Sequence-specific recognition of deoxyribonucleic acid. Chemical synthesis and nuclear magnetic resonance assignment of the imino protons of lambda OR3 operator deoxyribonucleic acid

Using solid-phase phosphite triester methods, we have synthesized both strands of the phage lambda OR3 DNA sequence, reannealed them, and studied the native operator duplex by high-resolution NMR at 500 MHz. At 7 degrees C the imino protons of the two terminal base pairs at each end have disappeared from the spectrum by exchange broadening. The 13 detectable imino resonances have been assigned to their respective base pairs in the duplex by using sequential nearest-neighbor NOE connectivity methods described previously. In cases where two imino protons overlap in the spectrum, spin diffusion was used to drive the cross-saturation further afield in order to produce second-order next-nearest-neighbor effects. The results show that the imino connectivity method can be used to unambiguously assign the imino proton spectrum of operator DNAs containing one to two full turns of the helix.     

1H NMR study of the exchangeable protons of the duplex d(GCGTTGCG).d(CGCAACGC) containing a thymine photodimer.

A comparison is presented of the imino proton NMR spectra of the double stranded octamer d(GCGTTGCG).d(CGCAACGC) and the same octamer in which the two central thymine residues occur as a cis-syn thymine dimer. Except for the terminal base pairs all imino protons were detected and assigned in the NMR spectrum. The spectra show that in the thymine dimer duplex, contrary to common belief, all base pairs occur in a hydrogen bonded form, although the hydrogen bonds of the two central AT base pairs are substantially weakened. The melting temperature decreases about 13 degrees C on thymine dimer formation.     

Behavior of the imino protons of the lambda OR3 17mer studied by high resolution NMR

Imino proton resonances of lambda OR3 17mer were observed with time-shared Redfield pulse method by using a JEOL 500 MHz NMR instrument. They show gradual broadening and disappearance with the elevation of temperature indicating the stepwise melting of the duplex. By the selective irradiation at each peak position nuclear Overhauser effects were observed between the imino and adenine C2H protons and between imino protons themselves. Combining these data, fifteen imino proton resonances could be assigned to each base pair except two terminal AT base pairs. Based on the assignment it can be said that AT rich regions near the terminal melt first, followed by the melting of the inside GC rich core. The two AT base pairs in the middle of the GC core are resistant to heat. Spin lattice relaxation times were also observed and the results are consistent with the melting profile.     

Properties of pseudouridine N1 imino protons located in the major groove of an A-form RNA duplex.

The exchangeable N1 imino protons of two pseudouridine (psi) bases located at adjacent internal positions within an undecamer RNA duplex (5'AUAC psi psi ACCUG/3'UAUGAAUGGUC) can report on the environment of the major groove of an A-form double-stranded nucleic acid. The psi N1 imino protons of these residues (which are not involved in interstrand Watson-Crick hydrogen bonding) are protected from chemical exchange with the solvent water and thus are observable in the proton NMR spectrum in H2O (1). These protons will exchange readily at increased pH values or upon thermal denaturation of the duplex. The longitudinal (T1) relaxation times of the psi N1 imino protons in 100 mM NaCl or in 10 mM MgCl2 and 100 mM NaCl are approximately two-fold faster than those of the psi N3 imino protons which are involved in Watson-Crick base pairing. With the addition of spermidine, the psi N1 imino protons become readily exchangeable at a temperature some 20 degrees C below the melting temperature of the duplex.     

High resolution NMR study of CAP binding site 22mer in H2O solution

High resolution proton NMR were measured for the deoxyoligonucleotide 22mer duplex corresponding to the CAP (catabolite gene activator protein) binding site of lac promotor. The spectra in the lower field region than the water resonance were taken with the time-shared Redfield pulse method by using a JEOL 500 MHz NMR spectrometer. In the imino proton region 18 peaks were separately observed, but the area intensity at 10 degrees C corresponds to 20 protons. By selective irradiation at each peak position NOEs (nuclear Overhauser effects) were observed between the imino and adenine C2H protons and between imino proton themselves. By tracing sequential NOE train carefully, 17 imino proton signals could be unambiguously assigned to each base pair except five AT base pairs at terminals. With the elevation of temperature the peaks showed gradual broadening and disappeared, which indicates the stepwise base pair opening of the duplex. Referring to the above peak assignments it can be concluded that GC20 and AT4 pairs close to terminals relax first and the base pair opening proceeds toward central GC13 and 14.     

Proton NMR investigation of the nucleosome core particle: evidence for regions of altered hydrogen bonding

Novel 1H nuclear magnetic resonance (NMR) resonances, arising from exchangeable protons and centered at approximately 11.2 and 10.1 parts per million (ppm), have been observed in the low-field spectrum (10-15 ppm) of the chicken erythrocyte core particle [145 +/- 2 base pairs (bp)]. These peaks are located upfield from the normal adenine-thymine (A-T) and guanine-cytosine (G-C) imino peaks characteristic of B-form deoxyribonucleic acid (DNA) and are not observed in free DNA under identical conditions. The appearance of the new peaks is ionic strength dependent and temperature-reversible below 75 degrees C. At 25 degrees C, the upfield peak area represents 5% of the DNA base pairs (7 bp), while between 45 and 55 degrees C, the area increases to 18%, affecting approximately 25 bp. Area increases in the upfield resonances result in a complementary decrease in the A-T and G-C imino peaks found between 12 and 14 ppm. We believe these novel proton signals represent a histone-induced DNA conformational change which involves localized alteration of base pairing in the core particle.     

Dynamic behavior of the imino protons of the gamma OR3 17mer in H2O solution studied by high-resolution NMR

The imino proton resonances of gamma OR3 17mer in water were observed at 500 MHz with the time-shared Redfield pulse train. All of the 17 imino proton resonances could be assigned specifically to individual base pairs by utilizing the trace of NOE connectivities between the imino and adenine C2H protons and between imino protons themselves. AT1 and 17 showed abnormally high chemical shifts in comparison with the other AT pairs. On raising the temperature, broadening of the signal occurred in a sequential manner from the terminals except for AT10 and AT11, which were broadened at a lower temperature than GC12. The relaxation rates of the imino protons were measured by the inversion recovery method. The rates at higher temperatures represent the exchange rates of the imino protons. From the temperature dependences, activation energies of about 15 kcal/mol for the AT imino protons and 23-26 kcal/mol for the GC imino protons were obtained.     

Proton exchange and base pair opening in a DNA triple helix

Nuclear magnetic resonance spectroscopy has been used to characterize opening reactions and stabilities of individual base pairs in two related DNA structures. The first is the triplex structure formed by the DNA 31-mer 5'-AGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3'. The structure belongs to the YRY (or parallel) family of triple helices. The second structure is the hairpin double helix formed by the DNA 20-mer 5'-AGAGAGAACCCCTTCTCTCT-3' and corresponds to the duplex part of the YRY triplex. The rates of exchange of imino protons with solvent in the two structures have been measured by magnetization transfer from water and by real-time exchange at 10 degrees C in 100 mM NaCl and 5 mM MgCl2 at pH 5.5 and in the presence of two exchange catalysts. The results indicate that the exchange of imino protons in protonated cytosines is most likely limited by the opening of Hoogsteen C+G base pairs. The base pair opening parameters estimated from imino proton exchange rates suggest that the stability of individual Hoogsteen base pairs in the DNA triplex is comparable to that of Watson-Crick base pairs in double-helical DNA. In the triplex structure, the exchange rates of imino protons in Watson-Crick base pairs are up to 5000-fold lower than those in double-helical DNA. This result suggests that formation of the triplex structure enhances the stability of Watson-Crick base pairs by up to 5 kcal/mol. This stabilization depends on the specific location of each triad in the triplex structure.     

NMR spectroscopy of RNA duplexes containing pseudouridine in supercooled water

We have performed NMR experiments in supercooled water in order to decrease the temperature-dependent exchange of protons in RNA duplexes. NMR spectra of aqueous samples of RNA in bundles of narrow capillaries that were acquired at temperatures as low as -18 degrees C reveal resonances of exchangeable protons not seen at higher temperatures. In particular, we detected the imino protons of terminal base pairs and the imino proton of a non-base-paired pseudouridine in a duplex representing the eukaryotic pre-mRNA branch site helix. Analysis of the temperature dependence of chemical shift changes (thermal coefficients) for imino protons corroborated hydrogen bonding patterns observed in the NMR-derived structural model of the branch site helix. The ability to observe non-base-paired imino protons of RNA is of significant value in structure determination of RNA motifs containing loop and bulge regions.     

Sequential NMR assignments of labile protons in DNA using two-dimensional nuclear-Overhauser-enhancement spectroscopy with three jump-and-return pulse sequences

Two-dimensional nuclear Overhauser enhancement (NOESY) spectra of labile protons were recorded in H2O solutions of a protein and of a DNA duplex, using a modification of the standard NOESY experiment with all three 90 degree pulses replaced by jump-and-return sequences. For the protein as well as the DNA fragment the strategically important spectral regions could be recorded with good sensitivity and free of artifacts. Using this procedure, sequence-specific assignments were obtained for the imino protons, C2H of adenine, and C4NH2 of cytosine in a 23-base-pair DNA duplex which includes the 17-base-pair OR3 repressor binding site of bacteriophage lambda. Based on comparison with previously published results on the isolated OR3 binding site, these data were used for a study of chain termination effects on the chemical shifts of imino proton resonances of DNA duplexes.     

Conformation and dynamics of the Pribnow box region of the self-complementary d(C-G-A-T-T-A-T-A-A-T-C-G) duplex in solution

Nuclear magnetic resonance (NMR) has been used to monitor the conformation and dynamics of the d(C1-G2-A3-T4-T5-A6-T6-A5-A4-T3-C2-G1) self-complementary dodecanucleotide duplex (henceforth called Pribnow 12-mer), which contains a TATAAT Pribnow box and a central core of eight dA X dT base pairs. The exchangeable imino and nonexchangeable base protons have been assigned from one-dimensional intra and inter base pair nuclear Overhauser effect (NOE) measurements. Premelting conformational changes are observed at all the dA X dT base pairs in the central octanucleotide core in the Pribnow 12-mer duplex with the duplex to strand transition occurring at 55 degrees C in 0.1 M phosphate solution. The magnitude of the NOE measurements between minor groove H-2 protons of adjacent adenosines demonstrates that the base pairs are propeller twisted with the same handedness as observed in the crystalline state. The thymidine imino proton hydrogen exchange at the dA X dT base pairs has been measured from saturation recovery measurements as a function of temperature. The exchange rates and activation barriers show small variations among the four different dA X dT base pairs in the Pribnow 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR study of a synthetic DNA hairpin

The secondary structure of the synthetic oligodeoxyribonucleotide d(CGCGCGTTTTCGCGCG) (I) has been demonstrated to be a unimolecular hairpin structure (hairpin I) over a wide range of oligonucleotide concentrations (2 X 10(-5) to 1.6 X 10(-3) M) and temperature (0-87 degrees C). The assignments of the resonances to specific protons were carried out by use of two-dimensional nuclear Overhauser effect and COSY spectra and by comparison with the spectra of the duplex formed by d(CG)3. Comparison of hairpin I and the hairpin of d(ATCCTATTTTTAGGAT) (II) reveals that the exchange of imino protons in stem base pairs with solvent is much slower in I than in II. However, the exchange of thymine imino protons in the loop region is much faster in I than in II even though both hairpins contain four unpaired thymine residues. The secondary structure of hairpin I contains only six G X C base pairs, yet it is more stable than the d(CG)8 duplex containing 16 G X C base pairs at all concentrations of duplex lower than 10(-3) M. These observations suggest that intramolecular hairpin formation may effectively compete with bimolecular duplex formations when the appropriate intramolecular base pairs can form.     

alpha-DNA-V. Parallel annealing, handedness and conformation of the duplex of the unnatural alpha-hexadeoxyribonucleotide alpha-[d(CpApTpGpCpG)] with its beta-complement beta-[d(GpTpApCpGpC)] deduced from high field 1H-NMR.

The beta-complementary hexamer, beta-d[GTACGC], to the alpha-sequence, alpha-d[CATGCG], was synthesized by the phosphotriester method. The non-exchangeable proton assignments were obtained using 1D- and 2D-NMR techniques, including NOE, COSY and NOESY. The beta-strand exists as a random coil at 21 degrees C; however, at 4 degrees C, it forms an antiparallel self-recognition duplex annealing at positions 1-4. The beta-strand was annealed to the alpha-strand, and confirmation of complete annealing was obtained by detection and assignment of the six base pair imino protons in H2O/D2O solution at 21 degrees C. 1D-NOE experiments of the alpha, beta duplex d[alpha-(CATGCG) X beta-(GTACGC)] reveal that (i) it exists in aqueous solution in a conformation that belongs to the B family, (ii) it is 70 +/- 10% right-handed, (iii) the sugar-base orientations of the beta-strand are anti, and the deoxyribose units exist predominantly in the 2'-endo-3'-exo conformation. NOE measurements of the imino proton signals in the alpha, beta duplex reveal that the duplex exhibits parallel polarity.     

Imino proton assignments in the proton nuclear magnetic resonance spectrum of the lambda phage OR3 deoxyribonucleic acid fragment

The 17 base pair duplex d(TATCACCGCAAGGGATAp) . d(TATCCCTTGCGGTGATAp) corresponding to the OR3 operator site of lambda phage has been synthesized and studied by 1H nuclear magnetic resonance spectroscopy at 470 MHz. The 13 imino proton resonances observed at 20 degrees C have been assigned to specific base pairs at positions 3-15 on the basis of nuclear Overhauser effect measurements and studies of the temperature dependence of peak intensities. Resonances from the A-T base pairs at positions 1, 2, 16, and 17 are assumed to be absent from the spectrum because of terminal fraying. Resonance from many of the base pairs suggested by Ohlendorf et al. [Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., & Matthews, B. W. (1982) Nature (London) 298, 718-723] to be involved in specific binding of the lambda phage cro repressor are well resolved.     

Phosphate catalyzed exchange rates of NH-N protons of DNA

We have studied the catalysis of the exchange of the hydrogen-bonded NH-N protons of the short DNA helix (d-CCAAGCTTGG)2 by phosphate addition. The NH exchange rates were monitored by the line widths of the corresponding NH resonances in the 1H nmr spectra. The exchange catalyst phosphate is most effective on the exchange rate of the terminal CG1 base pairs. However, all internal base pairs are also moderately affected by phosphate which suggests an exchange mechanism governed by a fast equilibrium between opened and closed states of the duplex. Within the limits of error the same effectiveness of phosphate on the exchange rate of all internal NH-N protons has been observed. With the exception of the terminal base pairs, no sequence and/or position specificity of the exchange rates of the NH-N protons of the base pairs has been found.     

Sequence-specific recognition of DNA: NMR studies of the imino protons of a synthetic RNA polymerase promoter

We have synthesized both strands of a DNA duplex containing the consensus Pribnow promoter sequence TATAATG , flanked by GC base pairs to stabilize the ends of the helix. The stability of this duplex has been studied by using 1H nuclear magnetic resonance. The imino protons have been assigned by using the sequential nuclear Overhauser effect approach. Exchange rates have been monitored by using selective inversion recovery measurements. The helix is relatively unstable in the center of the AT-rich region even when surrounded by GC base pairs, and there is considerable asymmetry in the melting of the helix.     

Helix-coil transition of the self-complementary dG-dG-dA-dA-dT-dT-dC-dC duplex.

The helix-coil transition of the octanucleotide self-complementary duplex dG-dG-dA-dA-dT-dT-dC-dC has been monitored at the Watson-Crick protons, the base and sugar nonexchangeable protons and the backbone phosphates by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The melting transition of the octanucleotide monitored by ultraviolet absorbance spectroscopy is characterized by the thermodynamic parameters delta H degree = -216.7 kJ/mol and delta S degree (25 degrees C) = -0.632 KJ mol-1 K-1 in 0.1 M NaCl, 10 mM phosphate solution. Correlation of the transition midpoint values monitored by the ultraviolet absorbance studies at strand concentrations below 0.2 mM and by NMR studies at 5.3 mM suggest that both methods are monitoring the octanucleotide duplex-to-strand transition. The NMR spectra of the Watson-Crick ring NH protons of the octanucleotide duplex have been followed as a function of temperature. The resonance from the terminal dG.dC base pairs broadens out at room temperature while the resonances from the other base pairs broaden simultaneously with the onset of the melting transition. The nonexchangeable base and sugar H-1' protons are resolved in the duplex and strand states and shift as average peaks through the melting transition. The experimental shifts on duplex formation have been compared with calculated values based on ring-current and atomic diamagnetic anisotropy contributions for a B-DNA base-pair-overlap geometry in solution. Several nonexchangeable proton resonances broaden in the fast-exchange region during the duplex-to-strand transition and the excess widths yield a duplex dissociation rate constant for the octanucleotide of 1.9 x 10(3) s-1 at 32 degrees C (fraction of duplex = 0.86) in 0.1 M NaCl, 10 mM phosphate buffer. The 31P resonances of the seven internucleotide phosphates are distributed over 0.6 ppm in the duplex state, shift downfield during the duplex-to-strand transition and undergo additional downfield shifts during the stacked-to-unstacked strand transition with increasing temperature.     

NMR study of the interaction between the lac repressor and the lac operator

Binding of the lac repressor headpiece, the N-terminal region of the lac repressor, to the lac operator of Escherichia coli was studied by 1H-NMR spectroscopy. Two DNA fragments, of 51 base pairs and 62 base pairs, containing the lac operator region, were investigated. The signals of their hydrogen-bonded imino protons were well resolved in the 500-MHz NMR spectra. The spectra of the free lac operator DNA are similar to those obtained from ring-current-shift calculations for a B-DNA structure. Complex formation with the headpiece led to small but nevertheless characteristic changes in the spectra. The fact that very few imino resonances shifted upon addition of headpiece, as well as the variety in direction and size of these chemical shifts, indicate the formation of a specific complex between the lac repressor and the lac operator. The observed changes in the resonance positions exclude the intercalation of tyrosine residues of the headpiece between adjacent base pairs of the lac operator as well as the formation of a cruciform structure. They rather reflect a small conformational transition in the DNA itself, caused for example by an alteration in the tilt of a few base pairs or a shift of the keto-enol tautomeric equilibrium of the bases towards the enolic form.     

Exploring a DNA Sequence for the Three-Dimensional Structure Determination of a Silver(I)-Mediated C-C Base Pair in a DNA Duplex By 1H NMR Spectroscopy

Recently, we discovered novel silver(I)-mediated cytosine–cytosine base pair (C–Ag^I–C) in DNA duplexes. To understand the properties of these base pairs, we searched for a DNA sequence that can be used in NMR structure determination. After extensive sequence optimizations, a non-symmetric 15-base-paired DNA duplex with a single C–Ag^I–C base pair flanked by 14 A–T base pairs was selected. In spite of its challenging length for NMR measurements (30 independent residues) with small sequence variation, we could assign most non-exchangeable protons (254 out of 270) and imino protons for structure determination.     

Base pair mismatches and carcinogen-modified bases in DNA: an NMR study of G.T and G.O4meT pairing in dodecanucleotide duplexes

High-resolution two-dimensional NMR studies have been completed on the self-complementary d(C-G-C-G-A-G-C-T-T-G-C-G) duplex (designated G.T 12-mer) and the self-complementary d(C-G-C-G-A-G-C-T-O4meT-G-C-G) duplex (designated G.O4meT 12-mer) containing G.T and G.O4meT pairs at identical positions four base pairs in from either end of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) spectra for the G.T 12-mer and G.O4meT 12-mer duplexes in H2O and D2O solution. The guanosine and thymidine imino protons in the G.T mismatch resonate at 10.57 and 11.98 ppm, respectively, and exhibit a strong NOE between themselves and to imino protons of flanking base pairs in the G.T 12-mer duplex. These results are consistent with wobble pairing at the G.T mismatch site involving two imino proton-carbonyl hydrogen bonds as reported previously [Hare, D. R., Shapiro, L., & Patel, D. J. (1986) Biochemistry 25, 7445-7456]. In contrast, the guanosine imino proton in the G.O4meT pair resonates at 8.67 ppm. The large upfield chemical shift of this proton relative to that of the imino proton resonance of G in the G.T mismatch or in G.C base pairs indicates that hydrogen bonding to O4meT is either very weak or absent. This guanosine imino proton has an NOE to the OCH3 group of O4meT across the pair and NOEs to the imino protons of flanking base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)