Search

Telomerase is an RNA-protein complex (RNP) that extends telomeric DNA at the 3' ends of chromosomes using its telomerase reverse transcriptase (TERT) and integral template-containing telomerase RNA (TER). Its activity is a critical determinant of human health, affecting aging, cancer, and stem cell renewal. Lack of atomic models of telomerase, particularly one with DNA bound, has limited our mechanistic understanding of telomeric DNA repeat synthesis. We report the 4.8 Å resolution cryoelectron microscopy structure of active Tetrahymena telomerase bound to telomeric DNA. The catalytic core is an intricately interlocked structure of TERT and TER, including a previously structurally uncharacterized TERT domain that interacts with the TEN domain to physically enclose TER and regulate activity. This complete structure of a telomerase catalytic core and its interactions with telomeric DNA from the template to telomere-interacting p50-TEB complex provides unanticipated insights into telomerase assembly and catalytic cycle and a new paradigm for a reverse transcriptase RNP., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Variations in the hydrogen bond network of the Oxy-1.5 DNA guanine quadruplex have been monitored by trans-H-bond scalar couplings, (h2)J(N2N7), for Na(+)-, K(+)-, and NH(4)(+)-bound forms over a temperature range from 5 to 55 degrees C. The variations in (h2)J(N2N7) couplings exhibit an overall trend of Na(+) > K(+) > NH(4)(+) and correlate with the different cation positions and N2-H2...N7 H-bond lengths in the respective structures. A global weakening of the (h2)J(N2N7) couplings with increasing temperature for the three DNA quadruplex species is accompanied by a global increase of the acceptor (15)N7 chemical shifts. Above 35 degrees C, spectral heterogeneity indicates thermal denaturation for the Na(+)-bound form, whereas spectral homogeneity persists up to 55 degrees C for the K(+)- and NH(4)(+)-coordinated forms. The average relative change of the (h2)J(N2N7) couplings amounts to approximately 0.8 x 10(-3)/K and is thus considerably smaller than respective values reported for nucleic acid duplexes. The significantly higher thermal stability of H-bond geometries in the DNA quadruplexes can be rationalized by their cation coordination of the G-quartets and the extensive H-bond network between the four strands. A detailed analysis of individual (h2)J(N2N7) couplings reveals that the 5' strand end, comprising base pairs G1-G9* and G4*-G1, is the most thermolabile region of the DNA quadruplex in all three cation-bound forms.

DNA A-tracts have been defined as four or more consecutive A.T base pairs without a TpA step. When inserted in phase with the DNA helical repeat, bending is manifested macroscopically as anomalous migration on polyacrylamide gels, first observed >20 years ago. An unsolved conundrum is why DNA containing in-phase A-tract repeats of A(4)T(4) are bent, whereas T(4)A(4) is straight. We have determined the solution structures of the DNA duplexes formed by d(GCAAAATTTTGC) [A4T4] and d(CGTTTTAAAACG) [T4A4] with NH(4)(+) counterions by using NMR spectroscopy, including refinement with residual dipolar couplings. Analysis of the structures shows that the ApT step has a large negative roll, resulting in a local bend toward the minor groove, whereas the TpA step has a positive roll and locally bends toward the major groove. For A4T4, this bend is nearly in phase with bends at the two A-tract junctions, resulting in an overall bend toward the minor groove of the A-tract, whereas for T4A4, the bends oppose each other, resulting in a relatively straight helix. NMR-based structural modeling of d(CAAAATTTTG)(15) and d(GTTTTAAAAC)(15) reveals that the former forms a left-handed superhelix with a diameter of approximately 110 A and pitch of 80 A, similar to DNA in the nucleosome, whereas the latter has a gentle writhe with a pitch of >250 A and diameter of approximately 50 A. Results of gel electrophoretic mobility studies are consistent with the higher-order structure of the DNA and furthermore depend on the nature of the monovalent cation present in the running buffer.

NHP6A is a non-sequence-specific DNA-binding protein from Saccharomyces cerevisiae which belongs to the HMGB protein family. Previously, we have solved the structure of NHP6A in the absence of DNA and modeled its interaction with DNA. Here, we present the refined solution structures of the NHP6A-DNA complex as well as the free 15bp DNA. Both the free and bound forms of the protein adopt the typical L-shaped HMGB domain fold. The DNA in the complex undergoes significant structural rearrangement from its free form while the protein shows smaller but significant conformational changes in the complex. Structural and mutational analysis as well as comparison of the complex with the free DNA provides insight into the factors that contribute to binding site selection and DNA deformations in the complex. Further insight into the amino acid determinants of DNA binding by HMGB domain proteins is given by a correlation study of NHP6A and 32 other HMGB domains belonging to both the DNA-sequence-specific and non-sequence-specific families of HMGB proteins. The resulting correlations can be rationalized by comparison of solved structures of HMGB proteins.

The localization of Mn(2+) in A-tract DNA has been studied by (1)H NMR spectroscopy using a series of self-complementary dodecamer oligonucleotides that contain the sequence motifs A(n)(n) and T(n)A(n), where n = 2, 3, or 4. Mn(2+) localization in the minor groove is observed for all the sequences that have been studied, with the position and degree of localization being highly sequence-dependent. The site most favored for Mn(2+) localization in the minor groove is near the 5'-most ApA step for both the T(n)A(n) and the A(n)T(n) series. For the T(n)A(n) series, this results in two closely spaced symmetry-related Mn(2+) localization sites near the center of each duplex, while for the A(n)T(n) series, the two symmetry-related sites are separated by as much as one half-helical turn. The degree of Mn(2+) localization in the minor groove of the T(n)A(n) series decreases substantially as the AT sequence element is shortened from T(4)A(4) to T(2)A(2). The A(n)T(n) series also exhibits length-dependent Mn(2+) localization; however, the degree of minor groove occupancy by Mn(2+) is significantly less than that observed for the T(n)A(n) series. For both A(n)T(n) and T(n)A(n) sequences, the 3'-most AH2 resonance is the least broadened of the AH2 resonances. This is consistent with the observation that the minor groove of A-tract DNA narrows in the 5' to 3' direction, apparently becoming too narrow after two base pairs for the entry of a fully hydrated divalent cation. The results that are reported illustrate the delicate interplay that exists between DNA nucleotide sequence, minor groove width, and divalent cation localization. The proposed role of cation localization in helical axis bending by A-tracts is also discussed.

A two-dimensional, quantitative J-correlation NMR experiment for precise measurements of the proton-carbon vicinal coupling constants 3J(C2)/4-H1' and 3J(C6)/8-H1' in uniformly 13C-labeled nucleic acids is presented. To reduce loss of signal due to 1H-13C dipole-dipole relaxation, a multiple-quantum constant time experiment with appropriately incorporated band selective 1H and 13C pulses was applied. The experiment is used to obtain the 3J(C2)/4-H1' and 3J(C6)/8-H1' coupling constants in a uniformly 13C, 15N-labeled [d(G4T4G4)]2 quadruplex. The measured values and glycosidic torsion angles in the G-quadruplex, obtained by restrained molecular dynamics with explicit solvent using the previously published restraints, along with selected data from the literature are used to check and modify existing parameters of the Karplus equations. The parameterizations obtained using glycosidic torsion angles derived from the original solution and recently determined X-ray structures are also compared.

Scalar coupling constants and magnetic shieldings in the imino hydrogen-bonding region of Hoogsteen-Watson-Crick T.A-T and C(+).G-C triplets have been calculated as a function of the distance between proton donor and acceptor nitrogen atoms. The Fermi contact contributions to (h2)J((15)N-H...(15)N), (1)J((15)N-(1)H), and (h1)J((1)H...(15)N) were computed using density functional theory/finite perturbation theory (DFT/FPT) methods for the full base triplets at the unrestricted B3PW91/6-311G level. Chemical shifts delta((1)H) and delta((15)N) were obtained at the same level using the gauge including atomic orbital (GIAO) method for magnetic shielding. All three scalar couplings and all three chemical shifts are strongly interrelated and exhibit monotonic changes with base pair separation. These correlations are in conformity with experimental data for a 32-nucleotide DNA triplex. The results suggest that both chemical shifts and coupling constants can be used to gain information on H-bond donor-acceptor distances in nucleic acids. In addition to the DFT/FPT calculations, a simple three-orbital model of the N-H...H bond and a sum-over-states analysis is presented. This model reproduces the basic features of the H-bond coupling effect. In accordance with this model and the DFT calculations, a positive sign for the (h2)J(NN) coupling is determined from an E.COSY experiment.

The DNA sequence d(G(4)T(4)G(4)) [Oxy-1.5] consists of 1.5 units of the repeat in telomeres of Oxytricha nova and has been shown by NMR and X-ray crystallographic analysis to form a dimeric quadruplex structure with four guanine-quartets. However, the structure reported in the X-ray study has a fundamentally different conformation and folding topology compared to the solution structure. In order to elucidate the possible role of different counterions in this discrepancy and to investigate the conformational effects and dynamics of ion binding to G-quadruplex DNA, we compare results from further experiments using a variety of counterions, namely K(+), Na(+)and NH(4)(+). A detailed structure determination of Oxy-1.5 in solution in the presence of K(+)shows the same folding topology as previously reported with the same molecule in the presence of Na(+). Both conformations are symmetric dimeric quadruplexes with T(4)loops which span the diagonal of the end quartets. The stack of quartets shows only small differences in the presence of K(+)versus Na(+)counterions, but the T(4)loops adopt notably distinguishable conformations. Dynamic NMR analysis of the spectra of Oxy-1.5 in mixed Na(+)/K(+)solution reveals that there are at least three K(+)binding sites. Additional experiments in the presence of NH(4)(+)reveal the same topology and loop conformation as in the K(+)form and allow the direct localization of three central ions in the stack of quartets and further show that there are no specific NH(4)(+)binding sites in the T(4)loop. The location of bound NH(4)(+)with respect to the expected coordination sites for Na(+)binding provides a rationale for the difference observed for the structure of the T(4)loop in the Na(+)form, with respect to that observed for the K(+)and NH(4)(+)forms.

DNA oligonucleotides can form multistranded helices through either the folding of a single strand or the association of two, three or four strands of DNA. Structures of several new DNA triplexes, G-quartet DNA quadruplexes and I-motif DNA quadruplexes have been reported recently. These structures provide new insights into helix stability and folding, loop conformations and cation interactions.

Application of heteronuclear magnetic resonance pulse methods to 13C, 15N-labeled nucleic acids is important for the accurate structure determination of larger RNA and DNA oligonucleotides and protein-nucleic acid complexes. These methods have been applied primarily to RNA, due to the availability of labeled samples. The two major differences between DNA and RNA are at the C2' of the ribose and deoxyribose and the additional methyl group on thymine versus uracil. We have enzymatically synthesized a 13C,15N-labeled 32 base DNA oligonucleotide that folds to form an intramolecular triplex. We present two- and three-dimensional versions of a new HCCCH-TOCSY experiment that provides intraresidue correlation between the thymine H6 and methyl resonances via the intervening carbons (H6-C6-C5-Cme-Hme)., (Copyright 1999 Academic Press.)

Monovalent cation binding sites on nucleic acids in solution can be localized using the isotopically labeled ammonium ion (15NH4+) as a probe in high resolution NMR spectroscopy experiments. The application of this technique to a series of DNA duplexes reveals a preference for the binding of ammonium cations in the minor groove of A-tract sequences. These results are consistent with a recent report which indicates that some solvent electron densities previously identified as water molecules in DNA X-ray crystal structures are partially occupied by sodium ions. The sequence-specific nature of monovalent cation binding sites demonstrated here for A-tract DNA provides an explanation for the origin of sequence-directed bending., (Copyright 1999 Academic Press.)

Guanine quartets are readily formed by guanine nucleotides and guanine-rich oligonucleotides in the presence of certain monovalent and divalent cations. The quadruplexes composed of these quartets are of interest for their potential roles in vivo, their relatively frequent appearance in oligonucleotides derived from in vitro selection, and their inhibition of template directed RNA polymerization under proposed prebiotic conditions. The requirement of cation coordination for the stabilization of G quartets makes understanding cation-quadruplex interactions an essential step towards a complete understanding of G quadruplex formation. We have used 15NH4+ as a probe of cation coordination by the four G quartets of the DNA bimolecular quadruplex [d(G4T4G4)]2, formed from oligonucleotides with the repeat sequence found in Oxytricha nova telomeres. 1H and 15N heteronuclear NMR spectroscopy has allowed the direct localization of monovalent cation binding sites in the solution state and the analysis of cation movement between the binding sites. These experiments show that [d(G4T4G4)]2 coordinates three ammonium ions, one in each of two symmetry related sites and one on the axis of symmetry of the dimeric molecule. The NH4+ move along the central axis of the quadruplex between these sites and the solution, reminiscent of an ion channel. The residence time of the central ion is determined to be 250 ms. The 15NH4+ is shown to be a valuable probe of monovalent cation binding sites and dynamics., (Copyright 1999 Academic Press.)

A DNA molecule was designed and synthesized with three octanucleotide stretches linked by two hexakis(ethylene glycol) chains to form an intramolecular triplex in solution. The structural data obtained from a series of NMR NOESY spectra yielded interproton distances, and COSY experiments provided dihedral angle information for analysis of deoxyribose ring pucker. Using distance geometry followed by simulated annealing with restrained molecular dynamics and relaxation matrix refinement, a well-refined ensemble of conformations was calculated. Although some NOE cross-peaks involving protons of the hexakis(ethylene glycol) linker could be identified, most could not be assigned and the conformations of the linkers were not determined. The deoxyribose conformations are predominantly of the S type, except for the protonated cytosine residues in the third strand which show hybrid N and S character. Overall, the duplex part of the molecule resembles a B-DNA double helix with the third strand bound in its major groove by Hoogsteen hydrogen bonds. This structure provides a basis for comparison with triplexes containing noncanonical or nonnatural nucleotides.

Incorporation of the modified base 5-(1-propynl)-2'-deoxyuridine (propynylU) in the third strand of a triplex leads to enhanced triplex stabilization. To investigate effects of the propyne nucleotide on triplex structure and the factors underlying the increased stability, we have determined the solution structure of the intramolecular DNA pyrmidine-purine-pyrimdine d(AGAGAGAA-(EG)6-TTCTCTCT-(EG)6-PCPCPCPP) (PDD-EG), which contains 5-(1-propynl)-2'-deoxyuridine (P) in the third strand and hexakis(ethylene glycol) linkers [(EG)6]. The structure was calculated using X-PLOR with distance and dihedral angle restraints obtained from two-dimensional NMR experiments and refined with the direct relaxation matrix method. The structures show that the extended aromatic electron cloud of the propynylU nucleotide stacks well over the 5'-neighboring nucleotides, resulting in increased stabilization. The propynylU nucleotides also affect the overall structure of the triple helix. A comparison of the structure to that of the nonmodified intramolecular DNA triplex of the same sequence, d(AGAGAGAA-(EG)6-TTCTCTCT-(EG)6-TCTCTCTT) (DDD-EG), shows that PDD-EG has a more A-DNA like X displacement and inclination than DDD-EG yet still maintains predominantly S-type sugar puckers as found in DDD-EG and other DNA triplexes.

The three-dimensional structure of a pyrimidine-purine-pyrimidine DNA triplex containing an N7-glycosylated guanine (7G) in the third strand has been determined by NMR spectroscopy, restrained molecular dynamics calculations, and complete relaxation matrix refinement. Glycosylation of the guanine at the N7 position permits it to adopt a conformation such that the Hoogsteen face of the base mimics the arrangement of hydrogen bond donors seen in protonated cytosine. The NMR data confirm the previously proposed hydrogen bonding scheme of the 7G x G x C triplet. The three-dimensional structure of the triplex accommodates the 7G with less distortion of the phosphodiester backbone than would be required for an N9-glycosylated guanine in the same sequence position, but some changes in the positions of the phosphodiester backbone are present compared to a C+ x G x C triplet. The structure provides a rationale for the observations that 7G binds to Watson-Crick G x C base pairs with higher specificity and affinity than guanine, but with a lower stability at pH 5.2 than would be provided by a canonical C+ x G x C triplet.

We have studied the competition between Na+ and K+ for coordination by G quartets using the oligonucleotide d(G3T4G3) as a model system. d(G3T4G3) forms a dimeric foldback structure containing three G quartets in the presence of either NaCl or KCl. Proton chemical shifts, which are particular to the species of coordinated ion, have been used to monitor the conversion between the sodium and potassium forms under equilibrium conditions. Analysis of titration experiments indicates that at least two K+ are coordinated by the three quartets of the dimeric molecule, and perfect fits of the data are obtained for two Na+ being displaced by two K+. Our results also indicate that the conversion of [d(G3T4G3)]2 from the sodium to the potassium form is associated with a net free energy change (delta G degrees) of -1.7 +/- 0.15 kcal/mol. It has long been suggested that the greater thermal stability of DNA quadruplex structures in the presence of K+ is primarily a result of the optimal fit of this ion in the coordination sites formed by G quartets. However, a consideration of the relatively small change in free energy associated with the conversion from the sodium to the potassium form and the relatively large difference between the free energy of hydration for Na+ and K+ indicates that this cannot be correct. Rather, the preferred coordination of K+ over Na+ is actually driven by the greater energetic cost of Na+ dehydration with respect to K+ dehydration.

We have used NMR spectroscopy to study a pyrimidine-purine-pyrimidine DNA triplex containing a non-natural base, 1-(2-deoxy-beta-D-ribofuranosyl)-4-(3-benzamido)phenylimidazole (D3), in the third strand. The D3 base has been previously shown to specifically recognize T-A and C-G base-pairs via intercalation on the 3' side (with respect to the purine strand) of the target base pair, instead of forming sequence-specific hydrogen bonds. 1H resonance assignments have been made for the D3 base and most of the non-loop portion of the triplex. The solution structure of the triplex was calculated using restrained molecular dynamics and complete relaxation matrix refinement. The duplex portion of the triplex has an over-all helical structure that is more similar to B-DNA than to A-DNA. The three aromatic rings of the D3 base stack on the bases of all three strands and mimic a triplet. The conformation of the D3 base and its sequence specificity are discussed.

The thrombin-binding aptamer d(GGTTGGTGTGGTTGG) is one of a family of DNA oligonucleotides that were identified by in vitro selection to bind specifically and with high affinity to thrombin. Two groups independently determined the tertiary structure in solution by NMR and at about the same time, the X-ray crystal structure of the aptamer in complex with thrombin was reported. In all cases, the thrombin-binding aptamer was found to fold into a structure containing two planar guanine quartets as its core. The NMR and crystal structures, however, have fundamentally different folding patterns owing to differences in the way these central bases are connected. We discuss the distinctions between the refined crystal and solution structures and show that the NMR model is consistent with the X-ray diffraction data.

Hoogsteen base pairs have been demonstrated to occur in base pairs adjacent to the CpG binding sites in complexes of triostin A and echinomycin with a variety of DNA oligonucleotides. To understand the relationship of these unusual base pairs to the sequence specificity of these quinoxaline antibiotics, the conformation of the base pairs flanking the YpR binding sites of the 2:1 drug-DNA complexes of triostin A with [d(ACGTACGT)]2 and of the TpA specific [N-MeCys3, N-MeCys7] TANDEM with [d(ATACGTAT)]2 have been studied by 1H NMR spectroscopy. In both the 2:1 triostin A-DNA complex and the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the terminal A.T base pairs are Hoogsteen base paired with the 5' adenine in the syn conformation. This indicates that both TpA specific and CpG specific quinoxaline antibiotics are capable of inducing Hoogsteen base pairs in DNA. However, in both 2:1 complexes, Hoogsteen base pairing is limited to the terminal base pairs. In the 2:1 triostin A complex, the internal adenines are anti and in the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the internal guanines are anti regardless of pH, which indicates that the central base pairs of both complexes form Watson-Crick base pairs. This indicates that the sequence dependent nature of Hoogsteen base pairing is the same in TpA specific and CpG specific quinoxaline antibiotic-DNA complexes. We have calculated a low resolution three-dimensional structure of the 2triostin A-[d(ACGTACGT)]2 complex and compared it with other CpG specific quinoxaline antibiotic-DNA complexes. The role of stacking in the formation of Hoogsteen base pairs in these complexes is discussed.

The structure formed by the DNA oligonucleotide d(G3T4G3) has been studied by one- and two-dimensional 1H NMR spectroscopy. In NaCl solution, d(G3T4G3), like d(G4T4G4) (Oxy-1.5), forms a dimeric quadruplex with the thymines in loops across the diagonal of the end quartets. Unlike Oxy-1.5, the dimer is not symmetric, and both monomer strands are observed in NMR spectra. Three quartets are formed from the GGG tracts. Glycosidic conformations of the guanines are 5'-syn-syn-anti-(loop)-syn-anti-anti in one strand and 5'-syn-anti-anti-(loop)-syn-syn-anti in the other strand. Thus, the stacking of the quartets (tail-to-tail, head-to-tail) is unlike all previously described fold-back (tail-to-tail, head-to-head) and parallel-stranded (head-to-tail, head-to-tail) quadruplexes.

The binding of CysMeTANDEM and triostin A to DNA containing a CpI step has been studied by one- and two-dimensional 1H NMR spectroscopy. CysMeTANDEM binds sequence specifically to CpI steps as well as TpA steps as a bis-intercalator with the peptide backbone in the minor groove of the DNA. Only nonspecific, nonintercalative binding is observed between triostin A and DNA containing a CpI step. Comparison of the CysMeTANDEM-[d(GGACITCC)]2 complex to the CysMeTANDEM-[d(GGA-TATCC)]2 complex indicates that the structures of both complexes are very similar. However, CysMeTANDEM binds less tightly to [d(GGACITCC)]2 than to [d(GGATATCC)]2. The NMR evidence presented provides molecular insight into the role of stacking interactions and hydrogen bonding between the drug and the DNA in the sequence-specific binding of CysMeTANDEM to TpA sites and of triostin A to CpG sites.

The DNA oligonucleotide d(GGTTGGTGTGGTTGG) (thrombin aptamer) binds to thrombin and inhibits its enzymatic activity in the chain of reactions that lead to blood clotting. Two-dimensional 1H NMR studies indicate that the oligonucleotide forms a folded structure in solution, composed of two guanine quartets connected by two T-T loops spanning the narrow grooves at one end and a T-G-T loop spanning a wide groove at the other end. We present the assignment strategy used, methods for the structure determination, and the refined three-dimensional structure of the thrombin aptamer. The initial structures were generated by metric matrix distance geometry using distance and dihedral bond angle constraints from NOE and coupling constants, respectively, and refined by restrained molecular dynamics and direct NOE refinement. Knowledge of the three-dimensional structure of this thrombin aptamer may be relevant for the design of improved thrombin-inhibiting anti-coagulants with similar structural motifs.

We have used two-dimensional 1H NMR spectroscopy to study the conformation of the thrombin-binding aptamer d(GGTTGGTGTGGTTGG) in solution. This is one of a series of thrombin-binding DNA aptamers with a consensus 15-base sequence that was recently isolated and shown to inhibit thrombin-catalyzed fibrin clot formation in vitro [Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H. & Toole, J. J. (1992) Nature (London) 355, 564-566]. The oligonucleotide forms a unimolecular DNA quadruplex consisting of two G-quartets connected by two TT loops and one TGT loop. A potential T.T bp is formed between the two TT loops across the diagonal of the top G-quartet. Thus, all of the invariant bases in the consensus sequence are base-paired. This aptamer structure was determined by NMR and illustrates that this molecule forms a specific folded structure. Knowledge of this structure may be used in the further development of oligonucleotide-based thrombin inhibitors.

[N-MeCys3,N-MeCys7]TANDEM (CysMeTANDEM) is an octadepsipeptide quinoxaline antibiotic that binds specifically by bisintercalation to double-stranded DNA at NTAN sites [Addess, K. J., Gilbert, D. E., Olsen, R. K., & Feigon, J. (1992) Biochemistry 31, 339-350; Addess, K. J., Gilbert, D. E., & Feigon, J. (1992) in Structure and Function Volume 1: Nucleic Acids (Sarma, R. H., & Sarma, M. H., Eds.) pp 147-164, Adenine Press, Schenectady, NY]. We have determined the three-dimensional structure of a complex of CysMeTANDEM and the DNA hexamer [d(GATATC)]2 using two-dimensional 1H NMR derived NOE and dihedral bond angle constraints. This is the first structure of a TpA-specific quinoxaline antibiotic in complex with DNA. Initial structures of the complex were generated by metric matrix distance geometry followed by simulated annealing. Eight of these structures, refined by restrained molecular dynamics, energy minimization, and NOE-based relaxation matrix refinement, have an average pairwise RMSD of 1.11 A for all structures, calculated using all heavy atoms of the drug and the DNA except the terminal base pairs. CysMeTANDEM binds to and affects the structure of the DNA in a manner similar to that observed in complexes of the CpG-specific quinoxaline antibiotics triostin A and echinomycin with DNA [Ughetto, G., Wang, A. H.-J., Quigley, G. J., van der Marel, G. A., van Boom, J. H., & Rich, A. (1985) Nucleic Acids Res. 13, 2305-2323; Wang, A. H.-J., Ughetto G., Quigley, G. J., Hakoshima, T., van der Marel, G. A., van Boom, J. H., & Rich, A. (1984) Science 225, 1115-1121; Wang, A. H.-J., Ughetto, G., Quigley, G. J., & Rich, A. (1986) J. Biomol. Struct. Dyn. 4, 319-342]. The two quinoxaline rings bisintercalate on either side of the two central T.A base pairs and the peptide ring lies in the minor groove. The central A.T base pairs of the complex are underwound (average helical twist angle of approximately -10 degrees) and buckle inward by approximately 20 degrees. There are intermolecular hydrogen bonds between each of the Ala NH and the AN3 protons of the TpA binding site, analogous to those observed between Ala NH and GN3 in the crystal structures of the CpG-specific complexes of echinomycin and triostin A with DNA. However, the structure of the peptide ring of CysMeTANDEM in the complex differs from that of echinomycin and triostin A.(ABSTRACT TRUNCATED AT 400 WORDS)

Two-dimensional 1H n.m.r. spectroscopy has been used to study the 31-base DNA oligonucleotide 5'-dAGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3', which folds to form a stable intramolecular triplex in solution at acidic pH. This structure is considerably more difficult to assign than short B-DNA duplexes and requires new assignment methods. The assignment strategy and assignments of almost all of the exchangeable and nonexchangeable resonances are presented. Seven base triplets and one Watson-Crick base-pair form the core of the structure and are connected by a four C and four T loop at either end. The second pyrimidine "strand" (bases 24 to 31) in this intramolecular pyrimidine-purine-pyrimidine triplex binds via Hoogsteen base-pairs in the major groove and is parallel to the purine "strand" (bases 1 to 8). Analysis of the sugar puckers reveals that, contrary to widely accepted belief, the triplex sugars are not predominantly in the N-type (close to C3'-endo) conformation. Except for some of the C nucleotides, all sugars are predominantly S-type (close to C2'-endo). Thus, the duplex DNA does not assume N-type sugar conformations to accommodate a third strand in the major groove. A preliminary model of the triplex structure is presented.

A 32-base DNA oligonucleotide has been studied by one- and two-dimensional 1H NMR spectroscopy and is shown to form a stable, pyr.pur.pyr, intramolecular triple helical structure, with a four C loop and a TATA loop connecting the Watson-Crick- and Hoogsteen-paired strands, respectively. This triplex contains five T.A.T base triplets, two C+.G.C base triplets, and an unusual G.T.A base triplet which disrupts the pyr.pur.pyr motif. The G.T.A triplet consists of a Watson-Crick T.A base pair, with the T situated in the "purine strand" and the A situated in the "pyrimidine strand" and a G situated in the Hoogsteen-base-paired "pyrimidine strand" hydrogen bonded to the T. The base-pairing structure of the G.T.A triplet has been investigated and has been found to involve a single hydrogen bond from the guanine amino group to the O4 carbonyl of the thymine, leaving the guanine imino proton free. The specific amino proton involved in the hydrogen bond is the H2(2) proton. This orients the guanine such that its sugar is near the thymine methyl group. The guanine sugar adopts an N-type (C3'-endo) sugar pucker in this triplet. The stability of the G.T.A triplet within pyr.pur.pyr triplexes is discussed.

The telomeres of most eukaryotes contain a repeating G-rich sequence with the consensus d(T/A)1-4G1-8, of which 12-16 bases form a 3' single-strand overhang beyond the telomeric duplex. It has been proposed that these G-rich oligonucleotides associate to form four-stranded structures from one, two or four individual strands and that these structures may be relevant in vivo. The proposed structures contain Hoogsteen base-paired G-quartets, precedent for which has been in the literature for many years. Here we use 1H NMR spectroscopy to study the conformations of the DNA oligonucleotides d(G4T4G4) (Oxy-1.5) and d(G4T4G4T4G4T4G4) (Oxy-3.5) which contain the Oxytricha telomere repeat (T4G4). We find that these molecules fold to form a symmetrical bimolecular and an intramolecular quadruplex, respectively. Both structures have four G-quartets formed from nucleotides that are alternately syn and anti along each strand. This arrangement differs from earlier models in which the strands are alternately all syn or all anti. The T4 loops in Oxy-1.5 are on opposite ends of the quadruplex and loop diagonally across the G-quartet, resulting in adjacent strands being alternately parallel and antiparallel.

[N-MeCys3,N-MeCys7]TANDEM, an undermethylated analogue of Triostin A, contains two N-methyl groups on the cysteine residues only. Footprinting results showed that [N-MeCys3,N-MeCys7]TANDEM binds strongly to DNA rich in A.T residues [Low, C. M. L., Fox, K. R., Olsen, R. K., & Waring, M. J. (1986) Nucleic Acids Res. 14, 2015-2033]. However, it was not known whether specific binding of [N-MeCys3,N-MeCys7]TANDEM requires a TpA step or an ApT step. In 1:1 saturated complexes with the octamers [d(GGATATCC)]2 and [d(GGTTAACC)]2, [N-MeCys3,N-MeCys7]TANDEM binds to each octamer as a bis-intercalator bracketing the TpA step. The octadepsipeptide ring binds in the minor groove of the DNA. Analysis of sugar coupling constants from the phase-sensitive COSY data indicates that the sugar of the thymine in the TpA binding site adopts predominantly an N-type sugar conformation, while the remaining sugars on the DNA adopt an S-type conformation, as has been observed in other Triostin A and echinomycin complexes. The drug does not bind to the octamer [d(GGAATTCC)]2 as a bis-intercalator. Only weak nonintercalative binding is observed to this DNA octamer. These results show unambiguously that [N-MeCys3,N-MeCys7]TANDEM binds sequence specifically at TpA sites in DNA. The factors underlying the sequence specificity of [N-MeCys3,N-MeCys7]TANDEM binding to DNA are discussed.

Intramolecular DNA triplexes that contain eight base triplets formed from the folding of a single DNA strand tolerate a single X.G.C mismatch in the third strand at acidic pH. The structure and relative stability of all four triplets that are possible involving a G.C Watson-Crick base pair were determined with one- and two-dimensional proton nuclear magnetic resonance techniques. Triplexes containing A.G.C, G.G.C, or T.G.C triplets were less stable than the corresponding parent molecule containing a C.G.C triplet. However, all mismatched bases formed specific hydrogen bonds in the major groove of the double helix. The relative effect of these mismatches on the stability of the triplex differs from the effect assayed (under different conditions) by two-dimensional gel electrophoresis and DNA cleavage with oligonucleotide EDTA.Fe(II).

The complexes formed between the cyclic octadepsipeptide antibiotic echinomycin and the two DNA octamers [d(ACGTACGT)]2 and [d(TCGATCGA)]2 have been investigated by using one- and two-dimensional proton NMR spectroscopy techniques. The results obtained for the two complexes are compared to each other, to the crystal structures of related DNA-echinomycin complexes, and to enzymatic and chemical footprinting results. In the saturated complexes, two echinomycin molecules bind to each octamer by bisintercalation of the quinoxaline moieties on either side of each CpG step. Binding of echinomycin to the octamer [d(ACGTACGT)]2 is cooperative so that only the two-drug complex is observed at lower drug-DNA ratios, but binding to [d(TCGATCGA)]2 is not cooperative. At low temperatures, both the internal and terminal A.T base pairs adjacent to the binding site in the [d(ACGTACGT)]2-2 echinomycin complex are Hoogsteen base paired (Gilbert et al., 1989) as observed in related crystal structures. However, as the temperature is raised, the internal A.T Hoogsteen base pairs are destabilized and are observed to be exchanging between the Hoogsteen base-paired and an open (or Watson-Crick base-paired) state. In contrast, in the [d(TCGATCGA)]2-2 echinomycin complex, no A.T Hoogsteen base pairs are observed, the internal A.T base pairs appear to be stabilized by drug binding, and the structure of the complex does not change significantly from 0 to 45 degrees C. Thus, the structure and stability of the DNA in echinomycin-DNA complexes depends on the sequence at and adjacent to the binding site. While we conclude that no single structural change in the DNA can explain all of the footprinting results, unwinding of the DNA helix in the drug-DNA complexes appears to be an important factor while Hoogsteen base pair formation does not.

The bisintercalation complex present between the DNA octamer [d(ACGTACGT)]2 and the cyclic octadepsipeptide antibiotic echinomycin has been studied by one- and two-dimensional proton NMR, and the results obtained have been compared with the crystal structures of related DNA-echinomycin complexes. Two echinomycins are found to bind cooperatively to each DNA duplex at the CpG steps, with the two quinoxaline rings of each echinomycin bisintercalating between the C.G and A.T base pairs. At low temperatures, the A.T base pairs on either side of the intercalation site adopt the Hoogsteen conformation, as observed in the crystal structures. However, as the temperature is raised, the Hoogsteen base pairs in the interior of the duplex are destabilized and are observed to be exchanging between the Hoogsteen base pair and either an open or a Watson-Crick base-paired state. The terminal A.T base pairs, which are not as constrained by the helix as the internal base pairs, remain stably Hoogsteen base-paired up to at least 45 degrees C. The implications of these results for the biological role of Hoogsteen base pairs in echinomycin-DNA complexes in vivo are discussed.

The binding of three ethidium derivatives, ethidium (1), des-3-amino ethidium (2) and des-8-amino ethidium (3), to short (approximately 35 base pairs), random sequence DNA has been investigated using 300 MHz proton NMR. At 35 degrees C all three drugs cause upfield shifts of the resonances from the exchangeable imino protons, as expected for intercalative binding to DNA. However, the lineshapes vary significantly with the nature of the drug. The temperature dependence of the spectra of the DNA shows that differences between spectra observed at 35 degrees C with ethidium and with des-3-amino ethidium are primarily due to differences in the drug binding kinetics rather than to differences in mode of binding. Removal of the amino group at position 3, but not at position 8, on the parent ethidium shortens the lifetime of the intercalative state; this implies that the 3-NH2 group is involved in stabilization of the drug-DNA complex. Analysis of the drug-DNA spectra indicates that there is a preference for binding of the drugs adjacent to G.C base pairs.

In this study 1H NMR has been used to investigate the conformational state of DNA in nucleosome core particles. The nucleosome core particles exhibit partially resolved low field (10-15 ppm) spectra due to imino protons in Watson-Crick base pairs (one resonance per GC or AT base pair). To a first approximation, the spectrum is virtually identical with that of protein-free 140 base pair DNA, and from this observation we draw two important conclusions: (i) Since the low field spectra of DNA are known to be sensitive to conformation, the conformation of DNA in the core particles is essentially the same as that of free DNA (presumably B-form), (ii) since kinks occurring at a frequency at 1 in 10 or 1 in 20 base pairs would result in a core particle spectrum different from that of free DNA we find no NMR evidence supporting either the Crick-Klug or the Sobell models for kinking DNA around the core histones. Linewidth considerations indicate that the rotational correlation time for the core particles is approximately 1.5 X 10(-7) sec, whereas the end-over-end tumbling time of the free 140 base pair DNA is 3 X 10(-7) sec.

The conformation of the self-complimentary DNA dodecamer d(br5CGbr5CGAATTbr5CGbr5CG) has been investigated in a variety of salt and solvent conditions by one and two-dimensional 1H NMR. In low salt aqueous solutions, the molecule forms a regular B-DNA structure similar to the unmodified dodecamer. However, in aqueous solution containing high salt concentration and methanol, the dodecamer adopts a structure in which the br5CGbr5CG ends of the molecule are in a Z-DNA like conformation and the AATT region is neither standard B-DNA nor Z-DNA. The implications of these results for the structure of junctions between B and Z-DNA and the sequence specificity of Z-DNA are discussed.

In this study two-dimensional NMR techniques (COSY and NOESY) have been used in conjunction with one-dimensional NMR results to complete the assignment of the proton NMR spectrum of the double-stranded DNA decamer, d(ATATCGATAT)2, and to obtain qualitative information about numerous interproton distances in this molecule and some limited information about conformational dynamics. COSY and NOESY measurements have been combined to systematically assign many of the resonances from the H1' and H2',2" sugar protons to specific nucleotides in the double helix. This method relies on the fact that sugar protons within a specific nucleotide are scalar coupled and that base protons (AH8, GH8, TH6, and CH6) in right-handed helices can interact simultaneously with their own H2',2" sugar protons and those of the adjacent (5'-3') nucleotide attached to its 5' side (i.e., XpA not ApX). A COSY experiment is used to identify sugar resonances within a residue whereas the NOESY experiment allows the neighboring sugar to be connected (linked). The CH5 and CH6 resonances in the spectrum can immediately be identified by the COSY experiment. The methyl protons of thymine residues exhibit strong through-space interbase interactions both with their own TH6 proton and with AH8 proton on the adjacent (5'-3') adenine residue. These interactions are used both to make assignments of the spectra and to establish that the thymine methyl groups are in close proximity to the AH8 protons of adjacent adenine residues [Feigon, J., Wright, J. M., Leupin, W., Denny, W. A., & Kearns, D. R. (1982) J. Am. Chem. Soc. 104, 5540].(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of a stretch of alternating dA-dT to adopt the left-handed Z form has been assessed by examining the behavior of the sequence d(CG)6(TA)4(CG)6 contained in the plasmid pBR322. The structural transition occurring within this sequence as a function of negative superhelicity was analyzed by several methods, including (1) the supercoiling-dependent unwinding of the insert as determined by two-dimensional gel electrophoresis, (2) the binding of anti-Z-DNA antibodies to the insert, (3) the sensitivity of the sequence to a single strand specific endonuclease, and (4) the sensitivity of the insert to digestion by a restriction endonuclease that cuts within the d(CG)6 segments when in the right-handed form. These studies have shown that in negatively supercoiled DNA the two d(CG)6 portions of the insert adopt the Z form, while the central d(TA)4 segment forms an underwound structure with a helical repeat that is best approximated as being intermediate between the B form and the Z form. A statistical mechanical treatment of the unwinding of the insert as a function of negative superhelicity provides an estimate of the minimum free energy required to convert an A-T bp from the B form to the Z form, as well as the free energy associated with the conversion of an A-T bp from the B form to the unwound form. These results strongly indicate that Z DNA is an unfavored structural alternative for stretches of d(AT)n in negatively supercoiled DNA.

1H NMR has been used to study the interactions of over 70 clinical and experimental antitumor drugs with DNA. Spectra of the low-field (H-bonded imino proton) resonances of DNA were studied as a function of drug per base pair ratio. From the spectral changes observed, it was possible to distinguish three modes of drug binding (intercalation, groove binding, and nonspecific outside binding), to determine the kinetics of drug binding (approximate lifetime of the bound drug), and, in favorable cases, to determine the specificity of the drugs for A X T or G X C base pairs. This method is a useful assay for general drug-binding characteristics. For the intercalating compounds there appears to be a correlation between drug-binding kinetics and useful antitumor activity.

The binding of eight ethidium derivatives to short (approximately 35 base-pair), random sequence DNA has been investigated using 1H-NMR. At 35 degrees C, all drugs cause upfield shifts of the DNA imino proton resonances characteristic of intercalative binding to DNA, but the line shapes vary significantly with the nature of the drug. The results confirm our previous proposal that removal of the amino group at position-3, but not at position-8, on the parent ethidium shortens the lifetime of the intercalative state (less than 1-2 ms at 35 degrees C). These results suggest that hydrogen-bonding interactions with the 3-NH2 group are involved in stabilization of the drug-DNA complex or that changes in charge distribution that accompany removal of the 3-NH2 group reduce the complex stability. The magnitude of the shift of the drug-DNA spectra indicates a slight preference for binding of the drugs adjacent to G X C base-pairs.

Nuclear magnetic resonance spectra (proton and phosphorus-31) and ultraviolet absorption spectra of the DNA decamer d(br5CGbr5CGATbr5CGbr5CG), in which the central two adenine-thymine base pairs are out of order with the rest of the purine-pyrimidine alternation sequence, indicate that under appropriate solvent conditions (high salt and methanol) the molecule undergoes a structural transition from a right-handed B-DNA conformation to a left-handed Z-DNA conformation. Measurements of the two-dimensional nuclear Overhauser effect on the decamer indicate that all of the guanines as well as the two equivalent thymines adopt the syn conformation.

A variety of one-dimensional proton NMR methods have been used to investigate the properties of two synthetic DNA decamers, d(ATATCGATAT) and d(ATATGCATAT). These results, in conjunction with the results of two-dimensional NMR experiments, permit complete assignment of the base proton resonances. Low-field resonances were assigned by sequential "melting" of the A . T base pairs and by comparison of the spectra of the two decamers. Below 20 degree C spin-lattice relaxation is dominated by through-space dipolar interactions. A substantial isotope effect on the G imino proton relaxation is observed in 75% D2O, confirming the importance of the exchangeable amino protons in the relaxation process. A somewhat smaller isotope effect is observed on the T imino proton relaxation. At elevated temperatures spin-lattice relaxation of the imino protons is due to proton exchange with solvent. Apparent activation energies for exchange vary from 36 kcal/base pair for base pairs (3,8) to 64 kcal/mol for the most interior base pairs (5,6), indicating that disruption of part, or all, of the double helix contributes significantly to the exchange of the imino protons in these decamers. By contrast, single base pair opening events are the major low-temperature pathways for exchange from A X T and G X C base pairs in the more stable higher molecular weight DNA examined in other studies. The temperature dependence of the chemical shifts and line widths of certain aromatic resonances indicates that the interconversion between the helix and coil states is not in fast exchange below the melting temperature, Tm. Within experimental error, no differential melting of base pairs was found in either molecule, and both exhibited melting points Tm = 50-52 degrees C. Spin-spin and spin-lattice relaxation rates of the nonexchangeable protons (TH6, AH8, and AH2) are consistent with values calculated by using an isotropic rotor model with a rotational correlation time of 6 ns and interproton distances appropriate for B-family DNA. The faster decay of AH8 compared with GH8 is attributed to an interaction between the thymine methyl protons and the AH8 protons in adjacent adenines (5'ApT3'). The base protons (AH8, GH8, and TH6) appear to be located close (1.9-2.3 A) to sugar H2',2" protons.(ABSTRACT TRUNCATED AT 400 WORDS)

Interest in triple and quadruple DNA helices built from homopurine and homopyrimidine strands has recently intensified principally because such structures may occur in vivo but also because of the potential use of triplexes both in forming highly sequence-specific complexes for use in chromosome mapping and in repressing transcription. From fibre diffraction data, models for triplex structures with poly(U).poly(A).poly(U) and poly(dT).poly(dA).poly(dT) have been proposed, in which the purine and one pyrimidine strand are Watson-Crick paired in an A' helix, and the other pyrimidine strand is Hoogsteen base-paired parallel to the purine strand along the major groove. A similar base-pairing scheme involving G and C would require protonation of C for Hoogsteen base-pair formation, and models for such triplexes have been proposed by analogy to the single-sequence fibre diffraction data. To date, however, there have been no single crystal or NMR structural data on DNA triplexes, and no direct observation of the protonated C in such a context. We present here the first NMR evidence for triplex formation in DNA from the homopurine d(G-A) and homopyrimidine d(T-C) oligonucleotides, and report direct observation of imino protons from protonated cytosines in the triplex.

Presents the designing and synthesizing of a 28-base DNA oligomer with a sequence that could potentially fold to form a triplex containing both T.A.T. and C+.G.C triplets. Methods; Results; Discussion.