DNA octaplex formation with an I-motif of water-mediated A-quartets: reinterpretation of the crystal structure of d(GCGAAAGC).

Abstract

The crystal structure of the tetragonal form of d(gcGAAAgc) has been revised and reasonably refined including the disordered residues. The two DNA strands form a base-intercalated duplex, and the four duplexes are assembled according to the crystallographic 222 symmetry to form an octaplex. In the central region, the eight strands are associated by I-motif of double A-quartets. Furthermore, eight hydrated-magnesium cations link the four duplexes to support the octaplex formation. Based on these structural features, a proposal that folding of d(GAAA)n, found in the non-coding region of genomes, into an octaplex can induce slippage during replication to facilitate length polymorphism is presented.

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J. Biochem. 140, 759–762 (2006)
doi:10.1093/jb/mvj213
Rapid Communication
DNA Octaplex Formation with an I-Motif of Water-Mediated
A-Quartets: Reinterpretation of the Crystal Structure of
d(GCGAAAGC)
Yoshiteru Sato1, Kenta Mitomi1, Tomoko Sunami1, Jiro Kondo2and Akio Take ´naka1,*
1Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuda, Midori-ku,
Yokohama 226-8501 and2Institut de Biologie Mole ´culaire et Cellulaire du CNRS, Universite ´Louis Pasteur,
15 rue Rene ´Descartes, Strasbourg F-67084, France
Received September 24, 2006; accepted October 17, 2006
The crystal structure of the tetragonal form of d(gcGAAAgc) has been revised and
reasonably refined including the disordered residues. The two DNA strands form a
base-intercalated duplex, and the four duplexes are assembled according to the crystal-
lographic 222 symmetry to form an octaplex. In the central region, the eight strands are
associated by I-motif of double A-quartets. Furthermore, eight hydrated-magnesium
cations link the four duplexes to support the octaplex formation. Based on these struc-
tural features, a proposal that folding of d(GAAA)n, found in the non-coding region of
genomes, into an octaplex can induce slippage during replication to facilitate length
polymorphism is presented.
Key words: A-quartet, DNA octaplex, non-coding DNA, VNTR, X-ray analysis.
Human genome analyses have revealed that non-coding
regions occupy more than 98% of the genome (1). VNTR
(Variable number tandem repeat) is a family of non-coding
sequences organized into long repetitive units, and it is
dispersed throughout the genome of all vertebrates (2).
Polymorphism of VNTR occurs as a consequence of
mutational processes such as replication slippage and/or
unequal sister chromatid exchange (3). Thus, the analysis
of VNTR patterns has been employed as a method of DNA
fingerprinting.
A VNTR immediately adjacent to the human pseudo-
autosomal telomere contains the G-rich sequence d(ccGA
[G]4Agg)1(4), and exhibits a high degree of length poly-
morphism. The repetitive unit is repeated eight times,
suggesting that this VNTR forms a specific structure. To
examine the structural property of the sequence, DNA
fragments including several analogues were prepared.
X-Ray analysis revealed that DNA fragments with the
sequence d(gcGA[G]1Agc) (hereafter G1) form an octaplex
with I-motif of G-quartets, in which four base-intercalated
duplexes are assembled to interact at the central bases (5).
Furthermore, we found that DNA fragments with the
sequence d(gcGA[A]1Agc) (hereafter A1), mutated at the
5th residues from G to A, also form base-intercalated
duplexes. In the previous report (6), however, it was
difficult to ascertain the formation of the octaplex due to
disordering at the 5th residues. In the present study, the
A1 crystal structure (tetragonal form) was revised. In the
re-refined crystal structure, an octaplex structure with
I-motif of double water-mediated A-quartets has been
found. In this paper, the details of the octaplex structure
will be described by comparing with that of the I-motif of
double G-quartets, and its biological significance will be
discussed.
The crystal structure of the tetragonal form of A1 (6) was
previously refined with the space group I422. Only the 5th
residue was assumed to be disordered, with two different
conformers, 1 and 2. For the present study, the crystal-
lographic two-fold symmetry between the two strands of
the duplex was initially released to refine (with I222) the
disordered structure, and then the I422 symmetry was
applied in the final refinement. The X-ray diffraction pat-
terns, which were collected in the previous study, were
reprocessed with the space groups I222 and I422 using
the program HKL2000 (7). Statistics of data reprocessing
with I422 are summarized in Table 1.
Since the conformer 2 of A5in the previous structure did
not fit to the electron density map [see the Supplementary
Figure (a)], it was replaced with a new conformer, 3, which
was built on the 2|Fo| – |Fc| map with the program
QUANTA (Accelrys Inc.). Under the I222 symmetry, four
duplexes combined with the two strands containing con-
formers (1-3*, 3-1*, 1-1* and 3-3*)2are possible around the
two-fold axis. However, the 1-1* and 3-3* combinations
were ruled out because the distances between C40and
C40* and between O40and O40* of the A5and A5* residues
were abnormally shorter than the lower limits of the van
der Waals interactions. The two alternative structures con-
taining 1-3* and 3-1* were separately refined using the
programs CNS (8) and Refmac5 (9) through a combination
of crystallographic conjugate gradient minimization and
B-factor fitting techniques. Therefined 1-3* and3-1* struc-
tures were symmetric to each other, and their R values
were almost the same (19.7% and 19.8%). In the latter
stages of refinement, half of the duplex structure, which
Vol. 140, No. 6, 2006
759
? 2006 The Japanese Biochemical Society.
*To whom correspondence should be addressed. Tel: +81-45-924-
5709, Fax: +81-45-924-5748, E-mail: atakenak@bio.titech.ac.jp
1Lowercase characters indicate that they can form a Watson-Crick
G:C or C:G pair when the two fragments are aligned in an anti-
parallel fashion.
2Asterisk represents the counter strand of the duplex around the
two-fold axis.

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was averaged between 1-3* and 3-1*, was refined with the
space group I422. Statistics of the structure refinements
are summarized in Table 1. The electron density maps
around the A5and A5* residues are shown in Supplemen-
tary Figure, produced with the program O (11). All local
helical parameters including torsion angles and pseudor-
otation phase angles of ribose rings were calculated using
the program 3DNA (12). Figures 1 and 2b were drawn with
the program RASMOL (13).
The final R and Rfreevalues for the revised structure are
reduced to 19.0% and 21.1%, respectively (refer to Table 1
and Supplementary Table 1 for comparisons). The A5and
A5* residues are well fitted to the electron density map [see
Supplementary Figure (b)]. Moreover, the atomic distances
between C40and C40* and between O40and O40* of the A5
and A5* residues, which were questionable in the previous
structure, are improved (C40...C40: from 2.9 to 3.6 A˚and
O40...O40: from 2.5 to 2.8 A˚) to suit the van der Waals
interaction ranges. Although the ribose-phosphate back-
bones of the A5 residues are disordered, their adenine
bases are not and occupy almost the same position, as
shown in Supplementary Figure (c).
The local helical parameters and the sugar puckers of
the revised structure are given in the Supplementary
Table 2 (a). The overall structure of the present A1 duplex
is almost the same as the previous one; there are no sig-
nificant differences in the helical parameters. Only
the sugar puckers of the A5residues are changed from
C20-endo to C10-exo in one strand and to C30-exo in the
other [Supplementary Table 2 (b)]. These sugar conforma-
tions are still in the range of B-form conformations.
The two strands, 1 and 3*, form a base-intercalated
duplex similar to those described previously (6). When
the four duplexes are generated according to the crystal-
lographic 222 symmetry, however, it has been found that
they form an octaplex at the central part, as shown in
Fig. 1a. Only the central A5 residues are asymmetric.
The helical axis of the octaplex is parallel to the crystal-
lographic c axis.
Figure 1, b–f, shows the interactions among the four
duplexes around the helical axis. In the octaplex, the
eight A5 bases are close together to form two quartets
with water-mediated hydrogen-binding networks (here-
after each is designated as water-mediated A-quartet).
The two water-mediated A5-quartets stack on each other.
Above and below the double A5-quartets, the eight A4resi-
dues also form two other A-quartets by water-mediated
hydrogen binding. In each of the water-mediated A4-
quartets, the adenine bases are shifted in a radial direction
from the central helical axis to make a central space in
which other water molecules are disordered.
The G3and A6* residues form sheared pairs, and thus
place the guanine bases inside of the octaplex, where the
four G3s are linked together by water molecules. At one end
of the octaplex, the G1and C8* residues, as well as the C2
and G7* residues form the Watson-Crick type pairs. The
phosphate groups of the four G1residues, and likewise of
the four C2s, are projected inside the octaplex and interact
to each other also by water networks. These structural
features are the same at the other end of the octaplex
due to the crystallographic symmetry.
As shown in Fig. 1 (c and d), the eight hydrated magne-
sium cations are bound specifically to the eight G7residues,
the O6 and N7 atoms of which are directly hydrogen
bonded to the cations. In addition, the cations are bridged
to the N2 atom of G3* in the same duplex and to the phos-
phate oxygen atom of G3* in the adjacent duplex, through
hydrogen bonds. The octaplex formation is thus stabilized
by the magnesium linkages.
The G1 and A1 DNA fragments both form octaplex struc-
tures, suggesting the stability of octaplex formation. Here
it is interesting to compare the G1 and A1 octaplex struc-
tures (see Fig. 1, f and g). In the G1 octaplex, the central
double G-quartets are tightly formed through the two
direct hydrogen bonds (N1-H...O6 and N2-H...N7). In
addition, the G1 octaplex is stabilized by three potassium
cations. One is located at the center of the space between
the double G-quartets and is surrounded by the eight O6
atoms of guanine bases. The two remaining cations are
bound to the four O6 atoms above and below the double
G-quartets, respectively. In contrast, the A1 octaplex is
stabilized by the magnesium linkages, which are found
between the two duplexes. Furthermore, the A1 octaplex
is swollen at the central region, in which several water
molecules form hydrogen-bonded networks to stabilize
the octaplex formation. These differences are reflected in
the diagonal C10...C10distances at the X5residues (21.6 A˚
for A1 and 16.2 A˚for G1) and at the A4residues (23.1 A˚for
A1 and 19.2 A˚for G1). The swelling of the central space of
the A1 octaplex may allow the ribose-phosphate backbone
conformation to disorder.
The central sequence of A1, d(GAAA), is repeated in
human (14), canine (15), Meloidogyne artiellia (16)
and Oryza sativa (17) genomes and exhibit length
Table 1. Statistics of data processing and structure
refinement.
Data processinga
Resolution (A˚)
Observed reflections
Unique reflections
Completeness (%)
in the outer shell (%)
Rmergeb(%)
Structure refinement
Resolution range (A˚)
Used reflections
R-factorc(%)
Rfreed
Number of DNA atoms
Number of ions
Number of water molecules
R.m.s. deviation
Bond lengths (A˚)
Bond angles (?)
Improper angles (?)
aThe programs MOSFLMand HKL2000were used respectively for
thepreviousandpresentdataprocessings.Thereareno changesin
the unit cell dimensions regardless of the program used. However,
the Rmergevalue in the present processing is slightly higher due to
different computing techniques between the two programs.
bRmerge= 100·P
calculated structure factor amplitudes, respectively.
using a random set containing 10% of observations that were not
included throughout refinement (10).
50–1.58
68,468
3,359
99.9
99.4
6.3
8.0–1.60 (Fo> 3s)
2,764
19.0
21.1
164
1.25 Mg2+
72
0.013
2.1
1.5
hklj|Ihklj–<Ihklj>|/P
hklj<Ihklj>.cR-factor = 100·
P||Fo|–|Fc||/P|Fo|,where|Fo|and|Fc|aretheobservedand
dCalculated
760Y. Sato et al.
J. Biochem.

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polymorphism (14–16). This short sequence is essential in
the formation of the present octaplex, and is also already
known to form a stable hairpin structure under low mag-
nesium concentration conditions (18, 19). Using the two
structural motifs, we constructed an octaplex model for
d(GAAA) repeats, as shown in Fig. 2, a and b. It is possible
to speculate that when a long GAAA repeated region is
folded into a cluster likeanoctaplex, it can induce sequence
Fig. 1. The detailed structure of A1 octaplex. (a) A stereo pair
drawing of the A1 octaplex with the sequence d(GCGAAAGC). The
base-intercalated duplex is highlighted with thick lines. The eight
central A5 residues form an I-motif of double water-mediated
A-quartets. Gray spheres represent water oxygen atoms participat-
ing in the stabilization of octaplex formation. (b–f) Detailed struc-
tures inside of the octaplexes. The nucleotides of the 1st to 5th
residues in the A1 octaplex are drawn. The remaining parts are
omitted due to the crystallographic symmetry. Water oxygen
atoms and hydrated-magnesium cations bound for the octaplex
formation are drawn with gray spheres. (g) The I-motif of double
G-quartetsfoundintheG1octaplex(5)isshowntocomparewiththe
I-motif of double water-mediated A-quartets of the A1 octaplex. A
potassium cation (gray) is bound to stabilize the G-quartet
formations. Broken lines indicate possible hydrogen bonds. Values
indicate hydrogen bond distances (A˚).
DNA Octaplex with an I-Motif of Water-Mediated A-Quartets
761
Vol. 140, No. 6, 2006

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slippage during DNA replication. As illustrated in Fig. 2, c
and d, the number of repeats could increase when slippage
occurs on the growing strand, or decrease when slippage
occurs on the template strand. It is plausible that such
slippages frequently occur to regulate transcription and
translation of genes according to its number of repeats
(20). To confirm the validity of our hypotheses, more exten-
sive and intensive investigations including the structural
analysis of GAAA repeats are required.
The atomic coordinates have been deposited in the Protein
Data Bank (PDB) with the ID code 2DZ7. We thank M. Suzuki
and N. Igarashi for facilities and help during data collection
and E. C. M. Juan for proofreading of the original manuscript.
ThisworkwassupportedinpartbyGrants-in-AidforScientific
Research (Nos. 12480177 and 14035217) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.
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