doi:10.1046/j.1432-1033.2002.02837.x
Eur. J. Biochem. 269, 1886–1894 (2002) (cid:211) FEBS 2002
Structural basis for poor uracil excision from hairpin DNA An NMR study
Mahua Ghosh1, Nidhi Rumpal2, Umesh Varshney2 and Kandala V. R. Chary1 1Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India; 2Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
reason why the Km value is poor for U1- and U3-hairpins as it is for the U2-hairpin. Furthermore, the bases U12 and U14 in both U1- and U3-hairpins adopt an anti conformation, in contrast with the base conformation of U13 in the U2-hair- pin, which adopts a syn conformation. The clear discrepancy observed in the U-base orientation with respect to the sugar moieties could explain why the Vmax 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 Km value, whereas the unfavourable nucleotide con- formation results in a poor Vmax value. These two parame- ters therefore make the U1- and U3-hairpins better 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.
Two-dimensional NMR 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 1H 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 these 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, which partially influence the local conformation of U12 and U14 in U1 and U3-hairpins, respectively, are probably locked in the trans conformation as in the case of U13 in the U2-hairpin. Such a stretched-out backbone con- formation in the vicinity of U12 and U14 is thought to be the
DNA in cells is unceasingly subjected to damages that 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 cells have uracil DNA glycosylase (UDG), which excises U from DNA [1].
the affinity (Km) of UDG towards
The single-stranded regions which arise in DNA during various physiological processes such as replication may adopt complex secondary and tertiary structures. 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 offer an extra- helical situation, wherein U is sometimes in a (cid:212)flipped out(cid:213) 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 the (Scheme I), U2-hairpin (see Table 1 [2]) is found to be substantially lower that that of the 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 Km) towards the enzyme. Yet, U excision from these substrates is relatively more efficient (see Table 1).
In order to gain an insight into such discrepancies in U excision we have carried out structural characterization by NMR of 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 U13 in the U2-hairpin [4,5] is the reason for the enzyme not being able to make appropriate contacts with the backbone.
Correspondence to K. V. R. Chary, Department of Chemical Sciences, Tata Institute of Fundamental 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, uracil 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)
Structural basis for poor uracil excision (Eur. J. Biochem. 269) 1887
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U
T
5¢ CTAGAGGATCC T 3¢ TCCTAGG T
5¢ CTAGAGGATCC T 3¢ TCCTAGG U
T
T
U1-hairpin
U3-hairpin
T
T
5¢ CTAGAGGATCC U 3¢ TCCTAGG T
5¢ CTAGAGGATCC T 3¢ TCCTAGG T
U
T
U2-hairpin
U4-hairpin
reported three-dimensional U2- and U4-hairpin structures [4,5]. This study in turn provides an insight into the interaction of Escherichia coli UDG with U.
Scheme 1.
M A T E R I A L S A N D M E T H O D S
DNA Samples
the existence of
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 32P-labelling by end filling with Klenow polymerase, when required. The oligonucleotides were custom made by Ransom Hill Bioscience, Inc. (Ramona, CA) and purified from 18% polyacrylamide/8 M urea gels [3], desalted on Sep-pak columns and lyophilized. Purified hairpins were examined by gel electrophoresis, which these oligos as monomers. reveals Although the overhangs at the 5¢ ends can trigger the formation of dumb-bells, single hairpins are favoured by the efficient end-filling experiments [3]. Cooperative ther- mal dissociation curves are observed for both of the hairpins (data not shown) with UV (the melting point, Tm (cid:25) 45 (cid:176)C), indicating that the DNA adopts a distinct and ordered conformation below the Tm.
NMR
About 8 mg of purified oligomers were dissolved in 0.6 mL of appropriate solvent ((cid:25) 1.8 mM strand concen- tration or 40 mM in nucleoside residues) with no buffer.
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, U15 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 (Vmax) for U excision in the U2-hairpin is poor. For productive enzyme–substrate complex formation, it is essential that the 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 appropriate 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 Km value, whereas the unfavourable nucleotide conformation results in a poor Vmax 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 U2-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
a
c
Table 1. Kinetic parameters of uracil excision from various DNA substrates and their structural features as derived from NMR data.
M)
b Vmax (· 102)
Substrate Phosphate backbone in the vicinity of U Uracil glycosidic torsion angle v Km (· 10)7 Relative Vmax/Km
100
a Km (dissociation constant) values are for the U residue in the oligonucleotides. b Vmax (excision rate) values are in pmol product formedÆmin)1Ælg)1 protein. c Relative Vmax/Km are shown as percentage of that for SS-U4. d Single-stranded oligonucleotide with uracil at the fourth position from the 5¢ end.
– Anti Syn Anti Anti SS-U4d U1-hairpin U2-hairpin U3-hairpin U4-hairpin 6.57 39.9 40.3 22.7 2.5 675.7 132.0 14.5 127.9 173.5 3.21 0.35 5.9 66.8 – Partially stretched Stretched Partially stretched Resembles B-DNA
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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 connectivities. For all of these torsion constraints a force constant of 20 kcalÆmol)1Ærad)2 was used.
Molecular dynamics and energy minimization methods
For experiments in 2H2O, the oligomers were lyophilized three times from 2H2O to deprotonate all of the exchange- able protons, prior to dissolution in 0.6 mL 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. The spectra in a mixed solvent of 90% H2O/10% 2H2O include one-dimensional 1H NMR spectra recorded with P1¢1 pulse sequence [6] and two-dimensional NOESY [7] with P1¢1 detection pulse sequence and a mixing time of 200 ms. The two-dimensional experiments in 2H2O include exclusive (E)-COSY [8], clean TOCSY [9] with a mixing time of 80 ms and a set of NOESY spectra with different mixing times (ranging from 50 to 350 ms). A temperature of 32 (cid:176)C was used in most of the NMR experiments, although one-dimensional 1H experiments were carried out in the range of 15–55 (cid:176)C. In all the experiments, the 1H-carrier frequency was kept at water resonance. In two-dimensional experiments, time domain data points were 512 and 4096 along t1 and t2 dimensions, respectively. The data multiplied with sine bell window functions shifted by p/4 and p/8 along the t1 and t2 axes, respectively, was zero-filled to 1024 data points along the t1 dimension prior to two-dimensional Fourier transfor- mation (FT). 1H chemical shift calibrations were carried out with respect to the methyl signal (at 0.0 p.p.m.) of [3,3,2,2-2H] propionate-d4, which has 3-(trimethylsilyl) been used as an external reference.
Starting structure and structural restraints
nonexchangeable
involving
nOes
all
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 point 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 performed on the initial structure followed by conjugate gradient minimization of 1000 steps. 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-hundred structures were collected at 1 ps intervals 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 steepest 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 NMR-derived distance restraints were applied throughout with the upper and lower bounds of (cid:139) 0.05 nm and with force constants of 25 kcalÆmol)1ÆA˚ )2 for protons, 10 kcalÆmol)1ÆA˚ )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)1Æ rad)2 was used.
R E S U L T S A N D D I S C U S S I O N
1H NMR assignments and secondary structure of the U1- and U3-hairpins Sequence-specific 1H resonance assignments were achieved by established procedures [14–19]. Fig. 1A and B show illustrative examples of selected NOESY spectral regions of the U1- and U3-hairpins, respectively, with H2¢/H2¢/ CH3–H6/H8 nOe connectivities. Except for the serious overlap seen in the case of H6 resonances belonging to C10, C11, C20 and C21, the 1H resonance assignments were straightforward for both of the hairpins. The degeneracy between these H6 protons could be resolved by the intra-nucleotide and sequential nOes observation of
The starting structures for both U1- and U3-hairpins were generated using the molecular modelling package INSIGHT-II (MSI) on an Iris (Indigo II) workstation as discussed earlier [5]. Distances were estimated from the initial build-up rates of the build-up curves by 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 1H 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)1ÆA˚ )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 kcalÆmol)1ÆA˚ )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 constant of 20 kcalÆmol)1ÆA˚ )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-O5¢-C5¢-), b(-P-O5¢-C5¢-C4¢-), c(-O5¢-C5¢-C4¢-C3¢-), e(-C4¢-C3¢-O3¢-P-) and f(-C3¢-O3¢-P-
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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.
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.
Conformation-dependent characteristic multiplet struc- tures of H2¢-H1¢ and H2¢¢-H1¢ cross-peaks in the E-COSY spectra of both the hairpins have been used to estimate values 3J(H1¢-H2¢) and 3J(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 map with P ranging from C1¢-exo to C3¢-exo (P (cid:136) 90–198(cid:176)).
NMR structure determination of U1- and U3-hairpins
Restrained molecular dynamics simulation and energy minimization calculations were performed on both U1- and U3-hairpins following the procedure described in Materials and methods.
In the 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
Intra-base pair 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 hence the conformation of stems of both of the hairpins. Qualitative analysis of the relative NOESY cross-peak intensities established that 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(cid:176) to )120(cid:176). 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 C10, C11, C20 and C21 we could not establish the respective v-values for either of the hairpins because of the severe spectral overlap of H1¢/H2¢/H2¢-H6 cross-peaks. Most of the expected
Fig. 1. Selected regions of pure-absorption NOESY spectra of (A) the U1-hairpin and (B) the U3-hairpin recorded in 99.9% 2H2O at 305 K and pH 7, showing intra-strand inter-residue nOe connectivities: CH3/H2¢/H2¢¢ protons to H6/H8 protons. Experimental parameters were: sm (cid:136) 250 ms, recycle delay 1 s, 64 scans per t1 increment, time-domain data points were 800 and 4096 along t1 and t2 dimensions, respectively. The 1H-carrier frequency was kept at water resonance. The data were multiplied with sine-bell window functions sifted by p/4 and p/8 along t1 and t2 axes, respectively, and zero-filled to 1024 data points along the t1 dimension prior to two-dimensional Fourier transmformation. The digital resolution along x1 and x2 axes, corresponds to 5.84 and 1.46 HzÆpt)1, respectively.
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In the case of
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 31P chemical shifts and 31P–1H vicinal coupling constants, which would have helped in further restraining some of the backbone torsion angles (b, c and e), suffer from extensive spectral overlaps. The stereo-chemistry of all 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
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. 2A shows the best-fit super- imposition of these 10 structures. The corresponding PDB files have been deposited in the Protein Data Bank (PDB ID 1II1; RCSB ID RCSB013285). 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 from 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).
U1-hairpin. The a, b, c, and e 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 G16, which is at the 3¢ end of the tetra-loop. For this, a angle ranges from
Even though only three torsion angles, namely -C2¢-C3¢- C4¢-O4¢-, -C1¢-C2¢-C3¢-C4¢- and glycosidic torsion angle (v)
Fig. 2. Stereoviews showing a best-fit super- imposition of the final molecular dynamics and energy minimized simulated structures of (A) the U1-hairpin and (B) the U3-hairpin.
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adopt )96(cid:176) on average and range from )64 to )128(cid:176) for all of the residues. The d-values adopt 116(cid:176) on average ranging from 93 to 139(cid:176). In the case of the tetra-loop, the a, b and e of T12, T13, U14 and T15 nucleotides mostly get locked into g–, t, and t conformations, respectively, similar to the stem. The dihedral angles that facilitate the loop formation are b of T12 and T13, c of T13 and c, e and f of U14 and perhaps be t to a certain extent c of T15, all of which adopt conformation and thus stretch the backbone.
Sugar puckers, glycosidic torsion angles and turning phosphates in U1- and U3-hairpins
136 to 153(cid:176). The f-values adopt 104.5(cid:176) on average and range from 66 to 108(cid:176) for all of the residues. The d-values adopt 141.5(cid:176) on an average ranging from 127 to 156(cid:176). In the case of the tetra-loop, it is interesting to note that the b, c, and e of both the T14 and T15 nucleotide units get locked into t, g+ and t conformations, respectively, similar to the stem. On the other hand, for T14 and T15, the f is locked into g– conformation whereas the d adopts 148 and 110(cid:176) on average, respectively, similar to those observed in B-DNA. As far as T13 is concerned, the a, b, c, and e are locked into g+, g–, t, and t conformations, respectively. The most striking observation of the loop conformation concerns the backbone dihedral angles of U12 For this, a, b and f adopt unusual torsional angle values, namely 130, 88.5 and )87(cid:176), 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 T13 and a of T14, all of which adopt t conformation and thus stretch the backbone.
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(cid:176). The exception is the sugar of T13 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(cid:176). The exception is again in the case of T13, which adopts syn conformation, with an v-value of 38.5(cid:176) on
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 T19–C21. In this region, the a of T19, C20 and C21 adopt values within the range 131–148(cid:176), whereas the c of T19 and C20 are unusually in t conformation. The f-values
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.
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average. As mentioned earlier, the c of T13 and a of T14, are characteristically in (cid:212)t(cid:213) conformation. Because of this, the backbone takes a sharp bend near the phosphate linking T13 and T14. 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, the simulated model reveals that the turning phosphate is indeed in between T13 and T14.
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(cid:176))155(cid:176). 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 (cid:212)anti(cid:213) 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(cid:176). The exceptions are in the case of C11 and T13 which adopt )58.5 and )14.5(cid:176), respectively, on an average. As mentioned earlier, the c of T13, c, e and f for U14 and c of T15 are characteristically in (cid:212)t(cid:213) conformation. Because of this, the backbone takes a sharp swerve near the phosphate linking T13 and U14. Similar phosphodiester conformations were found for the turning phosphates in the case of U1, U2- and U4-hairpins [4,9] and CGTTTTCG-type hairpins [23,24]. In the present study, the simulated model reveals that the turning phosphate is indeed between T13 and U14.
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 (cid:212)c(cid:213) of the tetra-loop from the 3¢ top 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 findings are consistent with the observed inter- nucleotide nOes in each case. The most striking feature of U1- and U3-hairpin loops, however, is the base conforma- tion of U nucleotides (U12 and U14, respectively), which adopt an anti conformation with respect to their sugar moiety. As for U2-hairpin the U13 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).
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 sites 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.
Unfavourable nucleotide conformation results in poor uracil excision rate
The most striking feature of U1- and U3-hairpin structures, is in their in the vicinity of respective U, 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
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 U13 in the U2-hairpin could be as the
Fig. 4. Expanded loop regions of (A) the U1-hairpin and (B) the U3-hairpin.
Structural basis for poor uracil excision (Eur. J. Biochem. 269) 1893
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the observed values of Km are poor for both U1 and U3-hairpins as in the case of U2-hairpin.
Implications for studies of protein conformation. J. Am. Chem. Soc. 103, 3654–3658.
11. Wagner, G. & Wuthrich, K. (1979) Truncated driven nuclear Overhauser effect (TOE). A new technique for studies of selective 1H-1H 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., Gould, I.R., Merz, 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.
On the other hand, as described earlier both the U12 and U14 bases in both U1- and U3-hairpins adopt an anti conformation (Fig. 4) in contrast with the base conforma- tion of U13 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 Vmax is almost 10- to 20-fold lower for the U2-hairpin compared with the U1-, and U3-hairpins. it is worth mentioning here that U15 of the Further, 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 Km, whereas the unfavourable nucleotide conformation results in poor Vmax and jointly these parameters make U1- and U3-hairpins better substrates for UDG than U2-hairpins.
14. Feigon, J., Leupin, W., Denny, W.A. & Kearns, D.R. (1983) Two- dimensional proton nuclear magnetic resonance investigation of the synthetic 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 1H NMR spectra of 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.
A C K N O W L E D G E M E N T S
17. Reid, B.R. (1987) Sequence-specific assignments and their use in NMR studies of DNA structure. 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 spectroscopy. Biochemistry 26, 1315–1322. 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.
19. Chary, K.V.R., Hosur, R.V., Govil, G., Chen, C. & Miles, H.T. (1989) Quantification of DNA structure from NMR data: con- formation of d-ACATCGATGT. Biochemistry 28, 5240.
R E F E R E N C E S
20. Chary, K.V.R. (1991) Magnetic Resonance–Current Trends. Narosa Publishing House, New Dehli, 71–104.
1. Krokan, H.E., Standal, R. & Slupphaug, G. (1997) DNA glycosylases in the base excision repair of DNA. Biochem. J. 325, 1–16. 21. Chary, K.V.R. & Modi, S. (1988) Analysis of intrasugar inter- proton NOESY cross-peaks as an aid to determine sugar geo- metries in DNA fragments. FEBS Lett. 233, 319–325.
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. 22. Rinkel, L.J. & Altona, C. (1987) Conformational analysis of the deoxyribofuranose ring in DNA by means of sums of proton– proton coupling constants: a graphical method. J. Biomol. Struct. Dyn. 4, 621–649.
3. Kumar, N.V. (1999) PhD Thesis. Mechanism of uracil excision from different structural contexts of DNA oligomers by E. coli uracil DNA glycosylase and its applications’ submitted, Septem- ber 1997. Indian Institute of Science, Bangalore. 23. Hare, D.R. & Reid, B.R. (1986) Three-dimensional structure of a DNA hairpin in solution: two-dimensional NMR studies and distance calculations on d (CGCGTTTTCGCG). Biochemistry 25, 5341–5350.
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. 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.
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. Nucleic Acids Res. 27, 3938–3944. 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. 6. Hore, P.J. (1983) Solvent suppression in Fourier transform NMR. 26. Pearl, L.H. (2000) Structure and function in the uracil-DNA J. Magn. Res. 55, 283–300. 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.
S U P P L E M E N T A R Y M A T E R I A L
7. Kumar, A., Wagner, G., Ernst, R.R. & Wuthrich, K. (1980) Studies of J-connectives and selective 1H-1H Overhauser effects in H2O solutions of biological macromolecules by two-dimensional NMR experiments. Biochem. Biophys. Res. Commun. 96, 1156–1163. 8. Griesinger, C., Sorensen, O.W. & Ernst, R.R. (1986) Correlation of connected transitions by two-dimensional NMR-spectroscopy. J. Chem. Phys. 85, 6837.
The following material is available from http://www.black well-science.com/products/journals/suppmat/EJB/ EJB2837/EJB2837sm.htm Table S1. 1H NMR chemical shifts of exchangable and nonexchangable protons in the U1-hairpin.
9. Grieseinger, C., Otting, G., Wuthrich, K. & Ernst, R.R. (1988) Clean-TOCSY for 1H 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 of the nuclear Overhauser effects measured by spectroscopy: two-dimensional proton magnetic resonance
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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 of intranucleotide base-sugar (H6–H1¢/H2¢/H2¢) nOes seen in the respective NOESY spectra of U1 and U2 and U3-haiprins.
Table S2. 1H NMR chemical shifts of exchangeable and 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.
