Structure Of DNA In Solution Research Articles

Caught in the Loop: Binding of the [Ru(phen)2 (dppz)]2+ Light-Switch Compound to Quadruplex DNA in Solution Informed by Time-Resolved Infrared Spectroscopy.

Ultrafast time-resolved infrared (TRIR) is used to report on the binding site of the [Ru(phen)2 (dppz)]2+ "light-switch" complex with both bimolecular (Oxytricha nova telomere) and intramolecular (human telomere) guanine-quadruplex structures in both K+ and Na+ containing solutions. TRIR permits the simultaneous monitoring both of the "dark" and "bright" states of the complex and of the quadruplex nucleobase bases, the latter via a Stark effect induced by the excited state of the complex. These data are used to establish the contribution of guanine base stacking and loop interactions to the binding site of this biologically relevant DNA structure in solution. A particularly striking observation is the strong thymine signal observed for the Na+ form of the human telomere sequence, which is expected to be in the anti-parallel conformation.

Comparison of carboplatin and cisplatin effects on DNA structure in solution

Comparison of carboplatin and cisplatin effects on DNA structure in solution

Molecular Dynamics of a DNA Holliday Junction: The Inverted Repeat Sequence d(CCGGTACCGG)4

All-atom molecular dynamics (MD) computer simulations have been applied successfully to duplex DNA structures in solution for some years and found to give close accord with observed results. However, the MD force fields have generally not been parameterized against unusual DNA structures, and their use to obtain dynamical models for this class of systems needs to be independently validated. The four-way junction (4WJ), or Holliday junction, is a dynamic DNA structure involved in central cellular processes of homologous replication and double strand break repair. Two conformations are observed in solution: a planar open-X form (OPN) with a mobile center and four duplex arms, and an immobile stacked-X (STX) form with two continuous strands and two crossover strands, stabilized by high salt conditions. To characterize the accuracy of MD modeling on 4WJ, we report a set of explicit solvent MD simulations of ∼100 ns on the repeat sequence d(CCGGTACCGG)4 starting from the STX structure (PDB code 1dcw), and an OPN structure built for the same sequence. All 4WJ MD simulations converged to a stable STX structure in close accord with the crystal structure. Our MD beginning in the OPN form converts to the STX form spontaneously at both high and low salt conditions, providing a model for the conformational transition. Thus, these simulations provide a successful account of the dynamical structure of the STX form of d(CCGGTACCGG)4 in solution, and provide new, to our knowledge, information on the conformational stability of the junction and distribution of counterions in the junction interior.

Importance of Accurate DNA Structures in Solution: The Jun–Fos Model

Understanding the recognition of DNA sequences by proteins requires an accurate description of the structural dynamics of free DNA, especially regarding indirect readout. This involves subtle sequence-dependent effects that are difficult to characterize in solution. To progress in this area, we applied NMR and extensive simulations to a DNA sequence relevant to the Jun–Fos system. The backbone and base behaviors demonstrate that unrestrained simulations with major force fields (Parm98, Parmbsc0, and CHARMM27) are not reliable enough for in silico predictions of detailed DNA structures. More realistic structures required molecular dynamics simulations supplemented by NMR restraints. A new methodological element involved restraints inferred from the phosphate chemical shifts and from the phosphate dynamics. This provided a detailed and dynamic view of the intrinsic properties of the free DNA sequence that can be related to its recognition, by comparison with a relevant DNA–protein complex. We show how to exploit the relationship between phosphate motions and helicoidal descriptors for structure determination toward an accurate description of DNA structures and dynamics in solution.

A novel G-quadruplex-forming GGA repeat region in the c-myb promoter is a critical regulator of promoter activity

The c-myb promoter contains multiple GGA repeats beginning 17 bp downstream of the transcription initiation site. GGA repeats have been previously shown to form unusual DNA structures in solution. Results from chemical footprinting, circular dichroism and RNA and DNA polymerase arrest assays on oligonucleotides representing the GGA repeat region of the c-myb promoter demonstrate that the element is able to form tetrad:heptad:heptad:tetrad (T:H:H:T) G-quadruplex structures by stacking two tetrad:heptad G-quadruplexes formed by two of the three (GGA)4 repeats. Deletion of one or two (GGA)4 motifs destabilizes this secondary structure and increases c-myb promoter activity, indicating that the G-quadruplexes formed in the c-myb GGA repeat region may act as a negative regulator of the c-myb promoter. Complete deletion of the c-myb GGA repeat region abolishes c-myb promoter activity, indicating dual roles of the c-myb GGA repeat element as both a transcriptional repressor and an activator. Furthermore, we demonstrated that Myc-associated zinc finger protein (MAZ) represses c-myb promoter activity and binds to the c-myb T:H:H:T G-quadruplexes. Our findings show that the T:H:H:T G-quadruplex-forming region in the c-myb promoter is a critical cis-acting element and may repress c-myb promoter activity through MAZ interaction with G-quadruplexes in the c-myb promoter.

X-ray diffraction “fingerprinting” of DNA structure in solution for quantitative evaluation of molecular dynamics simulation

Solution state x-ray diffraction fingerprinting is demonstrated as a method for experimentally assessing the accuracy of molecular dynamics (MD) simulations. Fourier transforms of coordinate data from MD simulations are used to produce reciprocal space "fingerprints" of atomic pair distance correlations that are characteristic of the ensemble and are the direct numerical analogues of experimental solution x-ray diffraction (SXD). SXD experiments and MD simulations were carried out to test the ability of experiment and simulation to resolve sequence-dependent modifications in helix conformation for B-form DNA. SXD experiments demonstrated that solution-state poly(AT) and poly(A)-poly(T) duplex DNA sequences exist in ensembles close to canonical B-form and B'-form structures, respectively. In contrast, MD simulations analyzed in terms of SXD fingerprints are shown to deviate from experiment, most significantly for poly(A)-poly(T) duplex DNA. Compared with experiment, MD simulation shortcomings were found to include both mismatches in simulated conformer structures and number population within the ensembles. This work demonstrates an experimental approach for quantitatively evaluating MD simulations and other coordinate models to simulate biopolymer structure in solution and suggests opportunities to use solution diffraction data as experimental benchmarks for developing supramolecular force fields optimized for a range of in situ applications.

Assessment of the molecular dynamics structure of DNA in solution based on calculated and observed NMR NOESY volumes and dihedral angles from scalar coupling constants.

To assess the accuracy of the molecular dynamics (MD) models of nucleic acids, a detailed comparison between MD-calculated and NMR-observed indices of the dynamical structure of DNA in solution has been carried out. The specific focus of our comparison is the oligonucleotide duplex, d(CGCGAATTCGCG)(2), for which considerable structural data have been obtained from crystallography and NMR spectroscopy. An MD model for the structure of d(CGCGAATTCGCG)(2) in solution, based on the AMBER force field, has been extended with a 14 ns trajectory. New NMR data for this sequence have been obtained in order to allow a detailed and critical comparison between the calculated and observed parameters. Observable two-dimensional (2D) nuclear Overhauser effect spectroscopy (NOESY) volumes and scalar coupling constants were back-calculated from the MD trajectory and compared with the corresponding NMR data. The comparison of these results indicate that the MD model is in generally good agreement with the NMR data, and shows closer accord with experiment than back-calculations based on the crystal structure of d(CGCGAATTCGCG)(2) or the canonical A or B forms of the sequence. The NMR parameters are not particularly sensitive to the known deficiency in the AMBER MD model, which is a tendency toward undertwisting of the double helix when the parm.94 force field is used. The MD results are also compared with a new determination of the solution structure of d(CGCGAATTCGCG)(2) using NMR dipolar coupling data.

Determination of DNA structure in solution: enzymatic deuteration of the ribose 2' carbon.

An enzymatic solution to the problem of obtaining 13C/15N-labeled nucleotides that are deuterated uniquely at the H2' ' position within the ribose ring is presented. Selective deuteration occurs with an overall yield of >80%. The deuteron at the H2' ' position allows measurement of the scalar and residual dipolar couplings for the bond vectors attached to the C2' carbon of each ribose sugar. These data allow the accurate determination of sugar conformation. Interesting DNA double helices of 2-3 turns are now within the reach of solution NMR spectroscopy. As an example, these labeled nucleotides are incorporated uniquely at positions 6-14 in a 20-bp DNA sequence containing the adenovirus major late promoter.

Molecular dynamics simulations of B′-DNA: sequence effects on A-tract-induced bending and flexibility

Molecular dynamics (MD) simulations including water and counterions are reported on five examples of A-tract DNA oligonucleotide dodecamer duplexes for which crystal structures are available, the homopolymeric duplex sequences poly(dA) and poly(dG), and two related sequences that serve as controls. MD was performed using the AMBER suite of programs for 3 ns on each sequence. These results, combined with previously reported MDs on 25-mer and 30-mer oligonucleotides on sequences with phased A-tracts carried out under a similar simulation protocol, are used to examine salient issues in the structural chemistry of ApA steps and A-tract induced axis bending. MD modeling sucessfully describes the distinctive B′ structure of A-tracts in solution as essentially straight (wedge angles of <1°), more rigid than generic B-form DNA, with slight base-pair inclination, high propeller twist and a minor groove narrowing 5′ to 3′. The MD structures in solution agree closely with corresponding crystal structures, supporting the idea that crystal structures provide a good model for A-tract DNA structure in solution. From the collective MD results, bending and flexibility are calculated by step. Pyrimidine-purine steps are predicted to be most intrinsically bent and also most bendable, i.e. susceptible to bending. Pyrimidine-pyrimidine (∼ purine-purine) and purine-pyrimidine steps show less intrinsic deformation and deformability. The MD calculated flexibility correlates well with the protein-induced bendability derived independently from the protein DNA crystal structures. The MD results indicate that bending and flexibility of base-pair steps in DNA are highly correlated, i.e. steps that exhibit the most intrinsic deformation from B-form DNA turn are also the most dynamically deformable. The MD description of A-tract-induced axis bending shows most consistency with the non A-tract, general-sequence model, in which the sequence curvature originates primarily in base-pair roll towards the major groove in non-A-tract regions of the sequence, particularly pyrimidine-purine steps. The direction of curvature is towards the minor groove viewed from opposite the A-tracts, but the A-tracts per se exhibit only minor deformation. The MD results are found to be consistent with the directionality of bending inferred for DNA sequences from gel retardation and cyclization experiments.

Synthesis and characterization of fluorenone-, anthraquinone-, and phenothiazine-labeled oligodeoxynucleotides: 5'-probes for DNA redox chemistry.

A facile and automated procedure for the synthesis of oligodeoxynucleotides possessing derivatives of 9-fluoreneone, 9,10-anthraquinone, and phenothiazine is described. The phosphoramidite approach is used to attach these redox and spectroscopic probes to the 5'-terminus of oligodeoxynucleotides in high yield (>95%). Thermal denaturation studies of labeled oligodeoxynucleotides show a slight enhancement in duplex stability relative to the unlabeled control, and circular dichroism spectra confirm B-form helical DNA structure in solution.

Coumarin–fluorescein pair as a new donor–acceptor set for fluorescence energy transfer study of DNA

A method for introduction of the 2′-coumarin labeled nucleoside as a fluorescence energy donor into DNA duplexes has been described. Efficient FRET occurs between the coumarin–fluorescein pair in DNA owing to the high quantum yield of the donor. The present donor–acceptor pair may be useful as FRET indicator of DNA structures in solution.

A pseudosquare knot structure of DNA in solution.

We report a high-resolution NMR structure of a homodimer formed by a synthetic 25 residue DNA oligonucleotide GCTCCCATGGTTTTTGTGCACGAGC. This structure presents a novel structural motif for single-stranded nucleic acids, called a pseudosquare knot (PSQ). The oligonucleotide was originally designed to mimic a slipped-loop structure (SLS), another "unusual" DNA structure postulated as an alternative conformation for short direct repeats in double-stranded DNA. The design of the sequence is compatible with both SLS and PSQ structures, both of which possess identical sets of base-paired and unpaired nucleotides but different tertiary folds. We used deuteration of the H8 positions of purines to ascertain that the PSQ is actually formed under the conditions used. The PSQ structure was solved based on homonuclear proton nuclear Overhauser effect data using complete relaxation matrix methods. The structure essentially consists of two side-by-side helices connected by single-stranded loops. Each of the helices is well-defined; however, the relative orientation of the two remains undetermined by the NMR data. The sequences compatible with the PSQ formation are frequent in single-stranded genomes; this structure may play a role as a dimerization motif.

Modulation of cyclobutane pyrimidine dimer formation in a positioned nucleosome containing poly(dA.dT) tracts.

We have used a defined-sequence nucleosome to concomitantly investigate the generation and location of DNA lesions in nucleosomes and their influence on nucleosome positioning (translational and rotational setting). A 134 bp HISAT sequence from the yeast DED1 promoter, containing a polypyrimidine region (40 bp) with a T6-tract, two T5-tracts, and a T9-tract, was reconstituted in nucleosomes with a defined rotational setting. T-tracts adopt unusually rigid DNA structures in solution ("T-tract structure") and are hot spots of cyclobutane pyrimidine dimer (CPD) formation by UV light (254 nm). DNA was irradiated with UV light before or after reconstitution. The CPD yields and distribution were analyzed by cleavage with T4 endonuclease V. The rotational setting of nucleosomal DNA was characterized by DNase I digestion. With the exception of one T5-tract (1T5), the T6-, the 2T5-, and the T9-tracts formed T-tract structure in solution. T-tract structure was lost upon folding in nucleosomes, demonstrating a dominant constraint of DNA folding in nucleosomes over that of T-tract structure. CPD formation was strongly modulated by the nucleosome structure, but the CPD distribution differed from that reported for mixed-sequence DNA. CPD formation in the nucleosome had no effect on the rotational setting of nucleosomal DNA, but the rotational setting was affected when nucleosomes were assembled on damaged DNA. The toleration of DNA distortions imposed by CPDs in nucleosomes may have important implications for the recognition and repair of these damages in chromatin.

Sequence dependence of DNA structure in solution

Sequence-dependent structural variations in DNA can influence its binding by ligands and proteins. However, relatively little is known about sequence-structure relationship for arbitrary DNA sequences. The 1H two-dimensional NOESY data presented here for ten oligonucleotide duplexes show pronounced sequence-dependent changes in at least two types of internucleotide distances commonly used in sequential connectivity assignments, namely the H-6/H-8 to preceding (5') H-1' distance (S1) and the H-6/H-8 to preceding (5') H-2'' distance (S2). On the basis of these two measured distances, all dinucleotide steps can be divided into four different groups: Y-R, R-Y, R-R and Y-Y, where R is purine and Y is pyrimidine. These data suggest some rules for the variation in these distances along DNA duplexes. Correlations between these distances and helical parameters of DNA are discussed in comparison to well-resolved X-ray structures of B-type DNA.

Geometry of a branched DNA structure in solution.

An approximate geometry of a stable, four-way DNA junction has been determined in free solution by applying the technique of transient electric birefringence. The current approach consists of (i) construction of a set of six molecules in which two of the four arms of a synthetic junction are elongated by approximately 9-fold (in each of the six possible two-arm combinations), (ii) determination of the ratios of the longest birefringence decay time of each elongated junction to the decay time of a linear control molecule, and (iii) comparison of the experimental ratios with corresponding ratios computed as a function of the junction interarm angle. The result is a set of six angles that define the geometry of the junction. In the presence of magnesium ions, the junction adopts a geometry in which particular pairs of arms approach colinearity. Furthermore, the geometry of the junction is significantly altered in the absence of magnesium, adopting a more uniform structure, although such an effect is not apparent on gels. The application of transient electric birefringence, as described in the current study, should be useful for the characterization of a broad range of tertiary structures in both DNA and RNA.

Nuclear magnetic resonance and distance geometry studies of DNA structures in solution.

NMR AND DG METHODS . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . .... . . . . . . . . . . 424 Two-Dimensional NMR 424 Proton Assignments. . . . . . . . .... . . . . . . . . . . . .. . . . . .. . ... . . . . . ...... . . . . ... . . . . . . . . ...... . . . . . . ..... . . . . . . .... . . . . . . .... 425 Distance Constraints .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427 Distance Geometry Algorithm 429

Environment induced changes in DNA structure

AbstractInvestigations over the past decade have disclosed that the structure of DNA in solution is far from uniform and static. The present view of DNA structure is that it is: 1) dynamically variable, displaying relatively large flexibility in both bending and torsional modes, 2) it is not perfectly helical because of sequence dependent departures from a regular helix and 3) it is capable of adopting entirely different conformations, including a switch in the sense of the helix as is the case for the B to Z transition, as well as new folding patterns as is the case for cation induced DNA collapse.One of the principal problems in the field today is the detection and evaluation of structural changes in solution. Many experimental techniques are in use and a number of these will be reviewed. The discussion includes models of flexible DNA, the structural aspects revealed by several experimental techniques, and results for a number of systems in which solution environment brings about changes in structure.

Sequence-dependent anisotropic flexibility of B-DNA. A conformational study.

Bending flexibility of the six tetrameric duplexes was investigated d(AAAA):d(TTTT), d(AATT)2, d(TTAA)2, d(GGGG):d(CCCC), d(GGCC)2 and d(CCGG)2,. The tetramers were extended in the both directions by regular double helices. The stiffness of the B-DNA double helix when bent into the both grooves proved to be less than that in the perpendicular direction by an order of magnitude. Such an anisotropy is a property of the sugar-phosphate backbone structure. The calculated fluctuations of the DNA bending along the dyad axis, 5-7 degree, are in agreement with experimental value of the DNA persistence length. Anisotropy of the double helix is sequence-dependent: most easily bent into the minor groove are the tetramers with purine-pyrimidine dimer (RY) in the middle. In contrast, YR dinucleotides prefer bending into the major groove. Moreover, they have an equilibrium bend of 6-12 degree into this groove. The above inequality is caused by stacking interaction of the bases. The bend in the central dimer is distributed to some extent between the adjacent links, though the main fraction of the bend remains within the central link. Variation of the sugar-phosphate geometry in the bent helix is inessential, so that DNA remains within the B-family of forms: namely, when the helical axis is bent by 20 degree. the backbone dihedral angles vary by no more than 15 degree. The obtained results are in accord with x-ray structure of the B-DNA dodecamer; they further substantiate our early model of DNA wrapping in the nucleosome by means of "mini-kinks" separated by a half-pitch of the double helix, i.e. by 5-6 b.p. Sequence-dependent anisotropy of DNA presumably dictates the three-dimensional structure of DNA in solution as well. We have found that nonrandom allocation of YR dimers leads to the systematic bends in equilibrium structure of certain DNA fragments.

NMR studies of conformational states and dynamics of DNA.

The application of high resolution NMR techniques to the investigation of DNA double helices in solution is currently in a rapid state of change as a result of advances in three different fields. First, new methods (cloning, enzymatic degradation, sonication, and chemical synthesis) have been developed for producing large quantities of short DNA suitable for NMR studies. Second, there have been major advances in the field of NMR in terms of the introduction of new pulse techniques and improvements in instrumentation. Finally, as a result of recent X-ray diffraction studies on short DNA helices and the discovery of left-handed Z-DNA there is heightened interest in the study of DNA structures in solution and the effect of sequence on structure. In the present review, we discuss the way in which NMR techniques have been used to probe various aspects of the DNA properties, including base pairing structure, dynamics of breathing, effect of sequence on DNA structure, internal molecular motions, the effect of environment on the DNA, and the interaction of DNA with small ligands.

Salt‐ and sequence‐dependence of the secondary structure of DNA in Solution by 31P‐nmr spectroscopy

AbstractWe examined three sonicated, specific‐seqiemce polydeoxynucleotides in solution over a wide range of concentrations of several salts by 13P‐nmr spectroscopy, and we found that the alternating copolymer poly(dAdT)·poly(dAdT) exhibits a dinucleotide repeat unit in all five salts and at all concentrations studied, as indicated by the presence of a doubled in its 31P‐nmr spectra. The two components of the doublet show selective shift effects. The upfield component is assigned to dApdT in the gauche−‐gauche− conformation and shifts upfield in all four monovalent salts used, relative to a single‐stranded oligonucleotide control. The downfield component is assigned to dTpdA in the trans‐gauche− conformation and shifts downfield with increasing CsF concentration but remains essentially constant in LiCl, NaCl, and CsCl. These changes indicate a fast noncooperative transition for poly(dAdT)·poly‐(dAdT) from a presumed right‐handed dinucleotide‐repeat B‐form to another conformation with a dinucleotide‐repeat structure, via a continuum of structures that may differ in the extent of the winding of the double helix. Ethanol causes the upfield component to collapse into the other component, indicating conversion to a structure with a mononucleotide repeat unit and a trans‐gauche− conformation. Up to 1M Mg2+ appears to have no significant effect on the phosphodiester conformations of poly(dAdT)·poly(dAdT). By contrast, poly‐(dGdC)·poly(dGdC) gives a slow cooperative transition from what is considered to be a right‐handed regular B‐form to a left‐handed Z‐form on increasing MgCl2 and NaCl concentrations, although we observed no changes in chemical shifts below the transition points. The homopolymer poly(dA)·poly(dT) exhibits no unusual shift effects or transitions upon the addition of salts when compared to the oligonucleotide control and is considered to be a regular B‐form with a gauche−‐gauche− phosphodiester backbone conformation. These differences emphasize the distinct secondary structures of DNAs of different sequences and their selective responses to changes in solution conditions.