The structure of psi DNA

In concentrated solutions of neutral or anionic polymers (and an adequate cation concentration), DNA condenses into a compact state, which is of interest for its possible relevance to chromosome structure and the packing of DNA in viruses. The X-ray scattering of DNA condensed in this way has been examined with respect to the secondary structure of the helix and the tertiary structure of the compact state. Measurements have also been made with dense aqueous gels of DNA in the absence of poly(ethylene oxide) at ordinary salt concentrations and in 6·0 M-LiCl. All preparations exhibit a well-defined interhelical spacing, implying substantial parallelism, but no lattice spacings higher than first order are observed. Comparison with calculated scattering curves for disoriented helical segments indicates that a structure very close to the B fiber structure prevails in all preparations. No significant contribution from the A or C fiber structures can be detected in either the condensed preparations or the LiCl solutions. Thus there is no basis for attributing the origin of the strongly anomalous circular dichroism spectra to a secondary structure significantly different from that in dilute solution. The decrease in interhelix spacing with increasing polymer concentration is in reasonable accord with expectation on the basis of excluded volume interactions. There is improved short-range order in the polymer-induced compact state as compared with the simple solution having the same interhelix spacing.The results are in reasonably good agreement with expectation for a folded chain structure of the compact state, similar to the usual mode of crystallization of simple linear polymers. There is no evidence for supercoiling.     

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2′-fluoro-ANA/RNA and DNA/RNA hybrids

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2′-fluoro-ANA analog (2′F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2′F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3′-endo (north, A-form) conformation, whereas those of the ANA strand adopt a ‘rigid’ O4′-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2′F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2′F-ANA/RNA and DNA/RNA helices is 9.0 ± 0.5 Å, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2′F-ANA/RNA hybrids to elicit RNase H activity.     

Sequence-dependent variation in the conformation of DNA

The specificity of action of the enzyme DNAase I on double-stranded DNA polymers of defined sequence has been investigated. The results obtained with the alternating copolymers poly[d(A-T)] · poly[d(A-T)] and poly[d(G-C)] · poly[d(G-C)] support the suggestion of Klug et al. (1979) that regions of double-stranded DNA containing alternating purine-pyrimidine sequences may exist as structural variants of the classical B-form under physiological salt conditions. Digestion of defined oligomers containing alternating dG-dC sequences indicate that these too exist in some “alternating-B” structure in solution under similar conditions. The results obtained with the oligomers also provide a number of insights into the mode of action of DNAase I.In the case of the B-DNA dodecamer d(C-G-C-G-A-A-T-T-C-G-C-G), for which the crystal structure has been solved (Dickerson &; Drew, 1981), there is a very good correlation between the sites of rapid DNAase I cutting and positions of high local helical twist.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

The flow linear dichroism of DNA: comparison with the bead-spring theory

The flow linear dichroism if T7 bacteriophage DNA has been obtained as a function of velocity gradient in neutral aqueous solutions containing 0.15M Na⁺ or K⁺. The data are compared with the hydrodynamic bead-spring theory of Rouse and Zimm using currently available spectroscopy, structural, and hydrodynamic parameters. Good agreement between theoretical and experimental isocline angle behavior is found. The magnitude of the measured linear dichroism is not in exact agreement with theory when presently accepted stiffness and optical parameters are used. Agreement is improved either by assuming a shorter persistence length or a less negative value for the limiting dichroism of DNA.     

Structure and Dynamics of Double Helices in Solution: Modes of DNA Bending

Abstract

The long range structure of DNA restriction fragments has been analysed by electro-optical measurements. The overall rotation time constants observed in a low salt buffer with monovalent ions is shown to decrease upon addition of Mg²⁺ or spermine. Since the circular dichroism and also the limiting value of the linear dichroism remain almost constant under these conditions, the effect is attributed to a change of the long range structure. According to a weakly bending rod model, the persistence length decreases from about 600 Å in the absence of Mg²⁺ or spermine to about 350 Å in the presence of these ions. The persistence length measured in the presence of Mg²⁺ is almost independent of temperature in the range of 10 to 40 °C. The nature of DNA bending is analysed by measurements of bending amplitudes and time constants from dichroism decay curves. The observed absence of changes in the bending amplitudes upon addition of Mg²⁺ or spermine, even though addition induces changes of the persistence length by a factor of 2, is hardly consistent with simple thermal bending. The combined results, including the remarkably small temperature dependence of persistence length and bending amplitude, can be explained by the existence of two bending effects: inherent curvature of DNA dominates at low temperature, whereas thermal bending prevails at high temperature. Analysis of bending amplitudes from dichroism decay curves according to an arc model provides an approximate measure for the degree of bending in restriction fragments. The model is consistent with the observed chain length dependence of bending amplitudes and provides an approximate curvature corresponding to a radius of ab out400Å. Thus the curvature observed in restriction fragments is similar to that observed for high molecular DNA condensed into toroids by addition of ions like spermine.

Particularly strong bending of DNA is induced by [CO(NH₃)₆]³⁺, indicated by an apparent persistence length of 200 Å and an increased bending amplitude together with a reduced limit value of the linear dichroism. This effect is attributed to the high charge density of this ion and potential site binding.     

Natural DNA sequences can form left-handed helices in low salt solution under conditions of topological constraint

The conformation of DNA that originates from association of complementary single-stranded circles (form V DNA) is investigated in solution at low salt concentration. It is shown that circular dichroism extended to the far ultraviolet region (down to 165 nm) represents a powerful tool for determination of the handedness of double helical DNAs in solution. The positive intense band at 186 nm followed by a strong negative band around 170 nm is characteristic of all right-handed helical forms (B,A) of DNA, whereas the circular dichroism spectrum of the Z form of poly[d(G-C)] of opposite helical sense represents a quasi inversion of these far ultraviolet bands. Thus, form V DNA is found to represent a co-existence of left-handed Z-type and right-handed B double helical stretches in addition to negative superturns. The Raman spectrum of form V DNA provides further support for the contribution of a left-handed double helical conformation, as shown by comparison to the high resolution Raman spectra of poly[d(G-C)] in the Z and B forms.The analysis of present spectroscopic data and the analysis of occurrence of alternating [d(G-C)] purine-pyrimidine sequences in the form V DNA used strongly suggest that in DNA of natural sequence, topological constraint may generate left-handed double helices, a conformation thought so far to be limited to the alternating [d(G-C)] sequences. Such structure could play a role in recognition and regulation of gene expression.     

Macromolecular crowding-induced molten globule states of the alkali pH-denatured proteins

Structural and molecular properties extracted from circular dichroism (CD), tryptophan fluorescence and 1-anilino-8-napthalene sulfonate (ANS) binding experiments suggest that the high concentration of synthetic crowding agents (dextran 40, dextran 70 and ficoll 70) stabilizes and refolds the base-denatured ferricytochrome c (Ferricyt c) and lysozyme (Lyz) at pH 12.9 (±0.1) to molten globule (MG) states (C_B-states). These results further revealed that the C_B-states resemble the generic properties of MG-states. Thermodynamic analysis of thermal denaturation curves of base-denatured Ferricyt c and Lyz at pH 12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence increases the thermal stability of base-denatured proteins and also prevents the cold denaturation of Ferricyt c. The results further showed that the nature, size and shape of crowder influence the crowding-mediated increase in secondary structure stabilization and thermal stability of base-denatured Ferricyt c and Lyz. Analysis of kinetic and thermodynamic parameters measured for CO association reaction of alkaline ferrocytochrome c (Ferrocyt c) at pH 12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence reduces the level of structural fluctuation of M80-containing Ω-loop that control CO association to alkaline Ferrocyt c.     

Multiple four-stranded conformations of human telomere sequence d(CCCTAA) in solution

By detailed NMR analysis of a human telomere repeating unit, d(CCCTAA), we have found that three distinct tetramers, each of which consists of four symmetric single-strands, slowly exchange in a slightly acidic solution. Our new finding is a novel i-motif topology (T-form) where T4 is intercalated between C1 and C2 of the other duplex. The other two tetramers have a topology where C1 is intercalated between C2 and C3 of the other parallel duplex, resulting in the non-stacking T4 residues (R-form), and a topology where C1 is stacked between C3 and T4 of the other duplex (S-form). From the NMR denaturation profile, the R-form is the most stable of the three structures in the temperature range of 15–50°C, the S-form the second and the T-form the least stable. The thermodynamic parameters indicate that the T-form is the most enthalpically driven and entropically opposed, and its population is increased with decreasing temperature. The T-form structure determined by restrained molecular dynamics calculation suggests that inter-strand van der Waals contacts in the narrow grooves should contribute to the enthalpic stabilization of the T-form.     

Quasielastic light scattering by biopolymers. IV. Tertiary collapse of calf thymus DNA in 5.5M LiCl

Circular dichroism has been commonly employed to infer the conformation of DNA in solution. The basis of the conformational assignments is the work of Tunis-Schneider and Maestre, wherein CD spectra of DNA were obtained under conditions comparable to those employed in the x-ray diffraction studies of A-, B-, and C-DNA. It has recently been suggested that the CD spectrum of DNA in chromatin, which is similar to the CD spectrum of the C-form DNA, is a superposition of the normal B-DNA spectrum and a single negative band, centered at 275 nm. This negative band is qualitatively identical to the spectrum for condensed Ψ-form DNA. We have employed the hydrodynamic methods of quasielastic light scattering and sedimentation velocity to determine the extent of DNA tertiary structural alteration in 5.5M LiCl as a possible explanation of the C-form CD spectrum. These studies suggest an eightfold contraction of the Stokes hydrodynamic volume for calf thymus DNA in going from 0.4M NH₄Ac to 5.5M LiCl, with no change in molecular weight. The estimated maximum presistence length of DNA in 5.5M LiCl is estimated to be 20.0 nm compared to the “minimum” value of 44.7 nm in NaCl solutions. The value 20.0 nm corresponds to a maximum radius of 16.7 nm for a “continuously coiled” cylinder of DNA, which compares with the value 5.0 nm of DNA in the nucleosome unit of chromatin.     

On the molecular length of the replicative form DNA of bacteriophage phiX174

This paper describes an electron microscopic study of the circular replicative form DNA of bacteriophage φX174. The study has been carried out using a preparative technique in which the DNA molecules are adsorbed from solution on to the cleavage surface of mica and visualized in the electron microscope as a metal-shadowed replica (Gordon &; Kleinschmidt, 1969,1970). Contour lengths of open circular molecules were measured in samples obtained from preparations in which the following experimental parameters were varied: the ionic strength of the solution from which the DNA was adsorbed on the mica and the way in which the molecules were dried before shadowing. At the 0.05 significance level, varying these parameters had no effect on the mean length and variances of samples of molecules obtained from five experiments; the samples were therefore regarded as being drawn from the same molecular population with a mean length and variance of, respectively, 1.83 μm and 0.0117 μm².It was argued that the DNA molecules adsorbed on the mica are “frozen” into the molecular conformation present in solution at the time of adsorption and that, therefore, the experimentally determined contour lengths represent authentic molecular lengths in solution. Based on current estimates of the replicative form DNA molecular weight, the mean contour length obtained was slightly but significantly larger than the length predicted for molecules in an exact B configuration. The variance was larger than could be attributed solely to experimental error, indicating that the molecular population in aqueous solution is heterogeneous in contour length. These experimental results were shown to be consistent with a model for DNA structure in aqueous solution in which individual molecules are dynamic variants of a perturbed B form structure (von Hippel &; Wong, 1971).     

Structural polymorphism of liquid-crystalline dispersions of double-stranded DNA complexed with synthetic polycations

Linear double-stranded DNA molecules interact with positively charged polyconidine molecules in aqueous salt solutions to yield liquid-crystalline dispersions (LCDs) with a mean particle diameter of ～6000 Å. The packing density of (DNA-polycation) complexes differs among LCD particles formed at different ionic strengths. X-ray data on the liquid-crystalline phases of (DNA-polyconidine) complexes formed under different conditions were compared with a phase diagram, reflecting polymorphism of liquid crystals of linear double-stranded DNA. It was shown that LCD was hexagonal at 0.15 M ≤ C _NaCl < 0.4 M and cholesteric at 0.4 M ≤ C _NaCl < 0.55 M. Cholesteric LCD displayed abnormal optical activity in the circular dichroism spectrum. A similar situation was observed with poly(2,5-ionene), another polycation differing in chemical structure from polyconidine. The results demonstrated structural polymorphism of (DNA-polycation) LCDs. It was assumed that the packing mode of (DNA-polycation) complexes in LCD particles can be regulated by changing NaCl concentration. The mechanism generating the cholesteric liquid-crystalline state of DNA in a narrow range of NaCl concentrations is discussed.     

Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures.

Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules.Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10⁻² μm) is smaller than the random coil segment value b (7·17 × 10⁻² μm) was observed, while circular structures of the dimer of the same molecule (12 × 10⁻² μm) were detected.     

Comparison of the linear least mean squares procedure and the vector projection method of spectral analysis.

A mathematical analysis of two methods commonly used for the determination of fractions of secondary structure from circular dichroism spectral data—the vector projection method of Baker &; Isenberg [Biochem.15, 629 (1976)] and the classical linear least mean squares procedure-reveals that the two procedures handle errors of all types in an equivalent fashion and yield identical values of the fractional amounts of secondary structure present, as long as the same method of integration of the data is employed. Analyses of simulated data show that the method of integration [rectangular, trapezoidal or parabolic (Simpson's Rule)] has an insignificant effect on the calculated values of the fractions of secondary structure present. Our study also shows that the insensitivity of the fraction of α helix calculated using a given random coil reference spectrum is a natural consequence of certain intrinsic properties of the reference spectra of the α helix, β structures and random coil forms, rather than the method of calculation employed.     

Electrochemical nitration of myoglobin at tyrosine 103: Structure and stability

Nitration in proteins is a physiologically relevant process and the formation of 3-nitrotyrosine was first proposed as an in vivo marker of the production of reactive nitrogen species in oxidative stress. No studies have been published on structural changes associated with nitration of myoglobin. To address this deficiency the electrochemical nitration of equine skeletal muscle (Mb) at amino acid tyrosine 103 has been investigated for the evaluation and characterization of structural and thermal stability changes. Y103 in Mb is one of the most exposed tyrosine residues and it is also close to the heme group. Effects of Y103 nitration on the secondary and tertiary structure of Y103 have been studied by UV–Vis, circular dichroism, fluorescence and NMR spectroscopy and by electrochemical studies. At physiological pH, subtle changes were observed involving slight loosening of the tertiary structure and conformational exchange processes. Thermal stability of the nitrated protein was found to be reduced by 5 °C for the nitrated Mb compared with the native Mb at physiological pH. Altogether, NMR data indicates that nitrated Mb has a very similar tertiary structure to that of native Mb, although with a slightly open conformation.     

Multiple glycosylation of de novo designed α-helical coiled coil peptides

The aim of this study was to investigate the influence of multiple O-glycosylation in α-helical coiled coil peptides on the folding and stability. For this purpose we systematically incorporated one to six β-galactose residues into the solvent exposed positions of a 26 amino acid long coiled coil helix. Surprisingly, circular dichroism spectroscopy showed no unfolding of the coiled coil structure for all glycopeptides. Thermally induced denaturations reveal a successive but relative low destabilization of the coiled coil structure upon introduction of β-galactose residues. These first results indicate that O-glycosylation of the glycosylated variants is easily tolerated by this structural motif and pave the way for further functional studies.     

Alteration in the nucleosome and chromatin structures upon interaction with platinum coordination complexes

The interaction of various platinum coordination complexes with nucleosomes and chromatin has been investigated by ultraviolet absorption spectrophotometry, circular and electric linear dichroism, and thermal denaturation, at low binding ratios (r < 0.1–0.2). The general trend of the changes in these physicochemical properties is similar to that observed for the DNA-platinum complexes, which indicates that the same binding sites are involved in the platinum interaction with DNA and with its nucleoprotein complex. The cis-bidentate ligands, cis-dichlorodiammine, diaminocyclohexane and ethylenediamine platinum(II), showed a distinct behavior, with a more important destabilization of the DNA structure in the nucleoprotein than the trans-bidentate ligand, trans-dichlorodiammine-Pt(II), and monodentate ligand, diethylenetriamine-Pt(II). The drastic decrease of the negative electric dichroism in the 260 nm absorption band of the bases, observed with the five ligands, indicates a profound alteration of the DNA arrangement in chromatin and nucleosomes, attributed to a condensation of its superhelical structure. Some differences with previous observations on DNA complexes with the same platinum compounds indicate the possible formation of protein-DNA crosslinks in chromatin and nucleosomes. These could have some importance for the biological effects.     

Energetics of twisted DNA topologies

Our goal is to review the main theoretical models used to calculate free energy changes associated with common, torsion-induced conformational changes in DNA and provide the resulting equations hoping to facilitate quantitative analysis of both in vitro and in vivo studies. This review begins with a summary of work regarding the energy change of the negative supercoiling-induced B- to L-DNA transition, followed by a discussion of the energetics associated with the transition to Z-form DNA. Finally, it describes the energy changes associated with the formation of DNA curls and plectonemes, which can regulate DNA-protein interactions and promote cross talk between distant DNA elements, respectively. The salient formulas and parameters for each scenario are summarized in table format to facilitate comparison and provide a concise, user-friendly resource.