THe solution structure of a DNA hairpan containin a loop of three thymidines determined by nuclear manetic resonance and molecular mechanics (original) (raw)
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Conformational Analysis of DNA-Trinucleotide-Hairpin-Loop Structures Using a Continuum Solvent Model
Biophysical Journal, 2001
A number of trinucleotide sequences in DNA can form compact and stable hairpin loops that may have significance for DNA replication and transcription. The conformational analysis of these motifs is important for an understanding of the function and design of nucleic acid structures. Extensive conformational searches have been performed on three experimentally known trinucleotide hairpin loops (AGC, AAA, and GCA) closed by a four-base-pair stem. An implicit solvation model based on the generalized Born method has been employed during energy minimization and conformational search. In addition, energy-minimized conformers were evaluated using a finite-difference Poisson-Boltzmann approach. For all three loop sequences, conformations close to experiment were found as lowest-energy structures among several thousand alternative energy minima. The inclusion of reaction-field contributions was found to be important for a realistic conformer ranking. Most generated hairpin loop structures within ϳ5 kcal mol Ϫ1 of the lowest-energy structure have a similar topology. Structures within ϳ10 kcal mol Ϫ1 could be classified into about five structural families representing distinct arrangements of loop nucleotides. Although a large number of backbone torsion angle combinations were compatible with each structural class, some specific patterns could be identified. Harmonic mode analysis was used to account for differences in conformational flexibility of low-energy sub-states. Class-specific differences in the pattern of atomic fluctuations along the sequence were observed; however, inclusion of conformational entropy contributions did not change ranking of structural classes. For an additional loop sequence (AAG) with no available experimental structure, the approach suggests a lowestenergy loop topology overall similar to the other three loop sequences but closed by a different non-canonical base-pairing scheme.
A DNA hairpin with a single residue loop closed by a strongly distorted watson-crick G·C base-pair
Journal of Molecular Biology, 1999
Our previous NMR and modeling studies have shown that the single-stranded 19mer oligonucleotides d(AGCTTATC-ATC-GATAA GCT) -ATC-and d(AGCTTATC-GAT-GATAAGCT) -GAT-encompassing the strongest topoisomerase II cleavage site in pBR322 DNA could form stable hairpin structures. A new sheared base-pair, the pyrimidine-purine C ÁA, was found to close the single base -ATC-loop, while -GAT-displayed a¯exible loop of three/®ve residues with no stabilizing interactions. Now we report a structural study on -GAC-, an analog of -GAT-, derived through the substitution of the loop residue T by C. The results obtained from NMR, non-denaturing PAGE, UV-melting, circular dichroism experiments and restrained molecular dynamics indicate that -GAC-adopts a hairpin structure folded through a single residue loop. In the -GAC-hairpin the direction of the G9 sugar is reversed relative to the C8 sugar, thus pushing the backbone of the loop into the major groove. The G9Á C11 base-pair closing the loop is thus neither a sheared base-pair nor a regular Watson-Crick one. Although G9 and C11 are paired through hydrogen bonds of Watson-Crick type, the base-pair is not planar but rather adopts a wedge-shaped geometry with the two bases stacked on top of each other in the minor groove. The distortion decreases the sugar C1 H -C1 H distance between the paired G9 and C11, to 8 A Ê versus 11 A Ê in the standard B-DNA. The A10 residue at the center of the loop interacts with the G9 Á C11 base-pair, and seems to contribute to the extra thermal stability displayed by -GAC-compared to -GAT-. Test calculations allowed us to identify the experimental NOEs critical for inducing the distorted G ÁC Watson-Crick base-pair. The preference of -GAC-for a hairpin structure rather than a duplex is con®rmed by the diffusion constant values obtained from pulse-®eld gradient NMR experiments. All together, the results illustrate the high degree of plasticity of single-stranded DNAs which can accommodate a variety of turn-loops to fold up on themselves.
Nucleic Acids Research, 1997
The three-dimensional structure of the hairpin formed by d(ATCCTA-GTTA-TAGGAT) has been determined by means of two-dimensional NMR studies, distance geometry and molecular dynamics calculations. The first and the last residues of the tetraloop of this hairpin form a sheared G-A base pair on top of the six Watson-Crick base pairs in the stem. The glycosidic torsion angles of the guanine and adenine residues in the G-A base pair reside in the anti and high-anti domain (∼-60_) respectively. Several dihedral angles in the loop adopt non-standard values to accommodate this base pair. The first and second residue in the loop are stacked in a more or less normal helical fashion; the fourth loop residue also stacks upon the stem, while the third residue is directed away from the loop region. The loop structure can be classified as a so-called type-I loop, in which the bases at the 5′-end of the loop stack in a continuous fashion. In this situation, loop stability is unlikely to depend heavily on the nature of the unpaired bases in the loop. Moreover, the present study indicates that the influence of the polarity of a closing A·T pair is much less significant than that of a closing C·G base pair.
Journal of Molecular Biology, 2000
The solution structure of a DNA three-way junction (3H) containing two unpaired thymidine bases at the branch site (3HT2), was determined by NMR. Arms A and B of the 3HT2 form a quasi-continuous stacked helix, which is underwound at the junction and has an increased helical rise. The unstacked arm C forms an acute angle of approximately 55 degrees with the unique arm A. The stacking of the unpaired thymidine bases on arm C resembles the folding of hairpin loops. From this data, combined with the reported stacking behavior of 23 other 3HS2 s, two rules are derived that together correctly reproduce their stacking preference. These rules predict, from the sequence of any 3HS2, its stacking preference. The structure also suggests a plausible mechanism for structure-specific recognition of branched nucleic acids by proteins.
Nucleic Acids Research, 1987
We have determined the three-dimensional structure of a nonselfcomplementary nonanucleotide duplex which contains an abasic (apyrimidinic) site in the centre, i.e. a deoxyribose residue opposite an adenosine. The majority of the base and sugar proton resonances were assiggned by NOESY, COSY and 2DQF spectra in D20 and H20. We have measured the initial slope of buildup of NOEs in NOESY spectra at very short mixing times (25 to 50 ms), and from these were able to establish interproton distances for the central part of the duplex. We propose a different strategy for proton-proton distance determinations which takes into account the observed variations in correlation times for particular proton-proton vectors. A set of 31 measured interproton distances was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two structures were obtained which retain all aspects of a classical B DNA in which the unpaired adenine and the abasic deoxyribose lie inside the helix. We observe that the non-hydrogen bonded adenine is held well in the helix, the Tm of this base being the same as that of the A.T base pairs in the same duplex.
European Biophysics Journal, 1984
A 500-MHz 1H-NMR study on the single-stranded DNA undecamer (11-mer) 5'd AAGTGTGATAT is presented. Using a combination of one-dimensional pre-steady-state nuclear Overhauser enhancement (NOE) measurements and two-dimensional homonuclear J-correlated spectroscopy, virtually complete resonance assignments are obtained. The relative magnitudes of the intra-and internucleotide NOEs indicate that the overall structure of the single-stranded ll-mer is a right-handed B-type helix with extensive base stacking. Within this overall structure there is quite a large degree of variability, as exemplified by variations in glycosidic bond and sugar pucker conformations, most likely determined by base sequence.
Nucleic Acids Research, 1990
We have investigated loop-induced structural perturbation of the stem structure in hairpins d(GAATTCXnGAATTC) (X = A, T and n = 3, 4, 5 and 6) that contain an EcoRI restriction site in close proximity to the hairpin loop. Oligonucleotides containing either a T3 or a A3 loop were not hydrolyzed by the restriction enzyme and also showed only weak binding to EcoRI in the absence of the cofactor Mg2+. In contrast, hairpins with larger loops are hydrolyzed by the enzyme at the scission site next to the loop although the substrate with a A4 loop is significantly more resistant than the oligonucleotide containing a T4 loop. The hairpin structures with 3 loop residues were found to be thermally most stable while larger hairpin loops resulted in structures with lower mefting temperatures. The T-loop hairpins are thermally more stable than the hairpins containing the same number of A residues in the loop. As judged from proton NMR spectroscopy and the thermodynamic data, the base pair closest to the hairpin loop did form in all cases studied. The hairpin loops did, however, affect the conformation of the stem structure of the hairpins. From 31p and IH NMR spectroscopy we conclude that the perturbation of the stem structure is stronger for smaller hairpin loops and that the extent of the perturbation is limited to 2-3 base pairs for hairpins with T3 or A4 loops. Our results demonstrate that hairpin loops modulate the conformation of the stem residues close to the loop and that this in turn reduces the substrate activity for DNA sequence specific proteins.
How Flexible are DNA Constituents? The Quantum-Mechanical Study
Journal of Biomolecular Structure and Dynamics, 2011
Relaxed force constants (RFCs) and vibrational root-mean-square deviations have been evaluated by the original calculation method for conformational parameters of the DNA structural units and their constituents: nucleic acid bases (uracile, thymine, cytosine, adenine and guanine) and their 'building blocks' (benzene, pyrimidine, imidazole and purine molecules), as well as the DNA backbone structural units: tetrahydrofuran, 1,2-dideoxyribose, methanol and orthophosphoric acid. It has been found that the RFCs for nomenclature torsions b, g, e and sugar pseudorotation angle P in 1,2-dideoxyribose are sensible to the molecule conformation and their values are in the range of 1-25 kcal/(mole•rad 2) obeying the inequality K g. K e. K P. K b. The RFCs values for endocyclic torsions of nucleic acid bases six-member rings lie within 15-45 kcal/(mole•rad 2) in pyrimidines and within 20-60 kcal/(mole•rad 2) in purines. It is shown that the quantum zero-point motion effectively neglects the amino group non-planarity in cytosine, adenine and partially in guanine.
Biochemistry, 1997
The three-dimensional structural analysis of DNA ) 5′ duplex in which the X residue is a modified abasic site [3-hydroxy-2-(hydroxymethyl)tetrahydrofuran] has been performed using NOESY, DQFCOSY, TOCSY, and 31 P-1 H HSQC-TOCSY spectra in relation with molecular dynamics simulations. A total of 249 distances and 224 dihedral angles were used for construction. The optimal distances were calculated using the complete relaxation matrix method from hybrid matrices which were built with the experimental NOE intensities and additional data derived from either standard A-or B-DNA. Six independent refined structures starting from canonical A-and B-DNA were determined on the basis of the NMR data, and all converged to a single family with average rms deviations below 0.6 Å and final NOE R x factors of 0.055 † This work was supported by the Association pour la Recherche sur le Cancer (ARC) and the Ligue Nationale contre le Cancer (LIGUE).