Toward a dynamical structure of DNA: comparison of theoretical and experimental NOE intensities.

Comparisons of experimental and calculated interproton nuclear Overhauser effect (NOE) buildup curves for duplex d(CGCGAATTCGCG)2 have been made. The calculated NOEs are based on molecular dynamics simulations including counterions and water and on the single-structure canonical A, B, and crystal forms. The calculated NOE effects include consideration of the motions of individual interproton vectors and the anisotropic tumbling of the DNA. The effects due to inclusion of anisotropic tumbling are much larger than those due to the local motion, and both improve the agreement between calculated and experimental results. The predictions based on the dynamical models agree significantly better with experiment than those based on either of the canonical forms or the crystal structure.

Structures of the potassium-saturated, 2:1, and intermediate, 1:1, forms of a quadruplex DNA.

Potassium can stabilize the formation of chair- or edge-type quadruplex DNA structures and appears to be the only naturally occurring cation that can do so. As quadruplex DNAs may be important in the structure of telomere, centromere, triplet repeat and other DNAs, information about the details of the potassium-quadruplex DNA interactions are of interest. The structures of the 1:1 and the fully saturated, 2:1, potassium-DNA complexes of d(GGTTGGTGTGGTTGG) have been determined using the combination of experimental NMR results and restrained molecular dynamics simulations. The refined structures have been used to model the interactions at the potassium binding sites. Comparison of the 1:1 and 2:1 potassium:DNA structures indicates how potassium binding can determine the folding pattern of the DNA. In each binding site potassium interacts with the carbonyl oxygens of both the loop thymine residues and the guanine residues of the adjacent quartet.

Effect of magnesium and polyamines on the structure of yeast tRNAPhe.

The effect of magnesium and polyamines (spermine, spermidine, putrescine and cadaverine) on the structure of yeast tRNAPhe has been investigated. It is found that magnesium induces structural changes and stabilizes hydrogen bonds in the temperature range 22--44 degrees C in 0.17 M sodium. The number of Mg2+ which affect tRNA structure increases from 1 +/- 1 at 22 degrees C to 4 +/- 1 at 44 degrees C and the number of additional base pairs formed in the presence of magnesium increases from 1 +/- 1 at 22 degrees C to 4 +/- 1 at 44 degrees C. The spectral changes are more-or-less sequential. The polyamine spermine stabilizes some, but not all, of the structural features stabilized by magnesium at 44 degrees C, and the combination of magnesium and spermine, at low levels, is more effective than either cation alone in stabilizing tRNA structure. Comparison of the effects of spermine, spermidine, putrescine and cadaverine indicates that it is the asymmetric triamine unit which is important in the stabilization. Some spectral changes induced by magnesium can be assigned to stabilization of specific tertiary structure interactions and to alteration of stacking adjacent to U8-A14.

Tertiary structure in E. coli tRNA Arg and tRNA Val.

300 MHz proton NMR has been used to demonstrate that both Escherichia coli tRNA Arg and tRNA(1) Val have a tertiary structure base pair between 4-thiouridine (s-4U) at position 8 and A at position 14. Formation of this s-4U(8)-A(14) base pair leads to a very low field (14.8 ppm) resonance in the spectra of both molecules. When s-4U is converted to U the 14.8 resonance is replaced by a new resonance which appears at 14.3 ppm. The presence of the tertiary structure s-4U(8)-A(14) base pair greatly constrains the folding of these tRNAs in solution.

Determination of the number and location of the manganese binding sites of DNA quadruplexes in solution by EPR and NMR in the presence and absence of thrombin.

The interaction of a DNA quadruplex with thrombin has been studied by first determining the sites of manganese binding to the quadruplex in the absence of thrombin. This has been followed by determining if the interactions with thrombin displace the bound manganese. A different DNA quadruplex has also been studied as a control. The refined solution structures of two DNA quadruplexes have been used to predict the electrostatic potentials of these DNAs. The calculated electrostatic potentials have been used to predict the locations of the binding sites of the paramagnetic ion manganese to these DNAs. The enhanced relaxation of DNA protons due to the binding of the paramagnetic metal ion Mn2+ has been used to experimentally determine the locations of the binding sites. The NMR results and the predictions based on the electrostatic potentials both place the binding sites of the manganese in the narrow grooves of these quadruplex DNAs. The predicted locations are spatially close to those experimentally observed, and the predicted and experimental locations also have similar electrostatic potential energy. These results have allowed a validation of the predictions of electrostatic potentials from structure. The 15mer quadruplex has two strong Mn2+ binding sites with one in each narrow groove. Both Mn2+ are released when the 15mer is complexed with thrombin, indicating that both narrow grooves are involved in the 15mer-thrombin interactions. The dimer quadruplex has a different structural motif than the 15mer and the presence of thrombin does not appreciably affect its interactions with Mn2+.

Functional and dysfunctional roles of quadruplex DNA in cells.

A number of biological roles have been proposed for quadruplex, also referred to as G4 or tetraplex, DNA. The presence of quadruplex DNA may lead to errors in some biological processes and be required in others. Proteins that interact with quadruplex DNA have been identified including those that cause Bloom's and Werner's syndromes. There are small molecules that specifically bind to quadruplex DNA, inhibit telomerase, and are cytotoxic towards tumor cells indicating a role for quadruplex DNA in telomere function. It is now possible to make testable proposals for the possible biological implications of quadruplex DNA in replication, transcription, and recombination as well as possible routes to therapeutic intervention.

Structure determination and analysis of local bending in an A-tract DNA duplex: comparison of results from crystallography, nuclear magnetic resonance, and molecular dynamics simulation on d(CGCAAAAATGCG).

We have presented a detailed analysis for structure determinations for the DNA duplex d(CGCAAAAATGCG) obtained from X-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulation. Each of the structures for the duplex deviates from the structure of the canonical form of B-DNA in a number of observable characteristics. Specifically, the three determinations all contain DNA axis deflections at the junctions of the A-tract with the flanking sequences. The analysis provided shows that the general characteristics of the structures obtained for d(CGCAAAAATGCG) from X-ray, NMR, and MD methods turn out to be quite similar. The extent to which this result can be generalized remains to be established by consideration of similar cross-comparisons on diverse oligonucleotide structures.

Interresidue quiet NOEs for DNA structural studies.

The potential utility of long-range NOEs in DNA has not been exploited since the observed signals have contributions both from the direct magnetization route and from multiple diffusion pathways. The Quiet NOE approach can be used to select for the direct magnetization transfer pathway by suppressing spin diffusion. A single-band Quiet NOE, which allows detection of the direct NOEs between protons in a selected chemical shift window, has been demonstrated on two duplex DNAs, and the NOEs observed can contain important structural information.

Determination of internuclear angles of DNA using paramagnetic-assisted magnetic alignment.

Paramagnetic ions have been used to assist the magnetic alignment of DNA. The anisotropy of the binding sites is sufficient to give rise to significant alignment of the DNA with the observed proton-carbon dipolar couplings spanning a 70-Hz range. The dipolar couplings have been used to determine the positions of the axial and rhombic alignment axes. The positions of the alignment axes relative to the positions of the binding sites of the paramagnetic europium ions have also been determined.

Magnesium increases the curvature of duplex DNA that contains dA tracts.

Distinct structural features of DNA, such as the curvature of dA tracts, are important in the recognition, packaging, and regulation of DNA. Physiologically relevant concentrations of magnesium have been found to enhance the curvature of dA tract DNAs, as monitored by solution-state NMR, indicating that the structure of DNA depends on the cations present in solution. A model is presented which accounts for the sequence-dependent effects of magnesium on DNA curvature as well as for the previously known sequence-independent effect on DNA flexibility.

The curvature of dA tracts is temperature dependent.

The curvature of dA tracts has been proposed to be important in the recognition, packaging, and regulation of DNA. The effects of dA tracts on the gel mobility, rate of cyclization, and other properties of DNA have been extensively studied. The consensus value for the curvature induced by a single dA tract is about 18 degrees. There are two main competing models for the origin of the curvature of dA tracts. One model assigns the central role to sequence-dependent steric clashes and the other to sequence dependent interactions with cations. The temperature dependence of the shape functions, the molecule specific part of the diffusion coefficients, of a set of six DNAs has been examined here. The set contains DNAs with dA tracts in or out of phase with respect to the helical repeat as well as those with scrambled dA-dT regions. The results show that the curvature of dA tracts is highly temperature dependent and that the curvature is largely melted out by 40 degrees C. The curvature melts out before there is significant premelting, or breathing of the dA tracts or the scrambled dA-dT regions. The curvature does not appear to reach a plateau value at low temperatures. A qualitative model for the melting of the curvature of dA tracts is proposed.

Comparison of conformational features of staphylococcal nuclease in ternary complexes with pdTp, pdGp, and nitrophenyl-pdTp.

The conformations of wild-type staphylococcal nuclease (SNase) in the ternary complexes with thymidine 3',5'-bisphosphate (pdTp), 2'-deoxyguanine 3',5'-bisphosphate (pdGp), and thymidine 3'-phosphate 5'-(p-nitrophenylphosphate) (NpdTp) with Ca2+ were examined by two-dimensional NMR NOESY and ROESY experiments. The results of these experiments indicate that the conformational features of the SNase are quite similar in the three ternary complexes. This suggests that the conformational features of SNase, in these ternary complexes, are not strongly dependent on whether the 5'-phosphate is a mono- or diester. This is in contrast to our prior studies on substitutions of active site charged amino acids which indicated that the conformational features of SNase in the ternary complex are quite sensitive to substitutions for active site charged amino acids (Hibler et al., 1987; Wilde et al., 1988; Pourmotabbed et al., 1990). The similarity of the SNase conformational features in the ternary complexes with pdTp and pdGp indicates that the features of the nucleotide bound at the active site are not strong determinants of the enzyme conformation in the ternary complexes. These conclusions are in general agreement with the results on pdApdT ternary complexes with SNase which suggested that it is the conformational features of the bound nucleic acid which determine the differences in catalysis observed for SNase with different substrates (Weber et al., 1991), more so than the conformational features of the enzyme.

Structure of a duplex DNA containing a thymine glycol residue in solution.

Oxidative stress, ionizing radiation, and other events can induce the oxidation of the thymine in DNA to thymine glycol. The presence of thymine glycol can have significant biological consequences, and there are specific repair enzymes for thymine glycol in a wide range of organisms. The structure of a duplex DNA containing a single thymine glycol (5,6-dihydroxy-5,6-dihydrothymidine) has been determined by the combined use of NMR and restrained molecular dynamics. The duplex of d(C1G2C3G4A5Tg6A7C8G9C10C11) paired with d(G22C21G20C19T18A17T16G15C14G13G12), with Tg indicating thymine glycol, has been used for these studies. The structure shows that the thymine glycol induces a significant, localized structural change with the thymine glycol largely extrahelical. This structural information is consistent with the biological consequences of thymine glycol in DNA. This structure is compared with that of a DNA duplex with an abasic site in the same sequence context.

Conformational mobility of deoxyribonucleic acid, transfer ribonucleic acid, and poly(adenylic acid) as monitored by carbon-13 nuclear magnetic resonance relaxation.

The molecular motion of DNA, the native form of tRNA, and partially denatured poly(A) has been investigated by carbon-13 nuclear magnetic resonance (13C NMR). The nuclear Overhauser effect of the RNA samples was measured at 25.1 and 50.3 MHZ, and the spin-lattice relaxation time of all the samples was measured at 50.3 MHZ. The NMR data indicate that the local motion of the ribose carbons is much less restricted than that of the bases for DNA and tRNA. The local motion correlation times of the ribose carbons are in the range of 1-7 ns for the samples investigated. The local motion correlation times of the different nucleic acids are quite similar with the exception that the 2' carbon of DNA and poly(A) is apparently less restricted than that for tRNA. The local motion correlation times of the ribose carbons, except perhaps the 2', do not appear to be strongly coupled to the conformation of the polynucleotide. The 13C NMR results can be combined with those of other investigations to obtain a consistent picture of the internal and overall motions of polynucleotides which have a backbone that is much more flexible than that of the bases.

Porphyrins can catalyze the interconversion of DNA quadruplex structural types.

The binding of porphyrins to quadruplex DNAs provides a model system for the examination of drug binding to telomere, centromere, triplet repeat and other DNAs which may form quadruplex structures in vivo. Porphyrins, and certain other molecules that interact with quadruplex DNAs, have been shown to have significant biological activity. In this investigation the interactions of porphyrins with quadruplex DNAs have been examined by optical and NMR methods. The fluorescence of selected porphyrins can be used to discriminate between duplex and quadruplex DNAs. The fluorescence of the porphyrins can also be used to discriminate partially between the chair, basket and parallel stranded types of quadruplex DNA. At the relatively high DNA concentrations used in NMR, the porphyrins catalyze the conversion of both chair and basket type structures into parallel strand quadruplex DNAs. A DNA-porphyrin system has been found which appears to be a model for an intermediate of the catalytic pathway.

Hydrogen bonding interactions of polyamines with the 2' OH of RNA.

Polyamines and other charged amines bind to RNA by hydrogen bonding to the 3' phosphate and to the 2' OH. This mode of binding suggests a mechanism by which DNA and RNA might be distinguished by enzymes.