Structural basis for poor uracil excision from hairpin DNA An NMR study Mahua Ghosh 1 , Nidhi Rumpal 2 , Umesh Varshney 2 and Kandala V. R. Chary 1 1 Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India; 2 Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India Two-dimensional N MR and molecular dynamics simula- tions have been used to determine the three-dimensional structures of two hairpin DNA structures: d-CTAGAG GATCCUTTTGGATCCT (abbreviated as U1-hairpin) and d-CTAGAGGATCCTTUTGGATCCT (abbreviated as U3-hairpin). The 1 H resonances of both of these hairpin structures have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the three-dimensional structures of th ese hairpins. This study and concurrent NMR structural studies on two other d-CTAGAGGA TCCTUTTGGATCCT (abbreviated as U2-hairpin) and d-CTAGAGGATCCTTTUGGATCCT (abbreviated as U4-hairpin) have shed light upon various interactions reported between Echerichia coli uracil DNA glycosylase (UDG) and uracil-containing DNA. The backbone torsion angles, w hich partially influence the local conformation of U12 and U14 in U1 and U3-h airpins, respectively, a re probably locked in the trans conformation as in the case of U 13 in the U2-hairpin. Such a stretched-out backbone con - formation i n t he vicinity of U 12 and U 14 is thought to be the reason why the K m value is poor for U1- and U3-hairpins as it is for t he U2-hairpin. Furthermore, the bases U 12 and U 14 in both U 1- and U3-hairpins adopt an anti conformation, in contrast with the base conformation of U 13 in the U2-hair- pin, which adopts a syn conformation. The clear discrepancy observed in t he U-base orientation with respect to the sugar moieties could explain why the V max value is 10- to 20-fold higher for the U1- and U3-hairpins compared with the U2-hairpin. Taken together, these observations support our interpretation that the unfavourable backbone results in a poor K m value, whereas the unfavourable nucleotide con- formation r esults in a poor V max value. These two parame- ters the refore m ake t he U1 - a nd U3 -hairpins b etter substrates for UDG compared with the U2-hairpin, as reported earlier [Kumar, N. V. & Varshney, U. (1997) Nucleic Acids Res. 25, 2336–2343.]. Keywords: hairpin DNA; molecular dynamics; two-dimen- sional NMR spectroscopy; uracil DNA glycosylase; uracil excision. DNA in cells is unceasingly subjected to damages t hat occur even under normal physiological conditions. One such damage is the deamination of cytosine (C) to uracil (U). If left unrepaired, such damage can cause GC to AT mutations in the subsequent replication cycle. U may also be incorporated in place of T by DNA polymerase during replication. Such misincorporation may impend recognition of DNA by various regulatory proteins. Therefore, to maintain genomic integrity, the c ells have uracil DNA glycosylase (UDG), which excises U from DNA [1]. The single-stranded regions which arise in DNA during various physiological processes such as replication may adopt complex secondary and tertiary s tructures. During the formation of such higher-order stuctures, any unpaired C is prone to deamination. To understand the complex mechanism of U excision from such secondary structures, hairpin DNAs consisting of U in the loop provide useful model systems. At times, the hairpin loop can o ffer an extra- helical situation, wherein U is sometimes in a Ôflipped outÕ form. Thereby, U may be spontaneously recognized by UDG. Recently, it has been shown that the excision of U from such hairpin loops by UDG [2,3] is dependent on the U position in the loop. For a tetra-looped hairpin DNA (Scheme I), the affinity (K m ) of UDG towards the U2-hairpin (see Table 1 [2]) is found to be substantially lower that that of t he U4-hairpin. This suggests that poor excision of U from the U2-hairpin could be a consequence of its lower affinity to for the enzyme. A caveat to this interpretation, however, is that other substrates (U1 and U3) also ought to have poorer affinity (high K m ) towards the enzyme. Yet, U excision from these s ubstrates is relatively more efficient (see Table 1 ). In order to gain an insight into such discrepancies in U excision we h ave carried o ut structural characterization by NMR o f the four hairpin DNA structures shown in Scheme I. As reported earlier, comparison of the three- dimensional structures of U2- and U4-hairpins revealed that the stretched-out backbone conformation in the vicinity of U 13 in the U2-hairpin [4,5] is the reason for the enzyme not being a ble to make appropriate contacts with the backbone. Correspondence to K. V. R. Chary, Department of Chemical Sciences, Tata Institute of F undamental Research, Homi Bhabha Road, Bombay 400 005, India. Fax: + 91 22 215 2110/2181, Tel.: + 91 22 215 2971/2979, E-mail: chary@tifr.res.in Abbreviations: UDG, u racil DNA glycosylase; U, uracil. Dedication: This paper is dedicated to the memory of Prof. M . A. Viswamitra (1932–2001). (Received 25 July 2001, revised 16 November 2001, accepted 14 February 2002) Eur. J. Biochem. 269, 1886–1894 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02837.x In addition, the protrusion of the U towards the minor groove side of the hairpin stem may also lead to steric hindrance in the approach of UDG to DNA. On the other hand, U 15 in the U4-hairpin, the best substrate of the four, is located in an environment wherein both the backbone and the base conformation mimic the B-form of DNA. Thus the structural features of the U2-hairpin provided an explana- tion for its poor excision by the enzyme. However, this still did not explain why the catalytic rate (V max ) for U excision in the U 2-hairpin is poor. For productive enzyme–substrate complex formation, it is essential t hat t he U, which is facing the minor groove side of the stem, and is in syn configu- ration with respect to the sugar, be rotated into the major groove side of the DNA to make a ppropriate contacts in the active site of the enzyme. Presumably, the potential energy required for these structural changes to occur before a productive enzyme substrate complex is formed results in lower catalytic rates of U release from the U2-hairpin. This prompted us to suggest that the unfavourable backbone results in a poor K m value, whereas the unfavourable nucleotide conformation results in a poor V max value. This conclusion, however, raises the question of whether the conformation of dU in U1- and U3-hairpins is more favourable in comparison with that in the U 2-hairpin for its localization into the active site pocket. To address this question we have carried out the three-dimensional struc- ture determination of U1- and U3-hairpins by NMR and restrained molecular dynamics. This paper describes the intricate details of three-dimensional structures of U1- and U3-hairpins, as derived from two-dimensional NMR data and molecular dynamic simulation. This is followed by a comparison of these structures with that of the previously reported three-dimensional U2- an d U4-hairpin structures [4,5]. This study in turn provides an insight into the interaction of Escherichia coli UDG with U. MATERIALS AND METHODS DNA Samples The U1- and U3-hairpins (Scheme I) were designed such that a minimum of seven base pairs constitute the stem of the hairpins with four nucleotides in the loops. The four nucleotides overhanging at the 5¢ end of the hairpins was used to facilitate 32 P-labelling by end filling with Klenow polymerase, when required. The oligonucleotides were custom made by Ransom Hill Bioscience, Inc. (Ramona, CA) a nd purified from 18% polyacrylamide/8 M urea gels [3], desalted on Sep-pak columns and lyophilized. Purified hairpins were examined by gel electrophoresis, which reveals the existence of these oligos as monomers. Although the overhangs at the 5¢ ends can trigger the formation of dumb-bells, single hairpins are favoured by the e fficient end-filling experiments [3]. Cooperative ther- mal dissociation curves are observed for both of the hairpins (data not shown) with UV (the melting point, T m  45 °C), indicating that the DNA adopts a distinct and ordered conformation below the T m . NMR About 8 mg of purified oligomers were dissolved in 0.6 mL of appropriate solvent ( 1.8 m M strand concen- tration or 40 m M in nucleoside residues) with no buffer. 5′ CTAGAGGATCC T 5′ CTAGAGGATCC T 3′ TCCTAGG T 3′ TCCTAGG U U1-hairpin U3-hairpin 5′ CTAGAGGATCC U 5′ CTAGAGGATCC T 3′ TCCTAGG T 3′ TCCTAGG T U2-hairpin U4-hairpin U T T T U T T T Scheme 1. Table 1. Kinetic parameters of uracil excision from various DNA substrates and their structural features as derived from NMR data. Substrate K m a (· 10 )7 M ) V max b (· 10 2 ) Relative V max /K m c Phosphate backbone in the vicinity of U Uracil glycosidic torsion angle v SS-U4 d 6.57 675.7 100 – – U1-hairpin 39.9 132.0 3.21 Partially stretched Anti U2-hairpin 40.3 14.5 0.35 Stretched Syn U3-hairpin 22.7 127.9 5.9 Partially stretched Anti U4-hairpin 2.5 173.5 66.8 Resembles B-DNA Anti a K m (dissociation constant) values are for the U residue in the oligonucleotides. b V max (excision rate) values are in pmol product formedÆmin )1 Ælg )1 protein. c Relative V max /K m are shown as percentage of that for SS-U4. d Single-stranded oligonucleotide with uracil at the fourth position from the 5¢ end. Ó FEBS 2002 Structural basis for poor uracil excision (Eur. J. Biochem. 269) 1887 For experiments in 2 H 2 O, the oligomers were lyophilized three times from 2 H 2 O to deprotonate all of the exchange- able protons, prior to dissolution in 0.6 mL 99.9% 2 H 2 O. For experiments in H 2 Oamixtureof90%H 2 O and 10% 2 H 2 Owasused. 1 H NMR experiments were carried out on Varian Unity + 600 and Bruker AMX 500 spectrometers. The spectra in a mixed solvent of 90% H 2 O/10% 2 H 2 O include one-dimensional 1 HNMRspectra recordedwith P1¢1 pulse sequence [6] and t wo-dimensional NOESY [7] with P1¢1 detection pulse sequence and a mixing t ime of 200 ms. The two-dimen sional experiments in 2 H 2 O include exclusive (E)-COSY [8], clean TOCSY [ 9] with a mixing time of 8 0 ms and a set of NOESY spectra with different mixing times (ranging from 50 to 350 ms). A t emperature of 32 °C was used in most of the NMR experiments, although one-dimensional 1 H experiments were c arried ou t in the range of 15–55 °C. In all the experiments, the 1 H-carrier fr equency was kept at water resonance. In two-dimensional experiments, time domain data points were 512 and 4096 along t 1 and t 2 dimensions, respectively. The data multiplied with sine bell window functions shifted by p/4 and p/8 along the t 1 and t 2 axes, respectively, was zero-filled to 1024 data points along the t 1 dimension prior to two-dimensional Fourier transfor- mation (FT). 1 H chemical shift calibrations were carried out with respect to the methyl signal (at 0.0 p.p.m.) of 3-(trimethylsilyl) [3,3,2,2- 2 H] propionate-d 4 ,whichhas been used as an external reference. Starting structure and structural restraints The starting structures for both U1- and U3-hairpins were generated u sing the molecular mod elling p ackage INSIGHT - II (MSI) on an Iris (Indigo II) workstation as d iscussed earlier [5]. Distances were estimated from the initial build-up rates of the build-up curves b y the two spin-approximation as described earlier [10–12]. Six of the seven base pairs forming the stems of the hairpins showed evidence of hydrogen bonding in the 1 H NMR spectrum. Based on such data, the inter-atomic 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 kcalÆmol )1 ÆA ˚ )2 . On the other hand, the heavy atoms in these base pairs were r estrained within the range 0.28–0.32 nm with a force constant of 20 kcalÆmol )1 ÆA ˚ )2 . These constraints were relaxed during the final stages of the calculations. The strong Nuclear Overhausser enhancements (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, were restrained in the range 0.24–0.33 nm and 0.20–0.30 nm, respectively. For these constraints a force con stant of 20 kcalÆmol )1 ÆA ˚ )2 was used. The information about the range of pseudo-rotational phase angle (P) obtained from the knowledge of intra-sugar inter-proton vicinal coupling constants derived from the E-COSY spectrum, was used to define two of the five sugar ring torsion angles (-C2¢-C3¢- C4¢-O4¢-and-C1¢-C2¢-C3¢-C4¢-). This information was also used to define the lower and upper bounds for one of the backbone torsion angles, d(-C2¢-C3¢-C4¢-O4¢-). No restraints were used for the rest of the backbone [a(-O3¢-P-O 5¢-C5¢-), b(-P-O5¢-C5¢-C4¢-), c(-O5¢-C5¢-C4 ¢-C3¢-), e(-C4¢-C3¢-O3¢-P-) and f(-C3¢-O3¢-P- O5¢-)] torsion angles. The glycosidic torsion angles (v)were constrained based on the information derived from the intra-nucleotide H6/H8-H1¢/H2¢/H2¢ nOe c onnectivities. For all of these torsion constraints a force constant of 20 kcalÆmol )1 Ærad )2 was used. Molecular dynamics and energy minimization methods Molecular dynamics simulations were performed with DISCOVER software (MSI). AMBER force field was used to calculate the energy of the system. Electrostatic interactions were calculated using Coulomb’s law with p oint charges (6–31G* standard ESP charges) [13] and the distance- dependent dielectric constant. Van der Waals’ contribu- tions were calculated with a 6–12 Lennard–Jones potential. A time step of 1 fs was used. To obtain the starting structure, an initial steepest descent minimization of 100 steps was p erformed on the initial structure followed by conjugate gradient minimization of 1000 s teps. The best-fit structure thus obtained was used for restrained molecular dynamics simulations. Initial random velocities were assigned with a Maxwell–Boltzmann distribution for a temperature of 600 K. Two-hund red structures were collected at 1 ps i ntervals along the restrained molecular dynamics trajectory. These structures were significantly different from each other as evident by their pair-wise root mean square deviations (rmsd). 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 s teepest descent minimization and 1000 steps of conjugate gradient minimization 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 restraint. The molecule was then cooled to 300 K and energy minimized as mentioned before. This procedure was repeated three times, until well converged structures were obtained with zero violations. In these calculations, as discussed earlier [4], the N MR-derived distance restraints were applied throughout with the upper and lower bounds of ± 0.05 nm and with force constants of 25 kcalÆmol )1 ÆA ˚ )2 for all nOes involving nonexchangeable protons, 10 kcalÆmol )1 ÆA ˚ )2 for all nOes involving exchangeable protons and the atoms involved in H-bonds. For the dihedral angle restraints a force constant of 20 kcalÆmol )1 Æ rad )2 was use d. RESULTS AND DISCUSSION 1 H NMR assignments and secondary structure of the U1- and U3-hairpins Sequence-specific 1 H resonance assignments were achieved by established procedures [14–19]. Fig. 1A and B show illustrative examples of selected NOESY s pectral regions of the U1- and U3-hairpins, respectively, with H2¢/H2¢/ CH 3 –H6/H8 nOe connectivities. Except for the serious overlap seen in the case of H6 resonances belonging to C 10 ,C 11 ,C 20 and C 21 ,the 1 H r esonance assignments were straightforward for both of the hairpins. The degeneracy between these H6 protons could be resolved by the observation of intra-nucleotide a nd sequential nOes 1888 M. Ghosh et al. (Eur. J. Biochem. 269) Ó FEBS 2002 between their respective CH5 and H2¢/H2 ¢/CH6 protons. The stereospecific assignment of individual H2¢ and H2¢¢ could be achieved by intensity comparison of the H1¢-H2¢ and H1¢-H2¢¢ cross-peaks [19] in the NOESY spectrum, wherein the latter was found to be stronger than the former. Intra-base pair NOESY cross peaks G 6 (H1)–C 21 (H41/ H42), G 7 (H1)–C 20 (H41/H42), A 8 (H2)–T 19 (H3), T 9 (H3)– A 18 (H2), C 10 (H41/H42)–G 17 (1NH) and C 11 (H41/H42)– G 16 (H1) establish a hydrogen bonded base-pairing between G 6 :C 21 (G 6 and C 21 ), G 7 :C 20 ,A 8 :T 19 ,T 9 :A 18 , C 10 :G 17 and C 11 :G 16 and hence the conformation of stems of both of the hairpins. Qualitative analysis of the relative NOESY cross-peak intensities established t hat the stems of both the hairpins adopt a right-handed B-DNA duplex conformation. The nOe data further confirm the association of A : T and G : C base pairs through Watson and Crick base-pairing schemes with almost all of the individual bases in both the stems adopting the anti conformation with the glycosidic torsion angle, v, ranging from )80° to )120°. This is based on the observation of strong intra-nucleotide H2¢-H6/H8 cross-peaks compared with H2¢-H6/H8 cross-peaks, while H1¢-H6/H8 cross-peaks are relatively weak or absent. In the case of C 10 ,C 11 ,C 20 and C 21 we could not establish the respective v-values for either of the hairpins bec ause of the severe spectral overlap of H1¢/H2¢/H2¢-H6 cross-peaks. Most of the expected sequential nOes are seen all along both the nucleotide stretches. By the end of the assignment procedure, all of the major cross-peaks in the two-dimensional spectra could be assigned uniquely. The nOe interactions seen in individual loop regions essentially govern the folding pattern of respective loops, which will be discussed later. Conformation-dependent characteristic multiplet struc- tures of H2¢-H1¢ and H 2¢¢-H1¢ cross-peaks in the E-COSY spectra of both the hairpins have been used to estimate values 3 J(H1¢-H2¢)and 3 J(H1¢-H2¢¢) [19–22]. As discussed earlier [4], for both of the hairpins, these J-values qualita- tively indicate that the corresponding sugar rings adopt conformation in the S domain of the pseudo-rotational m ap with P ranging from C1 ¢-exo to C3¢-exo (P ¼ 90–198°). NMR structure determination of U1- and U3-hairpins Restrained molecular dynamics simulation and energy minimization calculations were performed o n both U1- a nd U3-hairpins following the procedure described in Materials and metho ds. In th e case of the U1-hairpin, a total of 227 inter-proton distance constraints ( 10 involving exchangeable protons and 217 involving nonexchangeable protons) and 64 dihedral angle restraints were used with the force constants described earlier. All of these constraints have been deposited in the Protein Data Bank (PDB accession no. 1II1; RCSB ID Fig. 1. Selected regions of pure-absorption NOESY spectra of (A) the U1-hairpin and (B) the U3-hairpin recorded in 99.9% 2 H 2 O at 305 K and pH 7, showing intra-strand inter-residue nOe connectivities: CH 3 /H2¢/H2¢¢ protons to H6/H8 protons. Experimental parameters were: s m ¼ 250 ms, recycle delay 1 s, 64 scans per t 1 increment, time-domain data points were 800 and 4096 along t 1 and t 2 dimensions, r espectively . The 1 H-carrier frequency was kept at water resonance. The data were multiplied with sin e-bell window functions sifted by p/4 and p/8 along t 1 and t 2 axes, respectively, and zero-filled to 1024 data p oints along the t 1 dimension prior to two-dimensional Fourier transmformation. The digital resolution along x 1 and x 2 axes, corresponds to 5.84 and 1.46 HzÆpt )1 , respectively. Ó FEBS 2002 Structural basis for poor uracil excision (Eur. J. Biochem. 269) 1889 RCSB013285; http://www.pdb.bnl.gov/). Of the 200 calcu- lated structures, there are nine structures lying within 2.5 kcalÆmol )1 of the minimum energy structure. These 10 structures are characterized by low all-atom pair-wise rmsds in the range 0.25–1.41. Fig. 2 A shows the best-fit super- imposition o f these 10 structures. The corresponding PDB files have been deposited in th e Protein Data Bank (PDB ID 1II1; RCSB ID RCSB013285). In the case of the U3-hairpin, a total of 132 inter-proton distance constraints (10 involving exchangeable protons and 122 involving nonexchangeable protons) and 64 dihedral angle restraints were used with the force constants described earlier. All of these constraints have been deposited in the Protein Data Bank (PDB accession no. 1IDX; RCSB ID RCSB013191). Of the 200 calculated structures, there are five structures lying within 2.5 kcalÆmol )1 above the minimum energy structure. These six structures are characterized by low all- atom pair-wise rmsds ranging f rom 0.45 to 1.30. Fig. 2B shows the best-fit superimposition of these six structures. The corresponding PDB files have been deposited in the PDB (PDB ID 1IDX; RCSB ID RCSB013191). Even though only three torsion angles, namely -C2¢-C3¢- C4¢-O4¢-, -C1¢-C2¢-C3¢-C4¢- and glycosidic torsion angle (v) were constrained both in the case of U1- and U3-hairpins, the structures still converged mostly into a narrow range of torsion angles at the end of molecular dynamics simulation. The 31 P chemical shifts and 31 P– 1 H vicinal coupling constants, which would have helped in further restraining some of the backb one torsion angles (b, c and e), suffer from extensive s pectral overlaps. The stereo-chemistry of a ll 10 of the U1-hairpin structures and all six U3-hairpin structures mentioned above, were critically examined for correct hydrogen-bond lengths and angles in the Watson– Crick base-pairs, stereochemical feasibility of the various dihedral angles and any sterically hindered nonbonded interatomic distances. All of these structures satisfied these criteria. Backbone torsion angles in U1- and U3-hairpins U1-hairpin. The a, b, c,ande for each nucleotide in the stem of the U1-hairpin DNA in all the 10 structures 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 G 16 ,whichis at the 3¢ end of the tetra-loop. For this, a angle ranges from Fig. 2. Stereoviews showing a best-fit super- imposition of the final molecular dynamics and energy minimized simulated structures of (A) the U1-hair pin and (B) the U3-hairpin. 1890 M. Ghosh et al. (Eur. J. Biochem. 269) Ó FEBS 2002 136 to 153°.Thef-values adopt 104.5° on average and r ange from 66 to 108° for all of the residues. The d-values adopt 141.5° on an average ranging from 127 to 156°.Inthecase of the tetra-loop, it is interesting to note that the b, c,ande of both the T 14 and T 15 nucleotide units get locked into t, g + and t conformations, respectively, similar to the stem. On the other hand, for T 14 and T 15 ,thef is locked into g – conformation whereas the d adopts 148 and 110 ° on average, respectively, similar to those observed in B-DNA. AsfarasT 13 is concerned, the a, b, c,ande are locked into g + , g – , t,andt conformations, respectively. The most striking observation of the loop conformation concerns the backbone dihedral angles of U 12 For t his, a, b and f adopt unusual torsional angle values, namely 130, 88.5 and )87°, respectively, whereas c, d, e adopt those values that are observed in B-DNA. The dihedral angles that facilitate the loop formation are a of U12, c of T 13 and a of T 14 ,allof which adopt t conformation and thus stretch the backbone. U3-hairpin. The a, b, c and e for each nucleotide in the stem of the U3-hairpin DNA in all six of the structures are mostly locked in g – , t, g + and t, conformations, respectively, similar to those observed in B-DNA. The exceptions are in the sequence T 19 –C 21 . In this region, the a of T 19 ,C 20 and C 21 adopt values within the range 131–148°,whereasthec of T 19 and C 20 are unusually in t conformation. The f-values adopt )96° on average and range from )64 to )12 8° for all of the r esidues. The d-values adopt 116° on average ranging from 93 to 139°. In the case of the t etra-loop, the a, b and e of T 12 ,T 13 ,U 14 and T 15 nucleotides mostly get locked into g – , t,andt conformations, respectively, similar to the stem. The dihedral angles that facilitate the loop formation are b of T 12 and T 13 , c of T 13 and c, e and f of U14 and perhaps be to a certain extent c of T 15 , all of which adopt t conformation and thus stretch the backbone. Sugar puckers, glycosidic torsion angles and turning phosphates in U1- and U3-hairpins U1-hairpin. In all 10 structures, the sugar puckers lie in the S domain of the pseudo-rotational wheel with the P angle in the range of 122–152°. The exception is the sugar of T 13 which adopts the O4¢-endo pucker. This is supported by the observation of strong intra-nucleotide nOes between H1¢ and H4¢ for these nucleotides [21]. A different behaviour for this nucleotide could be expected , as it is present in the loop region of the hairpin DNA. As far as the v is concerned, almost all nucleotide units are in the anti domain, as evident in the relative intensities of the resolved nOes between the base and the sugar protons. The v-values range from )100 to )127°. The exception is again in the case of T 13 ,which adopts syn conformation, with an v-value of 38.5° on Fig. 3. NOESY cross-peaks as seen in the individual NOESY spectra of U1-, U2- and U3-hairpins, each recorded with a mixing time of 100 ms. (A) H2¢/H2¢-UH6 cross peaks (B) H5/H1¢-UH6 cross peaks. Ó FEBS 2002 Structural basis for poor uracil excision (Eur. J. Biochem. 269) 1891 average. As mentioned earlier, the c of T 13 and a of T 14 ,are characteristically in ÔtÕ conformation. Because of this, the backbone takes a sharp bend near the phosphate linking T 13 and T 14 . Similar phosphodiester conformations were found for the turning phosphates in the case of U2- and U4-hairpins and CGTTTTCG-type hairpins [23,24]. In the present study, t he simulated model reveals that the turning phosphate is indeed in between T 13 and T 14 . U3-hairpin. In all the six structures, the sugar puckers lie in the S domain of the pseudo-rotational wheel and most of the nucleotides assume a sugar pucker in the range of 93°)155°. All those nucleotides which adopt O4¢-endo puckers show a strong intra-nucleotide nOe which is expected between the H1¢ and H4¢ [21]. As far as the v-value is concerned, almost all the nucleotide units are in the ÔantiÕ domain, as are evident in the relative intensities of the resolved nOes between the base and the sugar protons. The v-values range from )116 to )160°. The exceptions are in the case of C 11 and T 13 which adopt )58 .5 and )14.5°, respectively, on an average. As mentioned earlier, the c of T 13 , c, e and f for U 14 and c of T 15 are characteristically in ÔtÕ conformation. Because of this, the backbone takes a sharp swerve near the phosphate linking T 13 and U 14 . Similar phosphodiester conformations were found for the turning phosphates in the case of U1, U2- and U 4-hairpins [4,9] and CGTTTTCG-type hairpins [23,24]. In the present study, the simulated model reveals that the turning phosphate is indeed between T 13 and U 14 . Comparison of U1 and U3-hairpin structure with U2-hairpin DNA It is interesting to compare the three-dimensional structure of U1- and U3-hairpins with that of U2-hairpin [4]. All the stems of U1, U2 and U3 are found to contain Watson– Crick base pairs adopting a right-handed B-DNA confor- mation. Besides, interesting common features are noted regarding the conformation of the loop of these hairpins. In all the 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 loops are characterized by the stacking of individual bases (T) d (T/U) c (The nucleotide T or U at the position ÔcÕ of the tetra-loop from the 3¢ to p of the stem), and (U/T) b over the 5¢ top of the stem as seen earlier in the case of CGTTTTCG-type hairpins [23,24]. These fi ndings are c onsistent with the observed inter- nucleotide n Oes in each case. The most striking feature of U1- and U3-hairpin loops, however, is the base conforma- tion of U nucleotides (U 12 and U 14 , respectively), which adopt an anti conformation with respect to their sugar moiety. As for U2-hairpin the U 13 base adopts a syn conformation. These observations are supported by the volumes of intra-nucleotide base-sugar (H6–H1¢/H2¢/H2¢) nOes seen in respective NOESY spectra (Fig. 3A,B). Unfavourable nucleotide conformation results in poor uracil excision rate As mentioned earlier, while comparing the three-dimen- sional structures of U2- and U4-hairpins [4], it was suggested that the stretched-out backbone conformation in the vicinity of U 13 in the U2-hairpin could be as the reason for the enzyme not being able to make proper contacts with the backbone. Recent three-dimensional structural analysis of the UDGs from human and E. coli [25,26] have demonstrated that the UDG establishes contacts with the DNA backbone through several hydro- gen bonds to the highly conserved serine residues, which are present in the active site s of the enzymes. It is also of interest that in the conformation of DNA in the UDG– DNA cocrystal structure [27], the position where the U is located gets kinked. During this kinking the interphosphate (flanking the U residue) distance is compressed by 4 A ˚ [27]. This implies that the phosphates present on the either end (5¢ and 3¢) of U are important in substrate recognition by UDG. The m ost striking feature of U1- and U3-hairpin structures, in the vicinity of respective U, i s in their backbone conformations that are partially in stretched out form (Fig. 4) as was seen in the case of U2-hairpin [ 4]. Such stretched-out conformation could be the reason why Fig. 4. Expanded loop regions of (A) the U1-hairpin and (B) the U3-hairpin. 1892 M. Ghosh et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the observed values of K m are poor for both U1 and U3-hairpins as in the case of U2-hairpin. On the other hand, as described earlier both the U 12 and U 14 bases in both U1- and U3-hairpins adopt an anti conformation (Fig. 4) in contrast with the base conforma- tion of U 13 in the U2-hairpin, which adopts syn conforma- tion [4]. Thus, such marked discrepancy observed in the U-base orientation with respect to the sugar moieties could be the reason why the V max is almost 10- to 20-fold lower f or the U2-hairpin compared with the U1-, and U3-hairpins. Further, it is worth m entioning here that U 15 of the U4-hairpin, which is the best substrate of all of the four hairpin DNA structures, is located in an environment wherein the backbone as well as the base conformation mimic the B-form of DNA [5]. Thus, taken together, these observations support our interpretation that the unfavour- able backbone results in poor K m , whereas the unfavourable nucleotide conformation results in poor V max and jointly these parameters make U1- and U3-hairpins better substrates for UDG than U2-hairpins. ACKNOWLEDGEMENTS The f acilities 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. REFERENCES 1. Krokan, H.E., Standal, R. & Slupphaug, G. (1997) DNA glycosylases in the b ase excision r epair of DNA. Biochem. J. 325, 1–16. 2. Kumar, N.V. & Varshney, U. (1997) Contrasting effects of single stranded DNA binding protein on the activity of uracil DNA glycosylase from Escherichia coli towards different DNA sub- strates. Nucl. Acids Res. 25, 2336–2343. 3. Kumar, N.V. (1999) PhD Thesis. M echanism of uracil excision from different structural contexts of DNA oligomers b y E. coli uracil DNA glycosylase and its applicatio ns’ submitted, Septem- ber 1997. Indian Institute of Science, Bangalore. 4. Ghosh, M., Kumar, N.V., Varshney, U . & Chary, K.V.R. (2000) Structural basis for uracil DNA glycosylase interaction with uracil: NMR study. Nucleic Acids Res. 28, 1906–1912. 5. Ghosh, M., Kumar, N.V., Varshney, U. & Chary, K.V.R. (1999) Structural characterization of a uracil containing hairpin DNA by NMR and molecular dynamics. N ucleic Acids Res. 27, 3938–3944. 6. Hore, P.J. (1983) S olvent supp ression in Fourier transform NMR. J. Magn. Res. 55, 283–300. 7. Kumar, A., Wagner, G., Ernst, R.R. & Wuthrich, K . ( 1980) Studies of J-connectives and sele ctive 1 H- 1 HOverhausereffectsinH 2 O solutions of biological ma cromolecules by two-dimensional NMR experiments. Biochem. Biophys. Res. Commun. 96, 1156–1163. 8. Griesinger, C., Sorensen, O.W. & Ernst, R.R. (1986) Correlation of co nnec ted transitions by two-dimensional NMR-spectro scopy. J. Chem. Phys. 85, 6837. 9. Grieseinger, C., Otting, G., Wuthrich, K. & Ernst, R.R. (1988) Clean-TOCSY for 1 H spin system identification in macro- molecules. J. Am. Chem. Soc. 110, 7870–7872. 10. Kumar, A., Wagner, G., Ernst, R.R. & Wuthrich, K. (1981) Build-up rates o f the nuclear Ove rhauser effects measured b y two-dimensional proton magnetic resonance spectroscopy: Implications for studies of protein conformation. J. Am. C hem . Soc. 103, 3654–3658. 11. Wagner, G. & Wuthrich, K. (1979) Truncated driven nuclear Overhauser effect (TOE). A new technique for studies of selective 1 H- 1 H Overhausser effects in the presence of spin diffusion. J. Magn. Reson. 33, 675–680. 12. Chary, K.V.R., Hosur, R.V., Govil, G., Chen, C. & Miles, H.T. (1988) Sequence-specific solution structure of d -GGTACGC TACC. Biochemistry 27, 3858–3867. 13. Cornell, W.D., Cieplak, P., Bayly, C.I., G ould, I.R., M erz, K.M., Ferguson, D.M., Spellmeyer, D.C., Fox, T., Caldwell, J.W. & Kollman, P.A. (1995) A second generation force field for the simulation of proteins and nucleic acids. J. Am. Chem. Soc. 117, 5179–5197. 14. Feigon, J., Leupin, W., Denny, W.A. & Kearns, D.R. (1983) Two- dimensional proton nuclear magnetic resonance investigation of the s ynthetic deoxyribonucleic acid decamer d-(ATATCGATAT) 2. Biochemistry 22, 5943–5951. 15. Scheek, R.M., Boelens, R., Russo, N., van Boom, J.H. & Kaptein, R. (1984) Sequential resonance assignments in 1 HNMRspectraof oligonucleotides by two-dimensional NMR spectroscopy. Bio- chemistry 23, 1371–1376. 16. Wuthrich, K. (1986) NMR of Proteins and Nucleic Acids.John Wiley and Sons, New York. 17. Reid, B.R. (1987) Sequence-specific assignments and their use in NMR studies of D NA stru cture. Quart. Rev. Biophys. 20, 1–34. 18. Chary, K .V.R., Hosur, R.V., Govil, G., Tan, Z K. & Miles, H.T. (1987) Novel solution conformation DNA observed in d (GAATTCGAATTC) by two-dimensional NMR spectroscop y. Biochemistry 26, 1315–1322. 19. Chary, K.V.R., Hosur, R.V., Govil, G., Chen, C. & Miles, H.T. (1989) Quantification of D NA structure from NMR data: con- formation of d-ACATCGATGT. Biochemistry 28, 5240. 20. Chary, K.V.R. (1991) Magnetic Resonance–Current Trends. Narosa Publishing House, New Dehli, 71–104. 21. Chary, K.V.R. & Modi, S. (1988) Analysis of intrasugar inter- proton NOESY c ross-peaks a s an aid to determine sugar geo- metries in DNA fragments. FEB S Lett . 233, 319–325. 22. Rinkel, L.J. & A ltona, C. (1987) Conformational analysis of the deoxyribofuranose ring in DNA by means o f sums of proton– proton coupling constants: a graphical method. J. Biomol. Struct. Dyn. 4, 621–649. 23. Hare, D.R. & Reid, B.R. (1986) Three-dimensional structure of a DNA hairpin in solution: two-dimensional NM R studies and distance calculations on d (CGCGTTTTCGCG). Biochemistry 25, 5341–5350. 24. Baxter,S.M.,Greizerstein,M.B.,Kushlan,D.M.&Ashley,G.W. (1993) Conformational properties of DNA hairpins with TTT and TTTT loops. Biochemistry 32, 8702–8711. 25. Parikh, S.S., Putnam, C.D. & Tainer, J.A. (2000) Lessons learned from structural results on uracil-DNA glycosylase. Mutation Res. 460, 183–199. 26. Pearl, L.H. (2000) Structure and function in the uracil-DNA glycosylase superfamily. Mutation Res. 460, 165–181. 27. Parikh, S.S., Mol, C.D., Slupphaug, G., Bharati, S., Krokan, H.E. & Tainer, J.A. (1998) Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA. EMBO J. 17, 5214–5226. SUPPLEMENTARY MATERIAL The following material is available from http://www.black well-science.com/products/journals/suppmat/EJB/ EJB2837/EJB2837sm.htm Table S1. 1 H NMR chemical shifts of exchangable and nonexchangable protons in the U1-hairpin. Ó FEBS 2002 Structural basis for poor uracil excision (Eur. J. Biochem. 269) 1893 Table S2. 1 H NMR chemical shifts of exchangeable a nd nonexchangable protons in U3-hairpin. Table S3. All atom pair-wise rmsds among the 10 lowest energy structures of the U1-hairpin. Table S4. Mean values with SD of all the backbone torsion angles and glycosidic torsion angles for all the 10 structures of U1-hairpin. Table S5. All atom pair-wise rmsds among the six lowest energy structures of U3-hairpin. Table S6. Mean values with SD of all the backbone torsion angles and glycosidic torsion angles for all the six structures of U3-hairpin. Table S7. Integral volumes o f intranucleotide base-sugar (H6–H1¢/H2¢/H2¢) nOes seen in the respective NOESY spectra of U1 and U2 and U3-haiprins. 1894 M. Ghosh et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Structural basis for poor uracil excision from hairpin DNA An NMR study Mahua Ghosh 1 , Nidhi Rumpal 2 , Umesh Varshney 2 and Kandala V. R. Chary 1 1 Department. K m are poor for both U1 and U3-hairpins as in the case of U2 -hairpin. On the other hand, as described earlier both the U 12 and U 14 bases in both U1- and U3-hairpins adopt an anti conformation. of that for SS-U4. d Single-stranded oligonucleotide with uracil at the fourth position from the 5¢ end. Ó FEBS 2002 Structural basis for poor uracil excision (Eur. J. Biochem. 269) 1887 For experiments