A 300 MHz proton nuclear magnetic resonance investigation of deoxyribonucleic acid restriction fragments: dynamic properties

Early, Thomas A.; Kearns, David R.; Hillen, Wolfgang; Wells, Robert D.

doi:10.1021/bi00516a015

Cited by 80 publications

(35 citation statements)

References 27 publications

(29 reference statements)

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“…Inversion recovery NMR measurements on the imino protons of thymidine residues in DNA restriction fragments gave an activation energy of 15.7 kcal, which has been assigned by Early et al to single-base-pair opening without disruption of adjacent base pairs (13,14). The activation parameters measured directly from the saturation recovery for the AATT 12-mer and the TATA 12-mer are in this range but are =50% larger after subtraction of the magnetic contribution to obtain the activation energy for the exchange contribution.…”

Section: Bmentioning

confidence: 78%

“…The imino protons can recover their magnetization after saturation by spin-lattice relaxation (magnetic contribution) and by exchange with unperturbed solvent water (chemical contribution). Previous studies on transfer RNA (11,23,24) and DNA fragments (13)(14)(15)(16)(17) have demonstrated that the magnetic contribution predominates below room temperature and has a small activation energy, whereas the chemical contribution predominates above room temperature and has a large activation energy. We have determined the temperature dependence of the saturation recovery lifetimes of the AATT 12-mer duplex and the TATA 12-mer duplex between 0C and 60'C, and these are tabulated in Table 1.…”

Section: Bmentioning

confidence: 98%

“…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 transfer RNA (11,12), and recent applications to DNA (13)(14)(15)(16) and its helix interruption and base pair-mismatch analogs (16) and to antibiotic complexes (17) establish the versatility of this technique to probe the kinetics of helix-opening processes.…”

mentioning

confidence: 99%

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

Patel¹,

Ikuta²,

Kozlowski³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Bmentioning

confidence: 78%

Section: Bmentioning

confidence: 98%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Patel¹,

Ikuta²,

Kozlowski³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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“…Similar kinds of observations have been applied to short DNA fragments obtained from both restriction endonuclease digestion (22,23) and chemical synthesis (24-28).…”

Section: Introductionmentioning

confidence: 77%

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

Cheung¹,

Arndt²,

Lu³

1984

Proc. Natl. Acad. Sci. U.S.A.

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The kinetics for imino hydrogen exchange, at individual base pairs in the DNA sequence corresponding to the lactose operon operator ofEscherichia coli, has been examined by NMR saturation recovery measurements as a function of temperature. Three 17-base-pair subsections of the kac operator DNA were chemically synthesized for these studies. The results support our previous observations in the 36-base-pair complete lac operator DNA fragment that has been used in our previous NMR studies. The results indicate faster opening kinetics at a GTG/CAC that is also the site of operator mutations leading to the highest level of constitutive f8-galactosidase synthesis. The GTG/CAC sequence occurs frequently and often symmetrically in prokaryotic and eukaryotic DNA sites where one anticipates specific protein interaction for gene regulation or recombination.Diffraction studies (1-5) of double helical DNA have yielded a diversity of geometries that vary from uniform B-form DNA (6). In the double helical dodecamer, solved to atomic resolution, there are variations from B-form seen in the structural parameters along the double helical structure (7-9). Since the regular structure of DNA is sequence dependent (10, 11) and varies under physiological conditions (12, 13), these variations can be a part of what proteins recognize. The potential biological significance of this type of heterogeneity is exemplified by the recent interest in Z-DNA (3, 14-16). We report here the possibility of a local structural variation in the lactose operon operator DNA of Escherichia coli detected by a series of kinetic experiments.In our NMR studies of the interaction between lac repressor and lac operator (17), we used a 36-base-pair (bp) DNA fragment containing the lac operator sequence derived from a chemically synthesized sequence cloned in tandem on a plasmid (18). The imino proton region (19) of the NMR spectrum of the 36-bp lac operator fragment shows a nonsequential loss of resonance intensity occurs as the temperature is elevated (20); i.e., melting does not only start from the ends of the double helix. To simplify the examination of this DNA sequence, three 17-bp subsections were synthesized (Fig. 1).The imino proton resonances of the subsections were assigned by nuclear Overhauser enhancement (NOE) and their exchange kinetics were examined by saturation recovery methods developed to probe the structure and dynamics of tR.NA in solution (20,21). Similar kinds of observations have been applied to short DNA fragments obtained from both restriction endonuclease digestion (22,23) and chemical synthesis (24-28).We report here the saturation recovery rates for the resolved imino protons in the lac operator DNA sequence as a function of temperature. There is an interesting dynamic heterogeneity with a maximal opening rate centered about a GTG/CAC sequence that appears frequently and often symmetrically in a number of other prokaryotic and eukaryotic systems where a specific interaction occurs between protein and DNA. Our suggestion is that...

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“…To estimate the maximum contribution from magnetic relaxation, we assume that the measured lifetimes at low temperature are completly determined by magnetic spin-lattice relaxation and that these lifetimes are independent of temperature. The magnetic spin-lattice lifetimes for a similar oligonucleotide were found to increase with increasing temperature (Early et al, 1981 ), therefore our assumptions will lead to an upper limit for a correction to the chemical exchange rates. Figures 3, 4 and 5 show, that good estimates for the magnetic spin-lattice relaxation times can be determined for AT base pairs 5 and 6 in all the 12mer-antibiotic complexes; however it is more difficult to determine the lifetimes for GCbase pairs 3 and 4.…”

Section: Spin-lattice Relaxation Versus Themical Exchangementioning

confidence: 91%

Kinetics for exchange of the imino protons of the d(C-G-C-G-A-A-T-T-C-G-C-G) double helix in complexes with the antibiotics netropsin and/or actinomycin

Pardi¹,

Morden²,

Patel³

et al. 1983

Biochemistry

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A 300 MHz proton nuclear magnetic resonance investigation of deoxyribonucleic acid restriction fragments: dynamic properties

Cited by 80 publications

References 27 publications

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

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

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

Kinetics for exchange of the imino protons of the d(C-G-C-G-A-A-T-T-C-G-C-G) double helix in complexes with the antibiotics netropsin and/or actinomycin

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