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Nucleic Acids Research 27:3938-3944 (1999)
© 1999 Oxford University Press

Article

Structural characterisation of a uracil containing hairpin DNA by NMR and molecular dynamics

Mahua Ghosh, N. Vinay Kumar1, Umesh Varshney1 and K. V. R. Charya

Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India and 1Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Three-dimensional (3D) structure of a hairpin DNA d-CTAGAGGATCCTTTUGGATCCT (22mer; abbreviated as U4-hairpin), which has a uracil nucleotide unit at the fourth position from the 5" end of the tetra-loop has been solved by NMR spectroscopy. The 1H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimisation procedures have been used to describe the 3D structure of the U4 hairpin. This study establishes that the stem of the hairpin adopts a right handed B-DNA conformation while the T12 and U15 nucleotide stack upon 3" and 5" ends of the stem, respectively. Further, T14 stacks upon both T12 and U15 while T13 partially stacks upon T14. Very weak stacking interaction is observed between T13 and T12. All the individual nucleotide bases adopt ‘anti’ conformation with respect to their sugar moiety. The turning phosphate in the loop is located between T13 and T14. The stereochemistry of U15 mimics the situation where uracil would stack in a B-DNA conformation. This could be the reason as to why the U4-hairpin is found to be the best substrate for its interaction with uracil DNA glycosylase (UDG) compared to the other substrates in which the uracil is at the first, second and third positions of the tetra-loop from its 5" end, as reported previously.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
DNA, composed of four nucleotides: adenine (A), guanine (G), cytosine (C) and thymine (T), has to face constant challenges with its genomic integrity, not only from the internal process of replication but also from external agents such as chemicals and ionising radiation (1). This results in the production of a variety of modified bases, e.g. uracil (U), a constituent of RNA. As U differs from T only at position 5 where T is methylated and U is not, base-pairing capabilities of both these nucleotides are identical. Hence, U can form as good a Watson–Crick base pair as T does with A in DNA. Uracil can occur in DNA by either (i) misincorporation of dUMP during replication by DNA polymerases or (ii) as a product of spontaneous hydrolytic deamination of cytosine residues in DNA. Such incorporation results in G:C to A:T transition mutations, in two rounds of replication, unless U is repaired back to C, before the first round of replication. Such transition mutations are prevented by uracil DNA glycosylase (UDG), which recognises U and excises it from DNA so that the correct base is reinserted before the first round of replication.

Recently, the structural basis for the recognition of U by various UDGs has been demonstrated (2). The structures show that these enzymes can accommodate a non-specific DNA sequence along a channel, which has an active site pocket, tailored to admit only U. These studies reveal that recognition of U by UDGs requires insertion of U base into a specific pocket in the enzyme, which for double-stranded (ds) DNA can only be achieved by ‘flipping’ the deoxyuridine nucleotide into an extra-helical conformation. However, this poses fundamental questions regarding the recognition process (2). One of them is whether UDG locates uracils by scanning DNA in a ‘one-dimensional’ process or by a bimolecular collision. The second is whether the enzyme actively promotes the flipping of the uracil from a stacked conformation, or it recognises flipped out bases.

Hairpin DNA, consisting of U in the loop, provides a good model system for understanding UDG interaction with DNA. Further, the hairpin loop can offer an extra-helical situation, where U is already in a ‘flipped out’ form. Therein U may be spontaneously recognised by UDG. Besides, hairpin DNAs have a very important role in biology as they often act as regulatory sites in gene transcription and replication.

T

5' CTAGAGGATCC U

3' TCCTAGG T

T

U2-hairpin

T

5' CTAGAGGATCC T

3' TCCTAGG T

U

U4-hairpin

Recently, it has been shown that the excision of uracil from various hairpin loops by UDG is dependent on the uracil position in the loop (3). For a tetra-looped hairpin DNA, the excision efficiency is substantially less when uracil is present at the second position (U2-hairpin) compared to when present at the fourth position (U4-hairpin) from the 5" end of the tetra loop. This suggests structural dependence of the UDG–uracil inter­action. In order to understand the role of DNA structure in substrate binding to the UDG, we have initiated the elucidation of the 3D-structures of the aforementioned hairpin DNAs. In this paper we present the 3D structural details of U4-hairpin, derived from 2D NMR data and molecular dynamics simulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
DNA synthesis and purification
The DNA oligonucleotide (a 22mer; U4-hairpin) was designed such that a minimum of 7 bp form the stem of the hairpin with 4 nt in the loop. The 4 nt overhang at the 5" end of the hairpin was used to facilitate 32P labelling by end filling with Klenow polymerase. The oligonucleotide was custom made by Ransom Hill Bioscience, Inc. (Ramona, CA, USA) and purified from 18% polyacrylamide 8 M urea gels (4), desalted on sep-pak columns and lyophilised. Purified U4-hairpin was examined using gel electrophoresis which reveals the existence of oligo as a monomer. Though the overhang at the 5" end can trigger the formation of a dumbbell, the single hairpins are favoured by the efficient end filling experiments (1). Cooperative thermal dissociation curves are observed for U4-hairpin (not shown here) with UV (the melting point, Tm ~45°C), indicating that the DNA adopts a distinct and ordered conformation below the Tm. In order to rule out the formation of a dimer containing a double-stranded segment of DNA with an internal bubble, DNA has been chilled rapidly after heating it to 80°C, thus favouring intramolecular structure. Further, in an independent experiment on a parent oligo containing an extended stem region was 5"-phosphorylated and subjected to ligation. Predominant population (>90%) stopped at dimer formation (cohesive ends), indicating the formation of a dumbbell (5). If it was bubble structure, we would have got trimers and tetra­mers, etc., as seen in the case of a control oligo with inter­molecular antiparallel base-pairing. Another reason for us to believe that the U4-hairpin is in stem–loop form comes from the kinetics of uracil excision from the four looped substrates. If it was bubble structure we would not expect substantial differences in the excision efficiencies.

NMR
About 8 mg of purified DNA was dissolved in 0.6 ml of appropriate solvent (~5 mM strand concentration or 60 mM in nucleoside residues) with no buffer. For experiments in 2H2O, the DNA was lyophilised three times from 2H2O to deprotonate all the exchangeable protons, prior to its dissolution in 0.6 ml of 99.9% 2H2O. For experiments in H2O a mixture of 90% H2O and 10% 2H2O was used.

1H NMR experiments were carried out on Varian Unity+ 600 and Bruker AMX 500 spectrometers with the proton fre­quencies at 600 and 500 MHz, respectively. The spectra in a mixed solvent of 90% H2O + 10% 2H2O include 1D 1H NMR spectra recorded with P11 pulse sequence (6) and 2D nuclear Overhauser enhancement spectroscopy (NOESY) (7) with P11 detection pulse sequence and a mixing time of 200 ms. The 2D experiments in 2H2O include exclusive correlation spectro­scopy (E-COSY) (8), clean total correlation spectroscopy (clean TOCSY) (9) with a mixing time of 80 ms and a set of NOESY spectra with different mixing times (50, 80, 100, 150, 200, 250 and 300 ms). A temperature of 32°C was used in most NMR experiments, though 1D 1H experiments were carried out in the range of 15–55°C. In all the experiments, the 1H-carrier frequency was kept at water resonance. In 2D experiments, time domain data points were 512 and 4096 along t1 and t2 dimensions, respectively. The data were multiplied with sine bell window functions shifted by {pi}/4 and {pi}/8 along t1 and t2 axes, respectively, and zerofilled to 1024 data points along t1 dimension prior to 2D-FT.

Structural restraints
Distance restraints. The interproton distances have been estimated from NOESY spectra recorded with mixing times of 50, 80, 100, 150, 200, 250 and 300 ms. As a prelude to distance estimation, we have monitored the build up of the nOe volumes of each resolved cross-peak (10–12). Distances were estimated from the initial build-up rates of the build-up curves by the two spin-approximation formula rij = rref (Rij/Rref)1/6, where rij is the distance between i and j protons, rref is the reference distance and Rij and Rref are the initial build-up rates, respectively. The interproton distances were estimated using the volume integral of the intranucleotide T(CH3)–T(H6) cross-peak, except in the estimation of all intrasugar inter­proton distances. There are two other standard distances in DNA, namely CH5–CH6 and H2"–H2"". However, in the present study both these cross-peak regions suffer from extensive overlaps. For intrasugar interproton distances the volume integral of H2""–H1" cross-peak, which is independent of pseudo­rotation phase angle (P) (13) was used as a reference. All such estimated interproton distances have been used as constraints with the upper and lower bounds 0.05 nm in the energy minimisation and molecular dynamics calculations. In the case of nOes which were partially overlapping or weak at short mixing times (50 and 80 ms), no distance estimation was carried out. However, the information was used generously to restrain the corresponding proton pairs with lower and upper bounds as 0.20 and 0.50 nm, respectively. Six of the 7 bp forming the stem of the hairpin DNA showed evidence of hydrogen bonding in the 1H NMR spectrum. Based on such data, the interatomic distances G(O6)–C(H41), G(H1)–C(N3), G(H21)–C(O2), A(H61)–T(04) and A(N1)–T(H3) within each base pair were restrained in the range 0.17–0.20 nm with a force constant of 10 kcal mol–1 Å–2. On the other hand, the heavy atoms in these base pairs were restrained within the range 0.28–0.32 nm with a force constant of 20 kcal mol–1 Å–2. These constraints were relaxed during the final stages of the calculations. For the strong nOes observed between A(H2) and T(H3) belonging to A:T base pairs, and the analogous G(H1) and C(H41) belonging to G:C base pairs, they were restrained in the range 0.24–0.33 nm and 0.20–0.30 nm, respectively. For these constraints a force constant of 20 kcal mol–1 Å–2 was used.

Torsion angle restraints
The information about the range of pseudorotational phase angle (P) obtained from the E-COSY spectrum has been used to define two of the five sugar ring torsion angles (–C2"–C3"–C4"–O4"– and –C1"–C2"–C3"–C4"–). Further, this information was also used to define the lower and upper bounds for one of the backbone torsion angles, {delta}. In addition, information about glycosidic torsional angles ({chi}), derived from the intranucleotide H6/H8–H1"/H2"/H2"" nOe connectivities, has been used to constrain {chi}. For all these torsion constraints a force constant of 20 kcal mol–1 rad–2 was used.

Starting structure
An initial B-DNA duplex structure with a sequence of individual strands (as shown below) was generated using the molecular modelling package INSIGHT-II (MSI, San Diego, CA) on an Iris (Indigo II) workstation. Other steps involved in generating the starting structure are as follows: (i) replacement of A14 and A15 by T and U nucleotide units, respectively; (ii) capping of the A5–T13 strand; (iii) merging of the two strands; (iv) changing the hybridisation of P atom at the 5" end of the A14–T22 strand; and finally, (v) connecting the 3" end of the A5–T13 strand to the 5" end of the A14–T22 strand, which resulted in a hairpin DNA conformation with seven Watson–Crick (four C:G and three A:T) base pairs forming the stem of the hairpin and with 4 nt -TTTU- in the loop. As mentioned earlier, although the 5" end of the stem is protruding by 4 nt, also, there being an indication of dumbbell formation, this stretch was ignored for molecular dynamics and energy minimisation calculations, primarily because this stretch is ill-defined with few nOe constraints.

5" A5 G6 G7 A8 T9 C10 C11 T12 T13 3"

3" T22 C21 C20 T19 A18 G17 G16 A15 A14 5"

Molecular dynamics and energy minimisation methods
Molecular dynamics simulations were performed with DISCOVER (Molecular Simulations Inc., San Diego, CA). AMBER force field was used to calculate the energy of the system. Electrostatic interactions were calculated using Coulomb’s law with point charges. Distance-dependent dielectric constant of ‘1*r’ was used. Van der Waals contributions were calculated with a 6–12 Lennard–Jones potential. A time step of 1 fs was used. Initial random velocities were assigned in accordance with a Maxwell–Boltzmann distribution. To obtain the starting structure, an initial steepest descent minimisation of 100 steps was performed on the initial structure followed by conjugate gradient minimisation of 1000 steps. The good-fit structure thus obtained was followed by restrained molecular dynamics simulations. Initial random velocities were assigned with a Maxwell–Boltzmann distribution for a temperature of 600 K. Two hundred structures were collected at 1 ps intervals along the restrained molecular dynamics trajectory. These structures were significantly different from each other as evident from their pairwise root-mean-square deviations (RMSDs). Each of these structures was cooled to 300 K in steps of 50 K. After each temperature step, the system was allowed to equilibrate for 10 ps. This was followed by 500 steps of steepest descent minimisation and 1000 steps of conjugate gradient minimisation for monitoring the convergence and structure analysis. In the event of any constraint violation another round of dynamics was performed by varying initial temperature as well as the weight of the restraints. The molecule was then cooled to 300 K and energy minimised as mentioned previously. This procedure was repeated three times until well-converged structures were obtained with zero violations. In these calculations, the NMR derived restraints were applied all along with force constants of 25 kcal mol–1 Å–2 for all nOes involving non-exchangeable protons, 10 kcal mol–1 Å–2 for all nOes involving exchangeable protons and the atoms involved in H-bonds.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
SUPPLEMENTARY MATERIAL
 REFERENCES
 
1H NMR assignments
Sequence-specific 1H resonance assignments were achieved through established procedures (14–20). Figure 1 shows selected regions of NOESY spectra of U4-hairpin with imino–imino (Fig. 1a) and H2"/H2""/CH3–H6/H8 (Fig. 1b) connectivities. Except for the serious overlap seen in the case of H6 resonances belonging to C10, C11, C20 and C21, the assignments were straightforward. The degeneracy between these H6 protons could be resolved by the observation of intranucleotide and sequential nOes between their respective CH5 protons and H2"/H2""/CH6. The only missing connectivities were between G4 and A5 units. The stereospecific assignment of individual H2" and H2"" could be achieved by intensity comparison of the H1"–H2" and H1"–H2"" cross-peaks in the NOESY spectrum, where the latter is found to be stronger than the former. The chemical shifts of the protons thus obtained have been deposited in the Protein Data Bank (PDB) (PDB ID 1QE7; RCSB ID RCSB009334).



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  		Figure 1. (a) Selected region of NOESY spectrum of U4-hairpin recorded with a mixing time of 200 ms, in a mixed solvent of 90% H2O and 10% 2H2O at 22°C and pH 7. This shows imino–imino nOe interactions. (b) Selected region of NOESY spectrum of U4-hairpin recorded with a mixing time of 200 ms, in 99.9% 2H2O at 32°C and pH 7. This shows nOe connectivities from T(CH3)/H2"/H2"" to H2/H6/H8.

 
Sugar puckers as derived from the coupling constant information
E-COSY experiments can be used to resolve the conformational-dependent characteristic multiplet structures of H2"–H1" and H2""–H1" cross-peaks. The {omega}2 axis in these cross-peaks contains information about 3J(H1"–H2") and 3J(H1"–H2"") (21–23). Although these estimated Js are error prone, one can at least conclude accurately as to which one of them is larger for fixing a certain window to the sugar puckers. In the present study, even though there are 22 furanose rings in U4-hairpin, the H2"/H2""–H1" region of the E-COSY spectrum shows fairly well resolved cross-peaks. Wherever the cross-peaks are well resolved, the 3J(H1"–H2") and 3J(H1"–H2"") values were extracted and in all these cases the 3J(H1"–H2") is clearly found to be larger than 3J(H1"–H2""). These J values qualitatively indicate that the corresponding sugar rings adopt conformation in the S domain of the pseudorotational map with P ranging from C1"-exo to C3"-exo (P = 90–198). The 3J(H2""–H3") and 3J(H3"–H4") values which could have helped in further narrowing the domains of sugar puckers could not be estimated from this E- COSY spectrum because of the low intensity of the corresponding peaks.

Secondary structure of U4-hairpin
The imino and amino proton resonances indicate hydrogen bonded base pairs all along the stem of U4-hairpin. Intricate networks of interstrand NOESY cross-peaks G6(H1)–C21(H41/H42), G7(H1)–C20(H41/H42), A8(H2)–T19(H3), T9(H3)–A18(H2), C10(H41/H42)–G17(1NH) and C11(H41/H42)–G16(H1) establish a hydrogen bonded base pairing between G6:C21 (G6 and C21), G7:C20, A8:T19, T9:A18, C10:G17 and C11:G16 and thus the secondary structure of the stem of U4-hairpin. Qualitative analysis of the relative NOESY cross-peak intensities (Fig. 2) establishes that the stem of the hairpin adopts a right handed B-DNA duplex conformation. The nOe data further confirm the association of A:T and G:C base pairs through Watson–Crick base pairing schemes with almost all the individual bases in ‘anti’ conformation with the glycosidic torsion angle, {chi}, ranging from –80 to –120. This is based on the observation of strong intranucleotide H2"–H6/H8 cross-peaks compared to H2""–H6/H8 cross-peaks, while H1"–H6/H8 cross-peaks are relatively weak or absent. In the case of C10, C11, C20 and C21 we could not establish the respective {chi} values because of the severe spectral overlap of H1"/H2"/H2""–H6 cross-peaks. On the other hand, the loop residues T12, T13, T14 and U15 show interesting nOe connectivities (Fig. 2). Although most of the expected sequential nOes are seen all along this stretch of nucleotide units, the interactions between T13(H6/CH3) and T12(H1"/H2"/H2""/H3") are found to be weak. Further, T14(CH3) shows medium intensity nOes to T12(H1"/H2"/H2""/H6/CH3), indicating a partial stacking interaction between T12 and T14 bases. These nOe interactions essentially dictate the folding pattern of the loop, which will be discussed later. As for the stem, the individual bases of the loop residues adopt ‘anti’ conformation. By the end of the assignment procedure, all the major cross-peaks in the 2D spectra could be assigned uniquely. Although no resonances could be ascribed to another conformer, a lone cross-peak was seen between C1(H6) and T22(H2"/H2""), indicating a dumbbell formation. However, no extra imino proton resonance was observed under the conditions of NMR experiments mentioned above to substantiate the formation of dumbbell.



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  		Figure 2. Various internucleotide nOe connectivities seen in 200 ms NOESY spectrum of U4-hairpin.

 
NMR structure determination of U4-hairpin
Restrained molecular dynamics simulation and energy minimisation calculations were performed on the U4-hairpin following the procedure described in the Materials and Methods. A total of 190 interproton distance constraints (10 involving exchangeable protons and 180 involving non-exchangeable protons) and 68 torsion constraints were used with the force constants described earlier. All these constraints have been deposited in the PDB (PDB ID 1QE7; RCSB ID RCSB009334; http://www.pdb.bnl.gov/ ). Out of the 200 calculated structures, there are nine structures lying within 2.5 kcal/mol above the minimum energy structure. These 10 structures are characterised by low all atom pairwise RMSDs, which range from 0.09 to 0.63 (see supplementary Table S1). Figure 3 shows the best-fit superimposition of these 10 structures. The corresponding PDB files have been deposited in the PDB (PDB ID 1QE7; RCSB ID RCSB009334). Average values with standard deviation of all the backbone torsion angles and glycosidic torsion angles for all the 10 structures are listed in the supplementary Table S2. The stereo-chemistry of all these 10 structures were critically examined for proper hydrogen-bond lengths and angles in the Watson–Crick base pairs, stereo­chemical feasibility of the various torsional angles and any sterically hindered non-bonded inter-atomic distances. All the 10 structures satisfied these criteria.



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  		Figure 3. Stereoview showing a best-fit superimposition of the final 10 molecular dynamics and energy minimized simulated structures of U4-hairpin.

 
Backbone torsion angles in the stem of U4-hairpin
In all the 10 structures, the {alpha} (–O3"–P–O5"–C5"–), ß (–P–O5"–C5"–C4"–), {gamma} (–O5"–C5"–C4"–C3"–) and {varepsilon} (–C4"–C3"–O3"–P–) for each nucleotide in the stem of the hairpin DNA are mostly locked into gauche– (g–), trans (t), gauche+ (g+) and trans (t) conformations, respectively, similar to those observed in B-DNA. The only exception is the case of G16, which is at 3" end of the tetra-loop. For this, ß angle is close to g+ while {gamma} is in t conformation. The {zeta} (–C3"–O3"–P–O5"–) values adopt –96° on average and range from –70 to –123° for all the residues. The {delta} (–C5"–C4"–C3"–O3"–) values adopt 130° on average and range from 96 to 150°.

Backbone torsion angles in the tetra-loop of U4-hairpin
In the case of tetra-loop, it is interesting to note that the {alpha}, ß, {gamma} and {varepsilon} of T12, T13 and T14 nucleotide units are locked into g–, t, g+ and t conformations, respectively, similar to the stem. Two significant deviations are observed for these three residues in the loop. One is the {gamma} of T14 which prefers a t conformation ({gamma} = –174.0 ± 3.0). The other is the {zeta} of T13, which also prefers a t conformation ({zeta} = –179.5 ± 1.5), instead of g– conformation seen in B-DNA. These deviations are necessary because the backbone has to be stretched sufficiently to accommodate the loop formation. On the other hand, for U15 the {varepsilon} and {zeta} are locked in t and g– conformations, respectively, and the {delta} adopts 110° on average and ranges from 103 to 117°, similar to those observed in B-DNA. However, the {alpha}, ß and {gamma} show two distinct variations. In five of the 10 structures, which include the minimum energy structure, they adopt g–, t and g+ conformations, respectively, similar to those observed in B-DNA. For the rest of the five structures, all the three torsion angles are in t conformation.

Nucleotides have C2"-endo sugar puckers and anti conformation
In all of the 10 structures, the sugar puckers lie in the S domain of the pseudorotational wheel and most of the nucleotides assume a sugar pucker in the range of 90–150°. The sugars of T13 and U15 are however around O4"-endo and C1"-exo puckers, respectively. This is supported by the observation of strong intranucleotide nOes which are expected between the H1" and H4" for these nucleotide units (13). A slightly different behaviour for these two nucleotides can be expected since these are present in the loop region of the hairpin DNA. The {chi} for all the nucleotide units are in the anti domain, as are evident in the relative intensities of the resolved nOes between the base and the sugar protons. The {chi} values range from –83 to –133°.

Comparison of U4-hairpin structure with other tetra-looped hairpin DNA
Four hairpin DNAs with -TTTT- stretch in the loop have been structurally characterised previously with a combined use of high resolution NMR and molecular modelling procedures: (i) d-(CGCG TTTT CGCG) (24); (ii) d-(ATCCTA TTTT TAGGTA) (25); (iii) d-(CGATCG TTTT CGATCG) (26); and (iv) d-(GCGC TTTT GCGC) (27). The stems of these hairpins are found to contain Watson–Crick base pairs adopting right handed B-DNA conformation. Few interesting common features are noted regarding the conformation of the loop of these hairpins. The right handed backbone continued through the 3" top of the stem to the 5" top of the stem, by taking one sharp turn. The phosphate group where this sharp turn occurs is called the turning phosphate with the {zeta} and {gamma} locked in g+ and t conformations, respectively, while {alpha} prefers either g+ or t conformation. However, the position of the turning phosphate, the loop bases and their stacking interactions are not the same in the four structures.

Orientation of the loop bases with respect to the grooves
Hilbers et al. (28) classified tetra-looped hairpin DNA (with -TaTbTcTd- sequence as the loop) into three different kinds of folding patterns, namely FP1, FP2 and FP3. In FP1, found for -GC TTTT GC- type hairpin, Tb is turned into the minor groove, while Tc stacks over both Ta and Td bases. In FP2, seen in the case of -TA TTTT TA- hairpin, the stacking of individual bases Ta, Tb and Tc continues through the 3" end of the stem. On the other hand FP3, observed in the -CG TTTT CG- hairpins, is characterised by the stacking of individual bases Td, Tc and Tb over the 5" end of the stem.

In the present study, U4-hairpin adopts FP3 folding pattern (Fig. 3) as seen earlier in the case of -CG TTTT CG- type hairpins, which is consistent with the observed internucleotide nOes. For example, T12 and U15 stack upon the top of the stem of the U4-hairpin, T12 over C11 and U15 over G16. As reported earlier, this should have encouraged U15 and T12 to participate in hydrogen bonding. However, NMR data in the present case does not support any formation of T12–U15 wobble base pair. On the other hand, T14 shows partial stacking interaction with U15 and T12 bases. This interaction is responsible for pulling T14 base towards the helical stem axis, as is evident from the observation of medium intensity nOes between T14(CH3) and T12(H6/CH3/H1"/H2"/H2""). Further, T13 base is tilted away from T12 as evident from weak internucleotide base to sugar proton sequential connectivities [T13(H6)–T12(H1"/H2"/H2"")], which is also the characteristic feature of the FP3 folding pattern. Such folding pattern is substantiated by the T(H6/CH3) chemical shifts for the U4-hairpin, which are consistent with the minor/major groove orientation of the bases present in the loop. For the minor groove T13 base the H6 and CH3 resonances are down-field shifted while for the major groove T14 both H6 and CH3 resonances are relatively up-field shifted.

Turning phosphate
As mentioned earlier, the {zeta} and {gamma} for T13 and T14, respectively, are characteristically in t conformation. Because of this the backbone takes a sharp turn near the phosphate linking T13 and T14. Similar phosphodiester conformations were found for the turning phosphates in the case of -CG TTTT CG- type hairpins (23,25). In the present study, the simulated model reveals that the turning phosphate is indeed between T13 and T14. The 31P chemical shifts, which would have thrown more light upon this, suffer from extensive overlaps.

Structural basis for UDG–U4 hairpin interaction
As mentioned, it has been shown that the UDG–hairpin DNA interaction is dependent on the position of uracil in the tetra-loop. The relative excision efficiencies for ds-DNA, U1, U2, U3 and U4 hairpins are reported to be 40, 3.60, 0.35, 5.23 and 66.34%, respectively, compared to that of single-stranded DNA (ss-DNA) (3). Thus of all the four hairpin DNAs studied so far, U4 hairpin is found to be the best substrate (3). This characteristic feature is reflected in the 3D structure of U4-hairpin. As discussed earlier, all the backbone torsion angles in the stem and the tetra-loop of U4-hairpin are mostly locked in conformations similar to those observed for B-DNA. The exceptions are in the {gamma} value for T14 and {zeta} value for T13 both of which are far from the lone U present in the U4-hairpin. The {delta}, {varepsilon} and {zeta} of T14 and {delta}, {varepsilon} and {zeta} of U15 and {alpha}, ß, {gamma} and {delta} of G16, which influence the local conformation of U15, are locked in conformations similar to those found for B-DNA. In addition, in five of the 10 structures, which include the minimum energy structure, the {alpha}, ß and {gamma} of U15 are locked in conformations similar to those found in B-DNA. Further, in spite of U15 being part of the tetra-loop, it is fairly stacked with T14 and G16 on either side (Fig. 3), with its base in anti conformation. Thus, the stereochemistry of U15 mimics the situation in which U would stack in a ds-DNA. This may be the reason as to why the excision efficiency is not much reduced compared to ss-DNA, as it does, in the case of other hairpin DNA, where U is located at the first, second and third positions of the tetra-loop from its 5" end. The 3D structural elucidation of U2-hairpin, which will provide further insight of the UDG–uracil interaction, is in progress.


   ACKNOWLEDGEMENTS
 
The facilities provided by the National Facility for High Field NMR supported by the Department of Science and Technology (DST), Department of Biotechnology (DBT), Council of Scientific and Industrial Research (CSIR), and Tata Institute of Fundamental Research are gratefully acknowledged. Part of the work was supported by the DBT. N.V. was a CSIR Senior Research Fellow.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUPPLEMENTARY MATERIAL
REFERENCES
 
See Supplementary Material available in NAR Online.


   FOOTNOTES
 
a To whom correspondence should be addressed. Tel: +91 22 215 2971; Fax: +91 22 215 2110; Email: chary{at}tifr.res.in Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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Received April 19, 1999. Revised July 20, 1999; Accepted August 16, 1999.


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