non-canonical tandem base pairs in RNA helices: review

The structures of tandem non-canonical base pairs, a frequently recurring motif in RNA molecules, are reviewed and analysed. The tandem non-canonical base pair motifs can be roughly divided in three groups, containing seven subgroups based on their base pairing patterns and local geometries. Structural details and helical parameters that can be used to numerically distinguish between the subgroups are tabulated. Remarkably, while the individual helical twists of the tandem and adjacent base pair steps can be substantially smaller or larger than the typical A-form value of 32.7 degrees, the average value is close to A-form. This and other striking regularities resulting from compensating geometrical adjustments, important for understanding and predicting the configurations of non-canonical base pairs geometries are discussed.     

Mechanical properties of high-G.C content DNA with a-type base-stacking

The sequence of a DNA molecule is known to influence its secondary structure and flexibility. Using a combination of bulk and single-molecule techniques, we measure the structural and mechanical properties of two DNAs which differ in both sequence and base-stacking arrangement in aqueous buffer, as revealed by circular dichroism: one with 50% G·C content and B-form and the other with 70% G·C content and A-form. Atomic force microscopy measurements reveal that the local A-form structure of the high-G·C DNA does not lead to a global contour-length decrease with respect to that of the molecule in B-form although it affects its persistence length. In the presence of force, however, the stiffness of high-G·C content DNA is similar to that of balanced-G·C DNA as magnetic and optical tweezers measured typical values for the persistence length of both DNA substrates. This indicates that sequence-induced local distortions from the B-form are compromised under tension. Finally, high-G·C DNA is significantly harder to stretch than 50%-G·C DNA as manifested by a larger stretch modulus. Our results show that a local, basepair configuration of DNA induced by high-G·C content influences the stretching elasticity of the polymer but that it does not affect the global, double-helix arrangement.     

Dynamic bending rigidity of DNA

Rapidly relaxing components in the decay of the transient electric dichroism of DNA restriction fragments were reported by Diekmann et al. [(1982) Biophys. Chem. 15, 263-270] and P?rschke et al. [(1987) Biopolymers 26, 1971-1974]. These are analyzed using a new normal mode theory for weakly bending rods and assigned to bending. The longest bending relaxation times for fragments with 95-250 base pairs coincide with the theoretical curve calculated for a dynamic bending rigidity corresponding to a dynamic persistence length Pd = 2100 A. Analysis of the relative amplitudes of fast and slow components following weak orienting pulses is also consistent with a rather large dynamic persistence length. The enhancement of the relative amplitude of the fast component in large electric fields is attributed to steady-state bending of initially perpendicular DNAs by the field. Several reasons are proposed why the dynamic bending rigidity is 4 times larger than the apparent static bending rigidity inferred from equilibrium persistence length measurements on the same fragments.     

DNA length and concentration dependencies of anisotropic phase transitions of DNA solutions

Critical concentrations for the isotropic to cholesteric phase transitions of double-stranded DNA fragments in simple buffered saline (0.1 M NaCl) solutions were determined as a function of DNA contour length ranging from approximately 50 nm to 2700 nm, by solid-state 31P NMR spectroscopy and polarized light microscopy. As expected for semirigid chains, the critical concentrations decrease sharply with increasing DNA length near the persistence length in the range from 50 to 110 nm, and approach a plateau when the contour length exceeds 190 nm. The biphasic region is substantially wider than observed for xanthan, another semirigid polyelectrolyte approximately twice as stiff as DNA, primarily because of low critical concentrations for first appearance of the anisotropic phase, C(i)*, in DNA samples > or =110 nm (320 base pairs) long. The limiting C(i)* for DNA > or =490 nm long is exceptionally low (only 13 mg/ml) and is substantially lower than the C(i)* of approximately 40 mg/ml reported for the stiffer xanthan polyelectrolyte. The much higher values of the critical concentrations, C(a)*, for the disappearance of the isotropic DNA phase (> or =67 mg/ml) are modestly higher than those observed for xanthan and are predicted reasonably well by a theory that has been applied to other semirigid polymers, if a DNA persistence length in the consensus range of 50-100 nm is assumed. By contrast, the broad biphasic region and low C(i)* values of DNA fragments > or =190 nm long could only be reconciled with theory by assuming persistence lengths of 200-400 nm. The latter discrepancies are presumed to reflect some combination of deficiencies in current theory as applied to chiral, strong polyelectrolytes such as DNA, and sequence-dependent variations in DNA properties such as flexibility, curvature, or interaction potential. The propensity of DNA to spontaneously self-order at low concentrations well in the physiological range may have biological significance.