Báo cáo khoa học: Structural basis for poor uracil excision from hairpin DNA An NMR study pptx

Chary1 1 Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India;2Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalo

Structural basis for poor uracil excision from hairpin DNA

An NMR study

Mahua Ghosh1, Nidhi Rumpal2, Umesh Varshney2and Kandala V R Chary1

1

Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India;2Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India

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) The1H 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

U13in the U2-hairpin Such a stretched-out backbone

con-formation in the vicinity of U12and U14is thought to be the

reason why the Kmvalue is poor for U1- and U3-hairpins as

it is for the U2-hairpin Furthermore, the bases U12and U14

in both U1- and U3-hairpins adopt an anti conformation, in contrast with the base conformation of U13in 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 Vmaxvalue 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 Kmvalue, whereas the unfavourable nucleotide con-formation results in a poor Vmaxvalue 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

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 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 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 (Km) of UDG towards the 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

U13in 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)

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, U15in 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 Vmaxvalue 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

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

M A T E R I A L S A N D M E T H O D S

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 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/8Murea 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 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 45 °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 ( 1.8 mM strand concen-tration or 40 mM in nucleoside residues) with no buffer

5′ CTAGAGGATCC T 5′ CTAGAGGATCC T

5′ CTAGAGGATCC U 5′ CTAGAGGATCC T

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 ma

(· 10)7M )

V maxb

(· 10 2 )

Relative

V max /K mc

Phosphate backbone

in the vicinity of U

Uracil glycosidic torsion angle v

U1-hairpin 39.9 132.0 3.21 Partially stretched Anti

U3-hairpin 22.7 127.9 5.9 Partially stretched 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)1Ælg)1protein.cRelative V max /K m are shown as percentage of that for SS-U4.dSingle-stranded oligonucleotide with uracil at the fourth position from the 5¢ end.

three times from2H2O 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-dimensional1H 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 in2H2O 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°C was used in most of the NMR experiments,

although one-dimensional1H 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

two-dimensional experiments, time domain data points

were 512 and 4096 along t1and t2dimensions, 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-(trimethylsilyl) [3,3,2,2-2H] propionate-d4, which has

been used as an external reference

Starting structure and structural restraints

The starting structures for both U1- and U3-hairpins were

generated using the molecular modelling packageINSIGHT-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 the1H 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-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)2was used

Molecular dynamics and energy minimization methods Molecular dynamics simulations were performed with

DISCOVERsoftware (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

± 0.05 nm and with force constants of 25 kcalÆmol)1ÆA˚)2 for all nOes involving nonexchangeable 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)2was used

R E S U L T S A N D D I S C U S S I O N 1

H NMR assignments and secondary structure

of the U1- and U3-hairpins Sequence-specific1H 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, C20and C21, the1H resonance assignments were straightforward for both of the hairpins The degeneracy between these H6 protons could be resolved by the observation of intra-nucleotide and sequential nOes

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 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° 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 C10, C11, C20

and C21we 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

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 H2¢¢-H1¢ cross-peaks in the E-COSY spectra of both the hairpins have been used to estimate values3J(H1¢-H2¢) and3J(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 ¼ 90–198°) 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

Fig 1 Selected regions of pure-absorption NOESY spectra of (A) the U1-hairpin and (B) the U3-hairpin recorded in 99.9%2H 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, respectively The1H-carrier frequency was kept at water resonance The data were multiplied with sine-bell window functions sifted by p/4 and p/8 along t 1 and t 2 axes, respectively, and zero-filled to 1024 data points 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.

RCSB013285; http://www.pdb.bnl.gov/) Of the 200

calcu-lated structures, there are nine structures lying within

2.5 kcalÆmol)1of 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) 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 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)

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 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 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

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.

136 to 153° The f-values adopt 104.5° on average and range

from 66 to 108° for all of the residues The d-values adopt

141.5° on an average ranging from 127 to 156° In the case

of the tetra-loop, it is interesting to note that the b, c, and e

of both the T14and T15nucleotide 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° on

average, respectively, similar to those observed in B-DNA

As far as T13is 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 U12For this, 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 T13and a of T14, all of

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 T19–C21 In this region, the a of T19, C20and

C21adopt values within the range 131–148°, whereas the c

of T19and C20are unusually in t conformation The f-values

adopt)96° on average and range from )64 to )128° for all

of the residues The d-values adopt 116° on average ranging from 93 to 139° In the case of the tetra-loop, the a, b and e

of T12, T13, U14and T15nucleotides 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 andT13, c of T13and c, e and f of U14 and perhaps be

to a certain extent c of T15, 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 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° The exception is again in the case of T13, 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.

13 14

characteristically in ÔtÕ 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 T13and 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°)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 C11and T13which adopt)58.5 and )14.5°,

respectively, on an average As mentioned earlier, the c of

T13, c, e and f for U14and c of T15are characteristically in ÔtÕ

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 T13and 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 ÔcÕ of the tetra-loop from the 3¢ top of

the stem), and (U/T)bover 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)

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 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 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

The most striking feature of U1- and U3-hairpin structures, in the vicinity of respective U, is 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.

the observed values of Km are poor for both U1 and

U3-hairpins as in the case of U2-hairpin

On the other hand, as described earlier both the U12and

U14 bases in both U1- and U3-hairpins adopt an anti

conformation (Fig 4) in contrast with the base

conforma-tion of U13in 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 Vmaxis almost 10- to 20-fold lower for

the U2-hairpin compared with the U1-, and U3-hairpins

Further, it is worth mentioning here that U15 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 Km, whereas the unfavourable

nucleotide conformation results in poor Vmaxand jointly

these parameters make U1- and U3-hairpins better

substrates for UDG than U2-hairpins

A C K N O W L E D G E M E N T S

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.

R E F E R E N C E S

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S U P P L E M E N T A R Y M A T E R I A L

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

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

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