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

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

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:
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

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.

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.

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

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

)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

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¢

(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

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

)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

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
+

,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

(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.

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

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

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