4696–4705 Nucleic Acids Research, 1998, Vol. 26, No. 20
 1998 Oxford University Press
Intercalated cytosine motif and novel adenine clusters
in the crystal structure of the Tetrahymena telomere
Li Cai, Liqing Chen, Sridharan Raghavan, Robert Ratliff1, Robert Moyzis1 and
Alexander Rich*
Department of Biology, Room 68-233, Massachusetts Institute of Technology, Cambridge, MA 02139, USA and
1Center for Human Genome Studies, Los Alamos National Laboratories, Los Alamos, NM 87545, USA
Received June 12, 1998; Revised and Accepted August 9, 1998
ABSTRACT
The cytosine-rich strand of the Tetrahymena telomere
consists of multiple repeats of sequence d(AACCCC).
We have solved the crystal structure of the crystalline
repeat sequence at 2.5 Å resolution. The adenines form
two different and previously unknown clusters (A
clusters) in orthogonal directions with their counterparts from other strands, each containing a total of
eight adenines. The clusters appear to be stable
aggregates held together by base stacking and three
different base-pairing modes. Two different types of
cytosine tetraplexes are found in the crystal. Each
four-stranded complex is composed of two intercalated parallel-stranded duplexes pointing in opposite
directions, with hemiprotonated cytosine-cytosine
(C·C+) base pairs. The outermost C·C+ base pairs are
from the 5′-end of each strand in one cytosine tetraplex
and from the 3′-end of each strand in the other. The A
clusters and the cytosine tetraplexes form two alternating stacking patterns, creating continuous base
stacking in two perpendicular directions along the xand z-axes. The adenine clusters could be
organizational motifs for macromolecular RNA.
PDB accession no. 294D
other in an antiparallel fashion (9). The first crystal structure of
a C-rich sequence d(C4) confirmed the novel I motif and revealed
more detailed structural information (10). Subsequently, several
additional crystal studies of sequences with cytosine stretches
have also revealed the I motif and showed structural variation
among different sequences (11–13).
In these sequences, the bases attached to the cytosine tetraplex
have shown a great degree of structural variability. In the
metazoan telomeric sequence d(TAACCC), a stabilized loop was
formed by TAA. However, in the Tetrahymena telomeric
sequence, d(AACCCC), the structure displays a novel structural
motif: the adenine cluster (A cluster). The adenines located at the
5′-end of each strand form two different types of A clusters, with
three stacking base pairs in one and four stacking base pairs in
another. Three different base pairing modes are involved. The
stacked A·A base pairs in each A cluster also stack upon the two
different types of cytosine tetraplexes in orthogonal directions to
form alternating A cluster–C tetraplex base stacking continuously
along the x- and z-axes. These features have some similarities
with another recently solved structure d(AACCC) (L.Chen,
L.Cai, Q.Gao and A.Rich, in preparation). There are two cytosine
tetraplexes in an asymmetric unit, however, there are significant
differences in their geometries.
MATERIALS AND METHODS
INTRODUCTION
Telomere DNA located at chromosome ends with many repeating
sequences plays a vital role in chromosomal stability (1,2). It is
important in both the normal control of cell proliferation and the
abnormal growth of cancer (3). The first telomere DNA was
isolated from the ciliate Tetrahymena thermophila in the early
1970s (4). Its G-rich strand contains repeats of a short sequence,
d(GGGGTT), and its complementary C-rich strand contains
repeated d(AACCCC). Both of these repeating segments can
exist as four-stranded molecules as well as in DNA duplex form.
It has long been known that polymers containing cytosine can
form three hydrogen bonds with another cytosine if they are
hemiprotonated (5–8). More recent NMR experiments on d(TC5)
and related sequences yielded an unusual structural motif: an
intercalated tetraplex (I motif), in which the same C·C+ pairings
were seen in two parallel-stranded duplexes intercalated into each
The oligodeoxyribonucleotide d(AACCCC) was synthesized on
an Applied Biosystem DNA synthesizer. It was then purified by
HPLC with a linear gradient of 5–40% acetonitrile in 0.1 M
triethylammonium acetate buffer, pH 7.0. Crystals were grown at
room temperature by vapor diffusion using the sitting drop
method from solutions containing 2.0 mM d(AACCCC) and
100 mM sodium cacodylate buffer adjusted to various pH values
and equilibrated with a reservoir of 70% ammonium sulfate. The
best crystal, measuring 0.3 × 0.2 × 0.1 mm, was obtained with
buffer at pH 7.5. The crystal diffracted to 2.5 Å resolution. It
crystallizes in space group P22121 with cell dimensions a = 35.93,
b = 52.33, c = 76.94 Å. All diffraction data were collected on a
Rigaku R-AXIS II imaging plate system at 4C and processed
with the PROCESS program provided by the Molecular Structure
Corporation. The data set was collected to 2.5 Å resolution, with
64 frames at a crystal-to-plate distance of 120 mm using 4
*To whom correspondence should be addressed. Tel: +1 617 253 4715; Fax: +1 617 253 8699; Email: [email protected]
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oscillations. There were 4628 independent reflections above the
1σ (I) level from 20 to 2.5 Å. Seventy-five percent of the
reflections were observed in the resolution shell between 2.75 and
2.5 Å. Overall completeness from 20 to 2.5 Å is 86.5%. See Table
1 for a summary of crystal data and data collection statistics.
Table 1. Crystallographic data
Crystal data for d(AACCCC)
Space group
P22121
Unit cell
a = 35.93 Å, b = 52.33 Å, c = 76.94 Å
Strands per unit cell
32
Strands per asymmetric unit
8
Summary of data collection statistics
Resolution
20–2.5 Å
Number of observations
33 551
Number of unique reflections
4628
Overall completeness
86.5%
Outermost shell
2.75–2.5 Å
Outermost shell completeness
75%
R-merge
6%
Refinement statistics
Resolution
10–2.5 Å
Number of reflections
4628
Number of non-hydrogen DNA
atoms
836
Number of water molecules
61
RMS bond length
0.016 Å
RMS bond angle
3.7
R-factor
0.21
Free R-factor
0.29
Several I motif crystal structures have been solved using
molecular replacement techniques (11–13). This structure was
also solved by that method using XPLOR (14). The starting
model used the I motifs from the crystal structure of d(AACCC),
which was solved by the single isomorphous replacement and
single anomalous scattering method as the crystal soaked with
HgCl2 was isomorphous to the native crystal (L.Chen, L.Cai,
A.Gao and A.Rich, in preparation). Rotation and translation
searches with that model at various resolution ranges of the
d(AACCCC) diffraction data always led to the same orientation
of the molecule in the lattice. This clearly showed that the
asymmetric unit contained eight independent strands of
d(AACCCC), enough to form two independent cytosine tetraplexes.
The position of the molecule showed that orientation of the helical
axis of one tetraplex was parallel to the x-axis and the helical axis
of the other parallel to the z-axis. This stacking pattern is in
agreement with the native Patterson map of the molecule. After
several cycles of rigid body refinement using 10–2.5 Å data, the
difference map allowed us to identify the missing adenines and
the extra cytosines. We then carried out simulated annealing
refinement, leading to an R-factor of 25.2%. Twenty cycles of
restrained individual isotropic B-factor refinement followed.
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Well-ordered water molecules were then located from the
difference Fourier map (Fo – Fc) and added as oxygen atoms to
the model only if they had a peak height of >3σ in the difference
density map. A total of 61 water molecules were found in this
way. A final round of refinement completed the structural
determination with an R-factor of 0.213 and root mean square
(RMS) deviations from ideal bond lengths and angles of 0.016 Å
and 3.744, respectively. The free R-factor (15) based on a
random subset of 10% of the reflections is 29%. The refinement
statistics are listed in Table 1. The coordinates have been
deposited in the Brookhaven Protein Data Bank (accession
no. 294D).
RESULTS
Two different cytosine tetraplexes
The oligonucleotide d(AACCCC) crystallizes in the orthorhombic
space group P22121. There are eight strands in the asymmetric
unit, enough to form two cytosine tetraplexes. Figure 1a and b
shows tetraplexes 1 and 2, respectively, together with the
adenines. The center of each figure shown is the four cytosines
from four different chains organized into an intercalation motif.
In Figure 1a the cytosine bases stack along the x-axis and in
Figure 1b they stack along the z-axis. There is an average stacking
distance of 3.2 Å between adjacent cytosines from different
strands. The stacking distance of 3.2 Å is in agreement with those
of previously solved C tetraplex structures and it occurs when
stacking is limited to the exocyclic amino and carbonyl groups
and does not involve the pyrimidine rings. The adenines from
each strand project out at the top and bottom, with the planes of
the adenine bases nearly perpendicular to those containing the
cytosine bases (with the exception of A52 and A71). Thus the
adenines in Figure 1a are perpendicular to the z-axis and the
adenines in Figure 1b are perpendicular to the x-axis.
Careful inspection clearly shows that the configurations of the
two cytosine tetraplexes differ in a subtle way. Tetraplex 1
(Fig. 1a), which is oriented along the x-axis, has the outermost
C·C+ layers coming from the 3′-end of the strands. However,
tetraplex 2 (Fig. 1b), which is oriented along the z-axis, has the
outermost C·C+ layers coming from the 5′-end of the strands.
Thus there is a significant variation among the two C tetraplexes.
In each tetraplex, the interaction of two parallel duplexes yields
a quadruplex with two wide and two narrow grooves which, as in
d(C4), are largely symmetrical about the helical axis. The narrow
groove is made up of two closely packed strands in antiparallel
orientation. The two backbone chains fit into each other
remarkably well in a zig-zag fashion. They are so close to each
other that some interchain P–P distances are even shorter than
intrachain ones. In tetraplex 1, the average intrachain P–P
distance is 6.33 Å. The average interchain P–P distance across the
minor groove is 6.36 Å, with the shortest being 5.62 Å. The
average interchain P–P distance across the minor groove for
tetraplex 2 is comparable at 6.81 Å. The minor groove is so
narrow that there is little room left to trap anything. Indeed, we
find no water molecules inside the minor groove.
In contrast, the major grooves are very wide. The average
interchain P–P distances across the major grooves of tetramers 1
and 2 are 16.09 and 15.19 Å, respectively. This symmetric feature
of two broad grooves is very different from that seen in the
metazoan telomeric structure d(TAACCC) (12), where one broad
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Figure 1. Structure of cytosine tetraplexes. (a) Cytosine tetraplex 1 of structure d(AACCCC). (Left) A schematic diagram illustrating the overall configuration of
tetraplex 1. The two strands that are parallel and form hydrogen bonds between their cytosine bases are colored black, while the other two are colored white. (Right)
View into the major groove of tetraplex 1. The major groove is wide and open. The center of the molecule is composed of intercalating cytosine residues held together
by C·C+ base pairs. Note that there are two adenine residues at the 5′-end of each strand and that they project away from the center of the molecule. The outermost
C·C+ base pairs of the tetraplex are from the 3′-end of each strand. (b) Cytosine tetraplex 2 of structure d(AACCCC). (Left) A schematic diagram illustrating the overall
configuration of tetraplex 2. The two strands that are parallel and form hydrogen bonds between cytosine bases are colored black, while the other two are colored white.
Residues with asterisks represent symmetry-related residues (equivalently, we could have chosen the asymmetric unit in such a way that four strands in the asymmetric
unit would form tetraplex 2). (Right) View into the major groove of tetraplex 2. The intercalating motif here is very similar to that of tetraplex 1. However, the outermost
C·C+ base pairs of the tetraplex are from the 5′-end of each strand.
groove is very flat and the phosphate groups in the other broad
groove are rotated away from the center and bend over towards
each other, stabilized by the bridging water molecules between
phosphate oxygens and cytosine N4 groups. Both major grooves
in d(AACCCC) are very flat. Figure 2 shows the flat nature of the
broad grooves with two stacked C·C+ base pairs from tetraplex 2
of d(AACCCC), together with two water molecules that are
within 3.3 Å of the base pairs. It also shows the two very wide,
flat grooves. The heavy hydration with bridging water molecules
between phosphate oxygens and cytosine N4 groups seen in other
structures (11–13) is clearly absent here. In both tetraplexes 1 and
2, the molecules twist slowly in a right-handed manner. The
average twist in both tetraplexes is 16.6, with a standard
deviation of 3.4. Thus, one cytosine base pair is on average
twisted 16.6 relative to its covalent neighbor. This is somewhat
larger than the twist value in d(C4), which is 12.4 (10).
Two adenine clusters
A novel feature of this structure is the presence of two groupings
containing only adenine residues. They provide the interactions
which hold the lattice together. The adenine bases, as shown in
Figure 1, project away from the direction of the cytosine tetraplex.
They form base pairs with adenine residues of neighboring
strands (some of them are symmetry-related), creating two
different kinds of adenine clusters in two orthogonal directions.
Figure 3a is a schematic diagram illustrating the origin of the eight
adenines in A cluster 1, which has bases perpendicular to the
z-axis. There are a total of four base pairs in the cluster, shown in
skeletal models in Figure 3b, stacking on top of each other with
an average stacking distance of 3.5 Å. The increase in stacking
distance from 3.2 Å in C·C+ base pair stacking to 3.5 Å when A·A
base pairs are involved is due to the involvement of aromatic rings
Figure 2. Two adjacent layers of C·C+ base pairs from tetraplex 2 along with two water molecules that are within 3.5 Å of the base pairs. The view is down the axis
of the molecule, which is the z-axis. Unlike structures such as d(AACCC) and d(TAACCC), the broad grooves of this structure are essentially flat and the phosphates
are not bent over. There is also no water molecule bridging the cytosine N4 amino group with the phosphate oxygens on the opposite side of the groove. The absence
of this feature shows the variability of cytosine tetraplexes.
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Figure 3. Adenine clusters of d(AACCCC). (a) A schematic diagram of adenine cluster 1 illustrating the formation of A cluster 1 and its relation to the cytosine residues
of the strands. There are two parallel backbone A·A base pairs, A20*-A30* and A21*-A31*. The other two A·A base pairs, A2*-A11 and A1*-A12, have antiparallel
backbones. Every cytosine portion of the four strands combines with three other symmetry-related cytosines strands (not shown) to form tetraplex 1. Thus, there are
four cytosine tetraplexes 1 connected by A cluster 1. (b) Skeletal view of A cluster 1 connecting four cytosine strands which belong to four different cytosine tetraplexes.
It consists of four stacking A·A base pairs. It stacks on two cytosine tetraplexes 2 (not shown), at both the top and bottom, forming a continuous stacking along the
z-axis. (c) A schematic diagram of A cluster 2. Note there are only three stacking base pairs. The other two bases stack on each other, tilted ∼38 from the other three
base pairs. Like A cluster 1, A cluster 2 connects four cytosine tetraplexes 2. Of the three A·A base pairs, A61-A72 and A41-A51 are parallel while A42-A62 is
antiparallel. (d) Skeletal view of A cluster 2 connecting four cytosine strands which belong to four different cytosine tetraplexes. It has three stacking A·A base pairs
shown at the top. It stacks on two cytosine tetraplexes 1 (not shown), at both the top and bottom, forming continuous stacking along the x-axis. At the lower right,
two stacking bases A52 and A71 are shown.
in stacking. When stacking interactions involve the aromatic
rings, such as is the case in B-DNA, the stacking distance is
generally 3.4–3.5 Å. These four base pairs are in turn sandwiched
between two symmetry-related cytosine tetraplexes running
along the z-axis, from the top to the bottom of the unit cell. The
adenine and cytosine bases effectively show continuous stacking
along the z-axis.
Another adenine cluster, A cluster 2, also made up of eight
adenine residues, has most of the bases perpendicular to the
x-axis. As shown in the schematic diagram of Figure 3c, there are
three A·A base pairs, stacking along the x-axis. The other two
adenine bases, A52 and A71, loosely stack upon each other and
are tilted 38 from the three paired adenines in the cluster.
Figure 3d shows a skeletal view. In a similar way, the three
stacking base pairs in this cluster are also sandwiched between
two symmetry-related cytosine tetraplexes along the x-axis and
this alternating C tetraplex–A cluster stacking pattern creates
continuous stacking along the x-axis.
Three modes of base pairing
Close inspection of Figure 3b and d reveals that the polarities of
the backbones holding the seven base pairs are not the same. In
Figure 3b, base pairs A1*-A12* and A2*-A11* are antiparallel,
while base pairs A20*-A30* and A21*-A31* are parallel. In
Figure 3d, base pairs A51-A41 and A61-A72 are parallel, while
A62-A42 is antiparallel. Among the four parallel A·A base pairs,
there exist all three possible different A·A base pairing modes.
Figure 4a shows base pair A20*-A30*. It is a symmetric A·A
N7–amino group base pairing of the type found in poly(A) fibers
(16) and in yeast tRNAPhe (17). Figure 4b shows base pair
A21*-A31*. It is a symmetric A·A N1–amino group base pairing.
Base pairs A51-A41 and A61-A72 adopt another paring mode,
which is asymmetric N1–amino group, N7–amino group, as
shown in Figure 4c. All the antiparallel base pairs adopt the
asymmetric N1–amino group, N7–amino group base pairing
mode, as illustrated in Figure 4d. The glycosyl conformations in
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the structure are anti for all the cytosine residues. Out of the 16
adenine residues, eight are anti, five are syn and three have almost
clinal conformations. Several modes of sugar pucker are present
in the structure. For cytosines, the most frequent one is C4′-exo,
in nine residues, followed by C2′-endo and C3′-endo, with six
residues apiece. For adenines, the most frequent puckers are
C3′-exo and C2′-endo. Further details will be published
elsewhere.
DISCUSSION
b
c
d
Figure 4. Various A·A base pairs are shown in an electron density map plotted
at 2σ. (a) Base pair A20*-A30* with parallel backbones. It is a symmetric A·A
N7–amino group base pairing. (b) Base pair A21*-A31* with parallel
backbones. It is a symmetric A·A N1–amino group base paring. (c) Base pair
A61-A72 with parallel backbones. It is an asymmetric A·A N1–amino group,
N7–amino group base pairing. Note that A61 is in the syn conformation.
(d) Base pair A42-A62 with antiparallel backbones. It is an asymmetric A·A
N1–amino group, N7–amino group base pairing.
Comparison with previous results
Compared with the previously solved C-rich crystal structures,
d(AACCCC) reveals many interesting features. The fourstranded, intercalated cytosine segment is an extremely stable and
predominant feature of the structure. It is interesting to note that
the crystals were grown over a wide range of pH, ranging from
pH 5.0 to 8.0. The formation of C·C+ base pairs depends on
hemiprotonation of the cytosines (18,6–8). In poly[d(C)], the
hemi-protonated structure was stable up to pH 7 (7). The fact that
crystals of d(AACCCC) can grow at pH 7.5 and 8.0 indicates that
the stable nature of the tetraplex and the packing forces raised the
pK for hemi-protonation to an even higher value. This reinforces
the possibility that the Tetrahymena telomere could adopt the
intercalation motif in vivo at physiological pH, possibly in the
presence of binding proteins.
Aside from the general structural similarity in I motifs, we have
found many variations. One notable difference is the presence of
two different conformations of cytosine tetraplexes. In one
tetraplex, as in all previously reported C tetraplex crystals, the
outermost base pairs are from the 5′-end of each strand; in the
other, however, the outermost base pairs are from the 3′-end of
each strand. This suggests that the two conformations are
energetically comparably favorable, leaving open the possibility
that telomere sequences might adopt either one of the two
conformations, depending on the contributions of the noncytosine residues.
Despite the apparent similarity of all cytosine tetraplex
conformations, each individual strand varies considerably from
structure to structure. The average twists between covalently
linked cytosines vary from 12.4 for d(C4) to 16.6 for
d(AACCCC). When the common I motif portion of the structures
are superimposed, the RMS differences are quite considerable,
especially among the sugar–phosphate backbones. For example,
the RMS difference between tetraplex 1 in d(AACCCC) and
tetraplex 1 in d(C4) is 1.26 Å, with the cytosine bases having an
RMS difference of 0.45 Å while the backbones have one of 1.55 Å.
In all the structures solved, the tetraplexes show considerable
differences from structure to structure and the differences are
mainly due to those between the sugar–phosphate backbones.
The positions of the cytosine bases are relatively stable and often
almost superimposable. This might be expected, given the less
flexible nature of the C·C+ base pairing associated with three
strong planar hydrogen bonds. In contrast, the sugar–phosphate
backbones are intrinsically more flexible, partly due to their lack
of torsional restraints and partly due to strong electrostatic
repulsion between phosphate groups in the narrow grooves.
Where the backbones are close together, they may be stablized by
C-H⋅⋅⋅O hydrogen bonds as well as van der Waals interactions
(19). These variable aspects of the cytosine tetraplex might be
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important if telomere sequences adopt different conformations
under differing biological conditions.
The bridging adenine clusters
Even though there is some variability in the cytosine tetraplex
among different structures, the major variation is seen in the
non-cytosine part of the structure. Unlike the other telomeric
sequence solved, namely the metazoan telomere d(TAACCC) (12),
where the adenine/thymine segment of the structure folds back on
itself to form a stable loop, the adenines in this structure adopt an
entirely different conformation. In this case, the adenines adopt three
different kinds of A·A base pairs and are an essential lattice building
block. There are two adenine residues per strand. In cytosine
tetraplex 1, which points along the x-axis, there are four stacked pairs
of adenine residues. As seen in Figure 1a, these four pairs project
away from the central C tetraplex and the planes of the bases are
perpendicular to the z-axis. Each pair forms an A cluster along with
six other symmetry-related adenine residues, stacking along the
z-axis, connecting two symmetry-related cytosine tetraplexes 2.
A rather interesting three-dimensional network is formed, in
which the adenine clusters play a key role in assembling the
complex (Fig. 5a). C tetraplex 2 and A cluster 1 form continuous
stacking along the z-axis, as illustrated in Figure 5b. The original
four pairs of adenine residues from tetraplex 1 are thus involved
in four A clusters at different symmetry-related locations,
creating four continuous columns of z-axis stacking. In a similar
manner, the four adenine pairs from cytosine tetramer 2 join
cytosine tetramer 1 along the x-axis, forming four continuous
stacking columns along the x-axis, as seen in Figure 5c. In contrast
to the rather rigid cytosine tetraplex, the non-cytosine part of the
telomeric sequences clearly show a great deal of variability and
versatility in forming different structural conformations.
Sequences containing stretches of cytosines and adenines are
found in telomeres (1) and also occur in segments scattered
throughout the genome. They may also exist in large RNAs such
as group I and group II introns and ribosomal and spliceosomal
RNAs. The recent crystal structure of the P4–P6 domain of the
T.thermophila intron (20,21) revealed adenosine platforms in
which two adjacent adenine residues contribute to key
components of the domain tertiary structure. The crystal structure
of the Tetrahymena telomeric sequence d(AACCCC) shows two
different novel adenine clusters that play a key role in building the
crystal lattice and stabilizing the structure. The abundance of
adenosine residues in internal loops of many RNAs and the ability
of A clusters observed in this structure to form stabilized tertiary
structures suggests the possibility that A clusters, like the
adenosine platforms observed in a group I intron fragment, could
be a motif present in large RNAs to facilitate folding and be
responsible for long range tertiary interactions.
This crystal structure shows that the telomeric sequence can
adopt a very different structural conformation from standard
B-DNA. Does this structural conformation occur in vivo? We do
not have the answer yet. The fact that both C-rich sequences and
complementary G-rich sequences can form tetraplexes (22–24)
makes it possible that the two structures could act in concert or
one could promote formation of the other. Such an event could
play an important role in DNA self-recognition, which is essential
in many biological systems (10).
4704 Nucleic Acids Research, 1998, Vol. 26, No. 20
Figure 5. The organization of adenine clusters and cytosine tetraplexes. (a) (Previous page) Stereo view of the three-dimensinal network formed by continuous stacking
along the x- and z-axes. The box shown is the unit cell of the crystal. (b) A cluster 1 stacks on two symmetry-related cytosine tetraplexes 2, at both the top and bottom,
creating continuous stacking along the z-axis. (c) A cluster 2 stacks on two symmetry-related cytosine tetraplexes 1, at both the top and bottom, creating continuous
stacking along the x-axis.
ACKNOWLEDGEMENTS
This research was supported by grants from the National
Institutes of Health, the National Science Foundation and the
Department of Energy through Los Alamos National Laboratories.
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