Molecular mechanical studies of DNA flexibility: Coupled backbone torsion angles and base-pair openings

Keepers, Joe W.; Kollman, Peter A.; Weiner, Paul K.; James, Thomas Leroy

doi:10.1073/pnas.79.18.5537

Cited by 76 publications

(50 citation statements)

References 19 publications

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“…Recent molecular mechanics calculations suggest that base sliding disrupts base pairing and permits imino proton exchange with solvent water without disruption of adjacent base pairs (28). The faster opening kinetics in the TATA box region compared to the AATT region in the two dodecanucleotides is in agreement with the relative stabilities of nearest-neighbor doublets in DNA deduced from fitting calculated to observed melting profiles (29).…”

Section: Bsupporting

confidence: 71%

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: Bsupporting

confidence: 71%

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 existence of a "base-opened state" about 5 kcal/mol higher in enthalpy than the Watson-Crick form for AFT pairs has been suggested on the basis of proton exchange kinetics by Mandel et aL (16). Molecular mechanics calculations in this laboratory have suggested that such a structure can be readily formed with little energy expenditure by non-Watson-Crick base pairing of AFT, but not of G-C pairs (17). It is tempting to speculate that part of the spectral changes in the AFT region may be attributed to such "partially opened" structures, which might be more readily observed in the spectrum of supercoiled (versus linear) DNA due to the larger population of higher energy structures anticipated from the reactivity of supercoiled DNA to singlestranded-specific reagents and endonucleases (1).…”

Section: Resultsmentioning

confidence: 88%

Structural and dynamic differences between supercoiled and linear DNA from proton NMR.

Bendel¹,

James²

1983

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

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Proton NMR experiments on an intact plasmid supercoiled DNA molecule are reported. Spectra of the hydrogen-bonded exchangeable imino protons and nonexchangeable aromatic protons of pIns36 (7,200 base pairs; Mr 5 X 106) were examined at 240 and 360 MHz. In contrast to linear DNA of comparable size in which the signals from these protons are broadened almost beyond detection, the supercoiled DNA exhibits a remarkably well-resolved spectrum in the range 12-15 ppm, with linewidths characteristic of linear DNA molecules with molecular weight nearly two orders of magnitude smaller. These results imply an extraordinary degree of mobility in accord with our earlier discovery of unusually long 31p spin-spin relaxation times in the supercoiled molecule. Alterations in the chemical shift of the imino proton resonance positions in supercoiled DNA compared to linear DNA imply that the structure is modified in the A T base pairs with at least two distinct structures apparent from the spectrum. However, little or no alteration is evident in the resonances due to the G'C base pairs. METHODS AND MATERIALS Plasmid pIns36 was derived from plasmid pBR322 by subcloning a 2.8-kb human DNA fragment into the HindIII site of pBR322. pIns36 was grown and amplified in Escherichia coli HB 101. The plasmid DNA was extracted by the alkali/NaDodSO4 method (3), and the closed circular DNA was isolated from an ethidium bromide/CsCl buoyant density gradient (4).(Typical yields were 2-4 mg of supercoiled DNA per liter of culture.) NMR samples were obtained by dialysis first against a pH 7 buffer containing 1 M NaCl, 0.05 M phosphate, and 1 mM EDTA; this was followed by a dialysis against 0.02 M NaCi, 0.01 M phosphate, and 0.5 mM EDTA. Samples were then reconstituted after lyophilization in 2H20 or 90% H20/10% 2H20 to final concentrations of 0.04 M NaCi, 0.02 M phosphate, and 1 mM EDTA; DNA concentration was determined by UV spectroscopy by usingA260 = 6,600. The DNA was shown to be >90% in the supercoiled form with a small amount of nicked, circular DNA by using 1.5% agarose gel electrophoresis before and after NMR experiments as described (2).Calf thymus DNA (Sigma) was sonicated at 00C in solutions containing about 1 mg of DNA per ml and 0.5 M NaCl. Size distributions were determined by 1.5% agarose gel electrophoresis (2). NMR samples were obtained and concentrations determined as above. RESULTS AND DISCUSSIONWe recorded the 1H NMR spectra of pIns36 as well as two samples of calf thymus DNA that had been sonicated to produce size distributions of 200-700 and 600-10,000 bp, respectively. The supercoiled pIns36 sample was sufficiently precious that we chose not to degrade it to its nicked, circular, and linear forms for the present 1H NMR study as we did for the 31P NMR study (2). Instead, we selected the two calf thymus DNA samples as representative linear DNA for comparison with supercoiled pIns36. The proton NMR of linear DNA samples reported thus far has revealed little diversity, with our calf thymus DNA samples displaying the same...

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“…The enzyme was originally proposed to facilitate a minor groove exit of the target cytosine into the active site, ∼180°from its base stacked position in B-form DNA 3 (Figure 1(a)). Some molecular dynamics (MD) simulations have agreed with this exit path [19][20][21][22] while others have proposed that a major groove path is energetically favored. 12,13 Recent MD simulations predict that a minor groove path is energetically unfavorable due to the solvent exposure of the cytosine required for this route.…”

Section: Introductionmentioning

confidence: 92%

The Role of Arg165 Towards Base Flipping, Base Stabilization and Catalysis in M.HhaI

Shieh

Youngblood

Reich

2006

Journal of Molecular Biology

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Molecular mechanical studies of DNA flexibility: Coupled backbone torsion angles and base-pair openings

Cited by 76 publications

References 19 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.

Structural and dynamic differences between supercoiled and linear DNA from proton NMR.

The Role of Arg165 Towards Base Flipping, Base Stabilization and Catalysis in M.HhaI

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