1H NMR studies of a 17-mer DNA duplex

Biochimica et Biophysica Acta 1574 (2002) 93^99

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1

H NMR studies of a 17-mer DNA duplex Weidong Liu, Hai M. Vu, David R. Kearns *

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA Received 5 June 2001 ; received in revised form 11 October 2001; accepted 18 October 2001

Abstract Transcription factor 1 (TF1), encoded by the Bacillus subtilis bacteriophage SPO1, is a DNA-binding protein of the HU family. In preparation for a determination of the structure of the DNA^TF1 complex, we have studied the conformation of one core 17-mer duplex d(5P-CACTACTCTTTGTAGTG-3P)-d(5P-CACTACAAAGAGTAGTG-3P). NOESY, DQF-COSY and TOCSY spectroscopy provide resonance assignments of non-exchangeable and exchangeable protons, internucleotide and interstrand proton^proton distances, and dihedral angle constraints. Restrained molecular dynamics calculations yield a family of NMR solution structures for which the RMSD is î (all atoms). The helical twist is 34.9‡ for the central 15 bp. Bends toward the major groove are located between the second and fourth 0.7 A base pairs from each end. The G12WC23 base pair, which is bounded on each side by consecutive AWT pairs, causes a local disturbance to the DNA helix that makes the conformations of the two end segments unsymmetrical. The pyrimidine rings at T9, T10 and T11 experience more extensive rotational movement than the rest of the structure. ß 2002 Elsevier Science B.V. All rights reserved. Keywords : Nuclear magnetic resonance; Transcription factor 1; DNA

1. Introduction

2. Materials and methods

Previous structure studies in our laboratory [1^3] have focussed on TF1 (transcription factor 1), a protein of the HU family [4,5] that is encoded by the Bacillus subtilis phage SPO1 [6]. In the SPO1 genome, hydroxymethyluracil (hmU) entirely replaces T [7], and TF1 shows a preference for hmU-containing DNA, as well as a preference for speci¢c DNA sequence [8]. Comparisons of the structures of 13 bp segments of hmU-DNA and the T-containing counterpart by nuclear magnetic resonance (NMR) have been performed in our laboratory [9,10]. A comparative analysis of these normal and hmU-DNA versions of one consensus sequence shows that while potential DNA £exibility may be important in TF1 binding [11], sequencedependent charge distribution and local structural £uctuations may also play an important role in the increased a⁄nity of hmU-DNA to TF1. Here we present the structure of a T-containing 17-mer DNA duplex that can serve as the core of a DNA-binding site for TF1.

2.1. Materials

Abbreviations : TF1, transcription factor 1; NOE, nuclear Overhauser e¡ect ; NOESY, nuclear Overhauser e¡ect spectroscopy; DQF-COSY, double-quantum-¢ltered correlation spectroscopy * Corresponding author. Fax: +1-858-534-0202. E-mail address : [email protected] (D.R. Kearns).

Oligodeoxynucleotide 17-mers 5P-CACTACTCTTTGTAGTG-3P and 5P-CACTACAAAGAGTAGTG-3P were purchased from Genset. Purity was veri¢ed and stoichiometry of duplex formation assured by appropriate gel electrophoresis assays. The samples were dissolved in 500 Wl of NMR bu¡er (50 mM KH2 PO4 , 50 mM K2 HPO4 , 100 mM NaCl, 1 mM NaN3 , 90% H2 O, 10% D2 O, pH 7.0) at a ¢nal duplex DNA concentration of 2.2 mM. The following numbering scheme is used : nucleotides are numbered 5PC3P, with CACTACTCTTTGTAGTG designated 1^ 17, and CACTACAAAGAGTAGTG designated 18^34. Thus, base pairs are C1G34, G12C23, etc. 2.2. NMR spectroscopy Proton NMR spectra were recorded at 10‡C and 38‡C on a Bruker DRX-600 spectrometer with a 5 mm inverse, triple resonance probe equipped with XYZ gradient coils. Two-dimensional total correlation spectroscopy experiments [12] were run with a mixing time of 50 ms. Twodimensional nuclear Overhauser e¡ect spectroscopy (NOESY) spectra [13] were collected with 50, 100, 150, 200, 300 ms mixing time, and with 100 ms, 200 ms mixing time,

0167-4781 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 1 ) 0 0 3 5 0 - 5

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for samples dissolved in D2 O and 90% H2 O/10% D2 O, respectively. For the sample dissolved in H2 O, the water peak was suppressed by a 3-9-19 pulse sequence with gradient [14]. For the sample dissolved in D2 O, presaturation with a 30 Hz ¢eld was applied. A double-quantum-¢ltered correlation spectroscopy (DQF-COSY) experiment [15] was carried out using a gradient ratio of 1:2 for selection of double quantum coherence transfer. The spectral width was 13 228 Hz with carrier frequency at the water resonance. All spectra were acquired with 4096 and 512 complex points in the t2 and t1 dimensions, respectively. Relaxation delays ranged from 2.5 s to 3 s. A onedimensional experiment investigating imino proton exchange with water utilized the technique developed by Gue¤ron and Leroy [16,17]. Time domain data were processed by XWINNMR 2.1 and analyzed using Felix 97.0 (Biosym Technologies). All spectra were referenced by 3-(trimethylsilyl)propionic-2,2,3,3,-d4 acid. 2.3. Restraint determination Nuclear Overhauser e¡ect (NOE) restraints were ¢rst obtained from 50 ms and 100 ms NOESY spectra using the isolated spin pair approximation, and the inverse sixth power dependence of NOE intensity on the interproton distance at short mixing time. Distances were given a 15% error range to cover the inaccuracy of NOE intensity due to peak overlaps, noise, and peak spread from scalar coupling. Because of peak overlaps, noise, and peak spread from scalar coupling, the maximum distance upper î . For extremely weak peaks only found limit was set at 5 A at 150 ms, 200 ms, and 300 ms mixing time due to spin di¡usion, and con¢rmed by NOE build-up curve, a maxî with a lower limit of imum upper limit was set at 6^8 A î 5 A. The analysis of DQF-COSY spectra and the intranucleotide NOE pattern showed that sugar pucker experienced a south-to-north conformational interconversion at 38‡C. DQF-COSY spectra recorded at 10‡C gave little information due to the large linewidth. For this reason, dihedral angle restraints were only applied to loosely constrain (O3P-C3P-C4P-C5P) from 80‡ to 145‡. Right-handed restraints [18] were added, comprising altogether 418 unique NMR restraints, 360 overlapped NMR restraints, 75 H-bond restraints and 224 loosely restrained dihedral angles.

equilibrium between the force ¢eld and NMR restraints; (iv) temperature decrease to 273 K in 5 ps with ramping force constants; (v) 4 ps dynamics for equilibrium ; (vi) 1500 steps of energy minimization or until a root î was mean square energy gradient of 0.01 kcal/mol A achieved. The 20 ¢nal structures converged to an RMSD î (all atoms). of 0.7 A

3. Results 3.1. Exchangeable protons Fig. 1 shows the imino proton region of the 200 ms NOESY spectrum recorded at 10‡C; 15 out of 17 imino protons were connected. Peak intensities from imino proton pairs (numbered in the upper strand for clarity) of base pair steps 6/7, 7/8, 8/9 and 9/10 are distinguishably greater than from steps 11/12 and 12/13; peaks from the imino proton pairs corresponding to T-A steps 4/5 and 13/ 14 vanished at 38‡C (data not shown). These NOE intensities provide an initial estimate that the conformation resembles B-DNA more than A-DNA. The chemical shifts of the protons from the three end pairs are degenerate, indicating a roughly symmetrical conformation re£ecting this partially palindromic sequence. Fig. 2 shows the exchange process between the imino protons and water. It is worth noting that after 100 ms of exchange with the inverted water resonance, all thymine imino proton resonances except those of T9 and T10 are inverted, and only those from G3 and G15 are inverted. At longer exchange times, only those from G6, G8 and G12 remain positive. This establishes that the central segment of the structure is relatively free of tendency toward

2.4. Molecular modeling Molecular modeling was performed on a SGI computer using DISCOVER Biosym software with AMBER force ¢eld. The simulated annealing protocol included : (i) energy minimization for 200 steps or until a root mean î was achieved; square energy gradient of 0.1 kcal/mol A (ii) temperature increase from 273 K to 1000 K in 5 ps with ramping force constants; (iii) 10 ps dynamics to reach

Fig. 1. Sequential NOE connectivity in the imino^imino region for the internal 15 bp of the 17-mer duplex. The NOESY spectrum was acquired at 283 K with 200 ms mixing time.

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Fig. 2. Data from the magnetization transfer from water. The imino proton regions of nine time points during the magnetization transfer from water are shown with the appropriate imino proton assignments.

fraying [19], that GWC pairs are, of course, more stable and that this is, in fact, a DNA duplex. Imino protons give unique cross peaks with amino protons, H2 and thymine methyl groups, all of which are assigned. However, the amino protons of guanine are only found at 38‡C, when they merge into one resonance, at around 6.5 ppm, due to fast rotation around the C^N bond. 3.2. Resonance assignment of non-exchangeable protons Two-dimensional 1 H NMR NOESY spectra recorded at 38‡C in D2 O (Fig. 3) demonstrate the intra- and internucleotide H1P-to-base H8/H6 connection. Sequential assignment followed established methods ([20,21], and e.g. [22]) and is presented along one strand in Fig. 3. (The tracking along the complementary strand is omitted for clarity of viewing.) In accordance with the analysis from DQFCOSY spectra, 50 ms NOESY gives most NOEs from H3P and H8/H6 with comparably strong intensities, excluding a pure C2P-endo sugar pucker conformation and specifying a mixture of C2P-endo and C3P-exo conformations. Complete analysis of intra-residue and sequential NOEs between H1P, H2P, H2Q, H3P and base H6/H8 again shows a conformation intermediate between A-DNA and B-DNA but closer to B-DNA. Another interesting phenomenon is that NOEs from intranucleotide H1P/H6 of T9, T10, T11 are all much weaker than their counterparts in the rest of the 17-mer duplex. However, the corresponding H1P^H6 distances do not vary over a range of greater î , and the imino proton exchange experiment than 0.3 A indicates a more rigid structure for this segment. It may

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Fig. 3. Sequential NOE connectivity in the base-sugar region of the 17mer duplex. For simplicity, only the top strand is traced. The spectrum was acquired at 311 K with 600 ms mixing time.

be tenable to imagine at this stage that this segment has some A-tract characteristics [23], but with a more extensive base rotation movement indicated by the scaled-down NOE intensity. Chemical shift is an important NMR parameter [24] and directly provides general information about the immediate environment. Most proton chemical shifts fall into a reasonable range for regular duplex DNA, but deviations for H1P of C23 and H5 of C8, and, to a lesser extent, H8 of A2 are observed. Considering the relatively shorter distances between H1P of A2/A19 and methyl groups of T4/T21 (as re£ected in relatively greater NOE buildup, Fig. 4 and data not shown), respectively, we anticipate that this DNA has some local bending and

Fig. 4. NOE buildup curves (50 ms, 100 ms, 150 ms, 200 ms and 300 ms mixing times) for three NOEs between H1P of A2 and methyl group of T4 (F), H1P of C3 and methyl group of T4 (b), H1P of A5 and methyl group of T7 (8), respectively. The obviously strong NOE between H1P of A2 and the methyl group of T4 indicates a close contact.

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Table 1 Proton chemical shifts of 17-mer DNA at 10‡C Base H1P

H2P

H2PP H3P

H4P

H5P/5Q

G-H1, A-H2, T-H3

C/T-H6, A/G-H8

H5, M

H41/H42

H61/H62

C1 C3 T4 A2 A5 C6 T7 C8 T9 T10 T11 G12 T13 A14 G15 T16 G17 C18 A19 C20 T21 A22 C23 A24 A25 A26 G27 A28 G29 T30 A31 G32 T33 G34

1.90 1.97 2.10 2.80 2.71 1.98 2.19 2.20 2.19 2.17 2.19 2.59 2.09 2.71 2.45 1.89 4.17 1.90 2.80 1.97 2.10 2.64 1.83 2.62 2.58 2.50 2.40 2.59 2.37 2.04 2.74 2.45 1.89 4.17

2.37 2.45 2.46 2.92 2.83 2.45 2.54 2.54 2.54 2.58 2.51 2.75 2.45 2.88 2.67 2.35 2.61 2.37 2.92 2.45 2.46 2.83 2.18 2.80 2.80 2.80 2.62 2.88 2.67 2.45 2.90 2.67 2.35 2.61

4.05 4.31 4.17 4.44 4.43 4.25 4.22 4.22 4.22 4.22 4.17 4.39 4.17 4.39 4.39 4.18 4.39 4.05 4.44 4.31 4.17 4.39 4.39 4.32 4.39 4.39 4.33 4.44 4.38 4.17 4.39 4.39 4.18 4.39

3.72/3.72 4.18/4.18 4.09/4.09 4.13/4.02 4.17/4.09 4.22/4.22 4.08/4.09 4.09/4.09 4.09/4.09 4.09/4.09 4.12/4.12 4.17/4.14 4.13/4.13 4.17/4.05 4.17/4.17 4.10/4.10 4.17/4.09 3.72/3.72 4.13/4.02 4.18/4.18 4.05/4.05 4.13/4.09 4.09/4.09 4.09/3.96 4.20/4.08 4.17/4.17 n/d 4.17/4.09 4.19/4.19 4.09/4.09 4.17/4.05 4.17/4.17 4.10/4.10 4.17/4.09

^a ^ 13.68 7.90 7.30 ^ 13.88 ^ 14.08 13.96 13.45 12.36 13.45 7.19 12.71 13.86 12.90 ^ 7.90 ^ 13.68 7.30 ^ 6.99 7.04 7.30 12.54 7.53 12.79 13.45 7.19 12.71 13.86 12.90

7.65 7.33 7.35 8.37 8.30 7.34 ^ 7.62 7.44 7.49 7.36 7.88 7.22 8.21 7.65 7.14 7.89 7.65 8.37 7.33 7.37 8.27 7.24 8.12 8.08 7.94 7.47 7.92 7.47 7.14 8.22 7.65 7.14 7.89

5.87 5.32 1.59 ^ ^ 5.20 1.51 5.55 1.59 1.59 1.68 ^ 1.41 ^ ^ 1.38 ^ 5.87 ^ 5.32 1.59 ^ 5.32 ^ ^ ^ ^ ^ ^ 1.25 ^ ^ 1.38 ^

n/db 6.79/8.06 ^ ^ ^ 6.74/7.90 ^ 7.03/8.26 ^ ^ ^ ^ ^ ^ ^ ^ ^ n/d ^ 6.79/8.06 ^ ^ 6.48/8.21 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^

^ ^ ^ 7.65/5.88 5.67/7.30 ^ ^ ^ ^ ^ ^ n/d ^ 5.67/7.30 n/d ^ n/d ^ 7.65/5.88 ^ ^ 5.83/7.70 ^ 6.03/7.70 5.88/5.88 6.03/7.45 n/d 5.77/7.50 n/d ^ 5.62/7.75 n/d ^ n/d

a b

5.60 5.84 5.71 6.26 6.13 5.75 6.02 6.03 6.02 6.10 5.83 5.88 5.62 6.03 5.84 5.84 6.13 5.60 6.26 5.84 5.66 6.13 5.25 5.72 5.75 5.86 5.42 6.03 5.78 5.67 6.06 5.84 5.84 6.13

4.69 4.68 4.86 5.03 5.04 4.56 4.86 4.81 4.86 4.89 4.91 4.95 4.86 5.04 4.91 4.84 4.69 4.69 5.03 4.68 4.86 5.00 4.75 4.99 4.99 4.99 4.91 5.00 4.83 4.87 5.04 4.91 4.84 4.69

Not applicable. Not detected.

sequence-dependent deviations of local conformation from canonical B structure. Resonances relating to T methyl groups are strong and with less overlap, making assignment more accurate and unambiguous. The chemical shift assignments are listed in Table 1. 3.3. Conformational analysis Twenty structures were calculated from initial A-DNA and B-DNA conformations [26]. Both ensembles conî . The RMSD between the verged to a RMSD of 0.7 A average structures from A-DNA and B-DNA was 0.68 î . However, because of minor di¡erences, 10 structures A were extracted from each group to comprise the mixed î , that is presented in ensemble, with RMSD of 0.73 A Fig. 5. The average structure from the ensemble presented in Fig. 5 was analyzed by the NEWHELIX91 program (implemented through Insight 97, MSI) and the results are shown in Fig. 6a^d. The overall shape of this average structure lies between the standard A-DNA and B-DNA structures, but closer to

Fig. 5. The ensemble of DNA structures, represented in two stereo views. Coordinates have been deposited in PDB (accession ID: 1IR5; structure RCSB005204).

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twist angles for the two T-A steps are relatively small. The T-A step is commonly found to have a high twist angle and is identi¢ed as a high twist low rise step [26] ; it is also the most thermally £exible step [27]. Its £exibility helps the bending of the two ends of our structure as it untwists and rolls to the major groove to keep the local conformation stable due to the relative rigidity of the sugar phosphate backbone. Consequently, this step is not suitable for intercalation by side chains of TF1, which is suggested to bind to DNA in the minor groove [3,4]. It should be noted that this bending is local and quite limited, and that it is stabilized by the less twisted T-A step. Fig. 6b shows the propeller twist angle g. Base pair steps 2/3 and 15/16 have quite large values of g due to bending; g values of the 8/9 and 9/10 base pair steps are also large, as the A-A-A segment may have some characteristics of the A-tract with its large propeller twist to keep it straight and rigid [23]. One particularly dramatic change of the twist angle, roll angle, and propeller twist involves the G12WC23 pairs. AWT pairs usually have larger twist angles for better base stacking, especially in A-tracts. On the other hand, GWC pairs are able to stack directly over each other. Therefore, the GWC pair located inside the consecutive AWT pairs does not favor a regular helix conformation. This is the major step that causes the two ends to be unsymmetrical. Evidence of this asymmetry is provided by the NOE between H6 of T4 and H8 of A5, which has a much stronger intensity than that from T21 and A22, supporting that these two T-A steps are not symmetrical. It can be imagined that this di¡erence propagates to the ends of the 17-mer duplex.

4. Discussion

Fig. 6. Variations in twist angle (a), propeller twist angle (b), roll angle (c) and rise (d) for the average structure.

B-DNA. The two ends are not symmetrical, although chemical shifts of the protons at the two ends are similar. The average twist angle for the internal 15 bp is 34.9‡. As indicated by the strong NOE between H1P of A2 and the methyl group of T4, the positive roll angle and lower rise parameters for the second and third step signal bending with compression of the major groove. The T-A step usually has a larger twist angle [25], but in this structure the

Compared to the previously analyzed hmU-DNA of nearly identical sequence [11], this T-DNA (i.e. unmodi¢ed DNA) is di¡erent, and it is also di¡erent from other T-DNA structures with regard to the two T-A steps. For linear DNA, NOEs can only re£ect proximities at short distance, at most involving residues separated by two bp. For global DNA structure determination and DNA bending, application of NMR residual dipolar coupling is necessary [28]. However, NOEs can still be used to study detailed local conformation in combination with chemical shift analysis. For this work, the strong NOEs between H1P of A2 and methyl group of T4 are ¢rm evidence that there is local bending. The chemical shift of H1P of C23 and the large distances between H6 and H2P and H2Q o¡er ¢rst indications of an abnormal M conformation [29]. Surprisingly, this unusual chemical shift is also manifested by a large local conformational disturbance. DNA has many degrees of freedom, so the speci¢cation of the force ¢eld plays a critical role in the calculations of conformation subject to the NMR-derived restraints. Information from crystallographic studies such as the characteristic propeller twist and base stacking is very useful. The £at-

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ness of the G12WC23 pair and the £exibility of the T-A step are therefore re£ected in the structure. Interestingly, the negative roll angle of the T-A step may explain the unfavorable minor groove for TF1 binding. TF1 binds to hmU-DNA preferentially and speci¢cally, relative to normal DNA. hmU di¡ers from thymine in the major groove. Assuming a minor groove binding mode, hmU-DNA binding to TF1 was explained by distortion and deformability compared to T-DNA. According to Pasternack et al. [9], for hmU-DNA, the presence of the hydroxyl group and intrastrand hydrogen bonding would render the interstrand base pair hydrogen bonding associated with the hmU residue less energetically favorable. This may cause the instability of the hmU-A base pair and result in local £exibility. This can be con¢rmed by the strong NOEs between imino groups of T9, T10 and T11, which are very weak for hmU9, hmU10 and hmU11 [10]. On the contrary, the NOE between hmU4 and A5 is strong, and is not detected for T-DNA at 38‡C. These results con¢rm that hmU causes £exibility in the central segment and changes the conformation for the two T-A steps. Comparing the chemical shift pattern of this T-DNA with the (nearly) corresponding hmU-DNA [10], it is certain that there is not a large global backbone conformation change. The di¡erence must be in the detailed local conformation, and it may not be the average conformation re£ected by NMR but the deformability or its dynamic character. The A24-A25-A26 segment has some properties of an A-tract, such as a large propeller twist angle, except for A26, whose base pair £attens because of its nearby GWC pair. The bases of T9, T10, T11 show high rotation movement despite the relatively rigid sugar phosphate backbone. We infer that this segment constructs a rigid part of this T-DNA. Finally, we have performed NMR experiments for the complex of TF1 with both hmU-containing and T-containing DNA. Dramatic chemical shift changes were observed only for hmU-DNA in its TF1 complex, suggesting that only hmU-DNA is highly distorted upon binding (cf. [17]). Surprisingly, imino protons in the hmU-DNA^TF1 complex were not detected, indicating that they may exchange more rapidly with the solvent in the DNA^protein complex than in free DNA. Further analysis will be required to explore this result. Acknowledgements We thank John Wright and Guiseppe Melacini for their help with NMR experiments. We also thank Mohamed Ouhammouch and George Kassavetis for most generous help in preparing materials, and Peter Geiduschek, Guiseppe Melacini and Regina Neves for editorial advice and assistance. This research was supported by grants from the National Institutes of Health (NIGMS-40635 and 39418).

References [1] T. Ha«rd, V. Hsu, M.H. Sayre, E.P. Geiduschek, K. Appelt, D.R. Kearns, Fluorescence studies of a single tyrosine in a type II DNA binding protein, Biochemistry 28 (1989) 396^406. [2] X. Jia, A. Grove, M. Ivancic, V.L. Hsu, E.P. Geiduscheck, D.R. Kearns, Structure of the Bacillus subtilis phage SPO1-encoded type II DNA-binding protein TF1 in solution, J. Mol. Biol. 263 (1996) 259^268. [3] W.D. Liu, H.M. Vu, E.P. Geiduschek, D.R. Kearns, Solution structure of a mutant of transcription factor 1: Implications for enhanced DNA binding, J. Mol. Biol. 302 (2000) 821^830. [4] P.A. Rice, Making DNA do a U-turn: IHF and related proteins, Curr. Opin. Struct. Biol. 7 (1997) 86^93. [5] P.A. Rice, S.W. Yang, K. Mizuuchi, H.A. Nash, Crystal structure of an IHF-DNA complex A protein-induced DNA U-turn, Cell 87 (1996) 1295^1306. [6] D.L. Wilson, E.P. Geiduschek, A template-selective inhibitor of in vitro transcription, Proc. Natl. Acad. Sci. USA 62 (1969) 514^520. [7] R. Kallen, M. Simon, J. Marmur, The occurrence of a new pyrimidine base replacing thymine in a bacteriophage DNA: 5-hydroxymethyl uracil, J. Mol. Biol. 5 (1962) 248^250. [8] J.R. Greene, L.M. Morrissey, E.P. Geiduschek, DNA binding by the bacteriophage SPO1-encoded type II DNA-binding protein, transcription factor 1. Site-speci¢c binding requires 5-hydroxymethyluracil-containing DNA, J. Biol. Chem. 261 (1986) 12828^12833. [9] L.B. Pasternack, J. Bramham, L. Mayol, A. Galeone, X. Jia, D.R. Kearns, H-1 NMR studies of the 5-(hydroxymethyl)-2P-deoxyuridine containing TF1 binding site, Nucleic Acids Res. 24 (1996) 2740^2745. [10] H.M. Vu, A. Pepe, L. Mayol, D.R. Kearns, NMR-derived solution structure of a 17mer hydroxymethyluracil-containing DNA, Nucleic Acids Res. 27 (1999) 4143^4150. [11] A. Grove, A. Galeone, L. Mayol, E.P. Geiduschek, Localized DNA £exibility contributes to target site selection by DNA-bending proteins, J. Mol. Biol. 260 (1996) 120^125. [12] A. Bax, D. Davis, MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy, J. Magn. Reson. 65 (1985) 355^360. [13] J. Jeener, B. Meier, P. Bachmann, R. Ernst, Investigation of exchange processes by two-dimensional NMR spectroscopy, J. Chem. Phys. 71 (1979) 4546^4553. [14] M. Piotto, V. Saudek, V. Sklenar, Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions, J. Biomol. NMR 2 (1992) 661^665. [15] R.E. Hurd, Gradient-enhanced spectroscopy, J. Magn. Reson. 87 (1990) 422^428. [16] M. Gue¤ron, J.L. Leroy, Studies of base pair kinetics by NMR measurement of proton exchange, Methods Enzymol. 261 (1995) 383^413. [17] G.M. Dhavan, J. Lapham, S. Yang, D.M. Crothers, Decreased imino proton exchange and base-pair opening in the IHF-DNA complex measured by NMR, J. Mol. Biol. 288 (1999) 659^671. [18] J.D. Baleja, R.T. Pon, B.D. Sykes, Solution structure of phage-lambda 1/2-operator DNA by use of NMR, restrained molecular dynamics, and NOE based re¢nement, Biochemistry 29 (1990) 4828^4839. [19] D. Patel, Nucleic Acid Geometry and Dynamics, Pergamon Press, Oxford, 1980, pp. 185^231. [20] K. Wu«thrich, NMR of Proteins and Nucleic Acids, John Wiley and Sons, New York, 1986, pp. 117^175. [21] C.M. Clore, A.M. Gronenborn, Two-, three-, and four-dimensional NMR methods for obtaining larger and more precise three-dimensional structures of proteins in solution, Annu. Rev. Biophys. Biophys. Chem. 20 (1991) 29^63. [22] P. Cuniasse, L.C. Sowers, R. Eritja, B. Kaplan, M.F. Goodman, J.A. Cognet, M. LeBret, W. Guschlbauer, G.V. Fazakerley, An abasic site in DNA. Solution conformation determined by proton NMR and molecular mechanics calculations, Nucleic Acids Res. 15 (1987) 8003^8022.

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W. Liu et al. / Biochimica et Biophysica Acta 1574 (2002) 93^99 [23] R.E. Dickerson, D. Goodsell, M.L. Kopka, MPD and DNA bending in crystals and in solution, J. Mol. Biol. 256 (1996) 108^125. [24] S.S. Wijmenga, M. Kruithof, C.W. Hilbers, Analysis of H-1 chemical shifts in DNA: Assessment of the reliability of H-1 chemical shift calculations for use in structure re¢nement, J. Biomol. NMR 10 (1997) 337^350. [25] N.B. Ulyanov, T.L. James, Statistical analysis of DNA duplex structural features, Methods Enzymol. 261 (1995) 90^120. [26] R. Dickerson, DNA structure from A to Z, Methods Enzymol. 211 (1992) 67^111.

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[27] J. SantaLucia, H.T. Allawi, P.A. Seneviratne, Improved nearestneighbor parameters for predicting DNA duplex stability, Biochemistry 35 (1996) 3555^3562. [28] A. Vermeulen, H.J. Zhou, A. Pardi, Determining DNA global structure and DNA bending by application of NMR residual dipolar couplings, J. Am. Chem. Soc. 122 (2000) 9638^9647. [29] S. Wijmenga, B. Buuren, The use of NMR methods for conformational studies of nucleic acids, Prog. NMR Spectrosc. 32 (1998) 287^ 387.

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