Sequence dependence of hydrogen exchange kinetics in DNA duplexes at the individual base pair level in solution.

Abstract

The kinetics for hydrogen exchange at individual base pairs in self-complementary deoxydodecanucleotide duplexes have been estimated from NMR saturation recovery measurements on the resolved imino protons as a function of temperature.The imino protons of dA-dT base pairs in the center of the fully alternating d(C-G-C-G-T-A-T-A-C-G-C-G) duplex exchange a factor of 2-to 3-fold faster than the corresponding protons at the same positions in the partially alternating d(C-G-C-G-A-A-T-T-C-G-C-G) duplex. These exchange parameters are a direct measure of the rate constants for transient opening of individual dA-dT base pairs in the dodecanucleotide duplexes and demonstrate faster opening kinetics for the "TATA" box region compared to the related "AATT" segment.Recent advances in oligonucleotide synthesis have led to the preparation of milligram quantities of DNA fragments of precise sequence containing at least one turn of helix (1). One such example is the self-Scheme I which has been extensively analyzed by single-crystal x-ray analysis in the solid state (2) and high-resolution NMR spectroscopy in aqueous solution (3). Both techniques have established that the central A-T-rich tetranucleotide segment shows conformational polymorphism in the duplex state as a function of temperature and solvent (2, 3).These observations initiated the present experiments, which probe the effect of sequence on the conformation and dynamics of DNA duplexes in solution. The completely alternating self-was synthesized by solid-state methods, and the effect of sequence variations in the center of the duplex was probed by comparative studies of the AATT 12-mer and TATA 12-mer duplexes.Redfield et al. have introduced nuclear Overhauser effect (NOE) and saturation recovery methods to probe the conformation and dynamics of transfer RNA in solution (4). They have demonstrated intra-and inter-base-pair NOEs involving exchangeable and nonexchangeable protons and have correlated the magnitude of the NOE effect with the inverse sixth power dependence of the interproton distance between the saturated and observed resonances. This distance-measuring methodology has wide applications, including its extension to elucidate conformational features of DNA fragments (3), synthetic DNAs (5), and antibiotic-DNA complexes (6) in solution.Hydrogen-deuterium exchange stop-flow measurements have demonstrated that the exchange kinetics of the Watson-Crick imino protons are sensitive markers of the dynamics of base pair opening in the interior of nucleic acid duplexes (7). NMR also can monitor hydrogen exchange, and the early research focused on line width changes of the exchangeable imino protons in the continuous wave mode to evaluate the kinetics of base pair-opening reactions (8, 9). The introduction of Fourier transform methods with H20 solvent suppression (10) readily permitted the incorporation of variable time delays between the saturation and observation pulses to monitor the kinetics of magnetization recovery. The initial measurements were made on...

Conformation, dynamics, and structural transitions of the TATA box region of the self-complementary d[C-G)n-T-A-T-A-(C-G)n] duplexes in solution

“…The imino protons of the central dA.dT base pairs 1 and 2 exhibit recovery lifetimes between 55 and 60 ms while those of flanking dG.dC base pairs 3 and 4 exhibit lifetimes between 180 and 195 ms ( Figure 4, Table 11). The hydrogen exchange contribution to the saturation recovery data predominates above room temperature as demonstrated from our detailed temperature- and pH-dependent studies of saturation recovery kinetics of the TATA 12-mer (Patel et al, 1983b).…”

“…We have previously demonstrated a sequence dependence to the hydrogen exchange kinetics in dA-dT base pairs in dodecanucleotide duplexes (Patel et al, 1983b). Specifically, the dA-dT base pairs in the center of the fully alternating TATA 12-mer duplex exchange a factor of 2-3 faster than the corresponding protons at the same positions in the AATT 12-mer duplex.…”

“…Specifically, the dA-dT base pairs in the center of the fully alternating TATA 12-mer duplex exchange a factor of 2-3 faster than the corresponding protons at the same positions in the AATT 12-mer duplex. These exchange parameters are a direct measure of the rate constants for transient opening of individual dA.dT base pairs in the dodecanucleotide duplexes and demonstrate faster opening kinetics for the TATA box region compared to the related AATT segment (Patel et al, 1983b). It is important to stress that these data were measured between 30 and 52.5 "C, conditions under which the TATA 12-mer is a fully paired duplex in 0.1 M phosphate.…”

Telomeres of the linear chromosomes of Lyme disease spirochaetes: nucleotide sequence and possible exchange with linear plasmid telomeres

“…9; see Hinnebusch and Barbour (1991) for a short similarity between Borrelia plasmids and African Swine Fever Virus telomeres); however, it is of possible significance that nearly all have a TATAAT within 10-20 bp of the terminal loop [it is missing only in the eukaryotic algal virus PBCV-1 (Rohozinski et al, 1989;Strasser et al, 1991) and in the Borrelia 28 kbp plasmid left end (Zhang et al, 1997)]. The sequence TATA has a particularly fast 'breathing' rate (Patel et al, 1983) and TATAAT resembles part of RNA polymerase binding sites, but the significance of both of these correlations is unknown.…”

Correlation of lac operator DNA imino proton exchange kinetics with its function.

“…2. The resonance assignments, indicated on the 250C spectra, were determined by inter-base-pair NOE measurements, which has now become routine in the analysis of short double-stranded DNA fragments (25)(26)(27)(28)33). The intensities of the imino proton resonances decrease with increasing temperature as one would expect for DNA helices that are unwinding due to melting, allowing exchange of these protons with solvent.…”