volume 15 Number 22 1987
Nucleic Acids Research
Crystal structure analysis of an A-DNA fragment at 1.8 A resolution: d(GCCCGGGC)
Udo Heinemann*, Hanspeter Lauble, Ronald Frank1 and Helmut Bldcker1
Abteilung Saenger, Institut fur Kristallographie, Freie Universitat Berlin, Takustrasse 6, D-1000 Berlin
33 and kjBF (Gesellschaft fur Biotechnologische Forschung mbH), Mascheroder Weg 1, D-33OO
Braunschweig, FRG
Received September 4, 1987; Revised October 20, 1987, Accepted October 27, 1987
ABSTRACT
Single crystals of the self-complementary octadeoxyribonucleotide d(GCCCGGGC) have been analysed by X-ray diffraction
methods at a resolution of 1.8 A. The tetragonal unit cell of
space group P4,2.2 has dimensions of a=43.25 A and c=24.61 A and
contains eight strands of the oligonucleotide. The structure was
refined by standard crystallographic techniques to an R factor of
17.1% using 1359 3a structure factor observations.
Two strands of the oligonucleotide are related by the crystallographic dyad axis to form a DNA helix in the A conformation.
The d(GCCCGGGC) helix is characterized by a wide open major
groove, a near perpendicular orientation of base pairs to the
helix axis and an unusually small average helix twist angle of
31.3° indicating a slightly underwound helix with 11.5 base pairs
per turn. Extensive cross-strand stacking between guanine bases
at the central cytosine-guanine step is made possible by a number
of local conforraational adjustments including a fully extended
sugar-phosphate backbone of the central guanosine nucleotide.
INTRODUCTION
Single crystal X-ray diffraction and NMR spectroacopic studies of synthetic oligodeoxyribonucleotides have demonstrated the
presence in DNA helices of a characteristic sequence-dependent
structure (1-3). These investigations have not only led to the
discovery of an entirely new form of DNA, left-handed Z-DNA (4-6),
but have also widened our understanding of the classical A and B
forms of DNA as previously defined by X-ray fiber diffraction
methods (7-9). In all principal helix forms, the fine spatial
structure depends on the base sequence in a partly predictable
manner (10,11).
Until this day, most individual DNA sequences studied by
X-ray methods have been found to adopt the A conformation in the
crystal, i.e. we have more information regarding the sequencedependent structure of A-DNA than for any other helix type.
© IRL Press Limited, Oxford, England.
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Crystal structure analyses at resolutions between 1.7 and 3 A
have been reported for d(GGTATACC) (12-14), d( I CCGG) (15.16),
d(GGCCGGCC) (17,18), d(GGGGCCCC) (19), d(GGATGGGAG) (20),
d(CCCCGGGG) (18), d(ACCGGCCGGT) (21) and d(GTGTACAC) (21a). In
addition, the three-dimensional structure of a DNA/RNA hybrid is
known (22) and several variants of the above sequences incorporating substituted bases (12-14,23) or non-Watson-Crick base pair
mismatches (24,25) have been investigated.
A comparison of these DNA fragments shows a considerable variation in both averaged and individual helical parameters. Here
the crystal structure of the octadeoxyribonucleotide d(GCCCGGGC)
is described. This octamer represents an extreme case of a
slightly underwound A-DNA helix with a wide major groove, a very
small base pair tilt and a concomitant large rise per base pair.
EXPERIMENTAL
DNA synthesis and crystallization
d(GCCCGGGC) was synthesized in solution by a modified phosphotriester approach (26). Crystals were grown at room temperature from 10 mg/ml DNA in 10 mM Tris-HCl or 20 mM Na-cacodylate
pH 7.5 in the presence of 10 mM MgCl- by microdialysis (27)
against an identical buffer solution containing 2-methyl-2,4pentanediol (MPD) or isopropanol. Large crystals in the form of
truncated tetragonal bipyramids appeared at concentrations of 28%
isopropanol or 30% MPD.
The crystals of d(GCCCGGGC) belong to the tetragonal space
group P4.2..2 (no. 9 6 ) . The unit cell constants of a=43.25 A and
c=24.61 A are similar to the crystal parameters of several octameric A-DNA fragments (15,17). One single strand of d(GCCCGGGC)
(or four base pairs) constitutes the crystallographic asymmetric
unit.
Data acquisition and processing
Diffraction data were collected from one crystal at room
temperature on a Stoe four-circle diffractometer using Ni-filtered C u K a radiation from a sealed tube X-ray generator. Peak intensities were measured in 0.8* oj-acans over 12 to 60 sec per reflection, depending on intensity and peak shape. A full quadrant
of reciprocal space with a limiting sphere of 1.8 A resolution
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was evaluated. The data set included 2599 reflection measurements
with intensities exceeding one standard deviation. The crystal
had suffered no observable radiation damage.
The diffraction intensities were corrected for Lorentz and
polarization factors and for the effect of absorption (28). Due
to the plate-like shape of the crystals, absorption scale factors
of up to 2.3 had to be applied. 2000 multiple measurements in 960
unique reflections yielded a symmetry R factor of 2.7% ( R
=
sym
El<F>-F(i)| /Ef(i), over all i measurements). The final diffraction data set contained 1553 unique reflections with F ^ lcr(F) .
Structure solution and refinement
The similarity in sequence and crystal parameters of the DNA
octamer under investigation with the oligonucleotides d( CCGG),
d(GGCCGGCC) and d(CCCCGGGG) immediately suggested that, as with
these DNA fragments, an A-DNA helix had formed where the complementary strands were related by the twofold axis of the unit cell.
A double-helical model of d(GCCCGGGC) was therefore generated
using idealized A-DNA coordinates based on fiber diffraction (79 ) . The four base pair asymmetric unit of the octaraer model was
least-squares fitted to the structure of d( CCGG) (15,16) and
placed into the unit cell accordingly. Owing to the differences
in cell constants, the resulting octamer helix showed a break between the two halves leading to a C3'-O3' bond length of 4.5 A.
The orientation of the four base pair fragment as a rigid body
was least-squares refined, slowly increasing the resolution from
8 to 3 A. After this refinement the crystallographic residual R =
LIF O -F c | /£F o was 44.4% and an intact helix with a C3'-O3' bond
length of 1.42 A had been reconstituted.
Constrained-restrained least squares refinement (29,30) was
subsequently used to improve the fit between calculated and observed structure factor amplitudes and to extend the resolution
from 3 to 2.5 A. After initial model idealization the octanucleotide double helix was divided into 8 nucleoside pairs with three
internal degrees of freedom and 14 connecting rigid phosphate
units and refined in space group P 4 3 (origin of P4,2 1 2). The refinement converged at R = 35.0%. Since no symmetry restraints had
been applied the two equivalent strands adopted considerably different conformations.
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Table 1
Crystallographic and stereocheraical refinement parameters.
Resolution range
Number of reflections (Iff on F )
Off on F°)
R factor (Iff reflections)
(3ff reflections)
F /F correlation coefficient
o c
Sugar-base bond distances
Sugar-base bond angle distances
Phosphate bond distances
Phosphate angle and H-bond distances
Planar groups
Chlral volumes
Single torsion contacts
Multiple torsion contacts
Isotropic thermal factors
sugar-base bonds
sugar-base angles
phosphate bonds
phosphate angles and H-bonda
6.0-1 .8 A
1475
1359
17.8%
17.1%
0.965
0.011
0.033
0.040
0.065
0.018
0.044
0.041
0.201
1.10
1.66
2.74
1.98
/
/
/
/
/
/
/
/
/
/
/
/
0.025
0.050
0.050
0.075
0.030
0.100
0.250
0.250
2.50
3.50
3.50
3.50
A
A
A
A
A
A3
A
A
AJ
AJ
A3
A*
The R factor is defined as £ [F -F I/EF_.. the correlation
coefficient is given by £[(F -<F 5)(F V » ] / [ j ; ( F -<F »*•
£(F -<F > J 2 ] a / 2 . For the stereocfiemical parameters the left number
givis tfie r.m.s. deviation from ideality and the right number is
the sigma value used in the refinement. The weight applied on the
corresponding restraint is the inverse square of the sigma value.
At this stage the constrained refinement was replaced by
stereochemically restrained least squares using the nucleic acids
version NUCLSQ (31) of the Hendrickson-Konnert program (32,33) in
conjunction with FRODO (34) model building on a vector graphics
system. Restraints were placed on bond lengths and angles, base
planes, deoryribose chiral centers, non-bonded contacts and thermal factors of linked atoms after these had been allowed to vary.
In order to retain reasonable stereochemistry the entire eight
base pair double helix rather than the asymmetric unit (one
strand) was used in the refinement with additional restraints on
hydrogen bonds in Watson-Crick base pairs. Initially, the two
strands were forced towards identical stereochemistry by a symmetry restraint. This was found unnecessary in the later stages
of refinement where the structure showed little tendency to deviate from ideal dyad symmetry.
In the first cycles of NUCLSQ refinement the resolution was
extended from 2.5 A to the limit of 1.8 A. The full 1CT data set
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including 1475 reflections was used subsequently. Individual
temperature factors were introduced into the refinement at R =
31.2% and further progress was achieved by the incorporation of
solvent molecules identified on the graphics system. Before each
FRODO session perfect dyad symmetry of the helix was reconstituted by averaging over the equivalent strands. Double difference
Fourier maps (2F -F ) and difference Fourier maps (F -F ) were
o c
o c
used for the identification of solvent and to check the model. No
manual revisions of the DNA helix model was necessary throughout
refinement but much care was invested into the identification of
solvent molecules. Only those difference Fourier peaks were assigned as water molecules that were nearly spherical in shape and
were located in hydrogen bonding distance to polar DNA atoms or
other waters. Despite the high resolution no cations bound to the
sugar-phosphate backbone of d(GCCCGGGC) could be located.
RESULTS AND DISCUSSION
Refinement results
The stereochemically restrained least-squares refinement of
d(GCCCGGGC) converged at R = 17.1% for 1359 3a structure factor
observations in the 6 to 1.8 A resolution range (R = 17.8% for
1475 la F o ) . The r.m.s. positional shift in the last cycle was
0.022 A and the r.m.s. temperature factor shift was 0.27 A 2 . In
the final difference Fourier map the highest feature appeared
with an electron density of 0.21 e/A3. Table 1 gives a summary of
stereochemical and crys,tallographic parameters at the 'end of refinement.
34 solvent molecules were identified per strand of the octadeoxyribonucleotide, 8 of which refined to temperature factors
between 50 and 60 A 2 . These water molecules were retained in the
coordinate set since they reappeared in the difference electron
density maps after having been omitted from refinement for five
cycles. All other water molecules had thermal factors below 50 A 2 .
The complete model for the d(GCCCGGGC) double helix thus consists
of 322 DNA and 68 water oxygen atoms. Atomic coordinates have
been deposited with the Brookhaven Protein Data Bank from which
copies are available.
'
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Global conformation
In the crystal, d(GCCCGGGC) forms an A-DNA double helix with
perfect dyad symmetry. The bases on the first strand are numbered
1-8 and on the stereochemically equivalent complementary strand
9-16. Figure 1 presents several views of the structure of the
octadeoxyribonucleotide helix. It is immediately apparent that
the conformation of the octamer deviates significantly from A-DNA
as defined by X-ray fiber diffraction (8,9).
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(c)
Figure 1. Stereo representation of the structure of d(GCCCGGGC).
Carbon, nitrogen, oxygen and phosphorus atoms are drawn with
increasing radius, (a) View into the minor groove, helix axis
vertical, (b) side-on view, dyad symmetry axis horizontally
across the helix, (c) view into the major groove, (d) view down
the helix axis.
The average helix twist angle of the DNA fragment is 31.3"
(Table 2 ) . Thus, there are 11.5 base pairs per complete turn of
the helix putting this structure halfway between the classical A
and A' helical conformations (38). The unusually small mean helix
twist angle is caused predominantly by a partial unwinding of the
helix between base pairs C4-G13 and G5-C12 which indicates a perturbation in the center of the helix structure where a poly(C)
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Table 2
Structural parameters for d(GCCCGGGC).
Helix
Rise per B.p.
B..p. Propel lor
twist
b.p.
roll
tilt
twist
5"
31
Gl - C16
C2 - G15
C3 - G14
C4 - G13
G5 - C12
G6 - Cll
G7 - CIO
C8 - G9
31
5'
Average
Fiber
X-ray max.
X-ray min.
(°)
30.4
33.9
33.6
23.2
33.6
33.9
30.4
(A)
3.51
3.24
2.86
3.18
2.86
3.24
3.51
(•)
C)
C)
(A)
-6.13
-3.83
2.46
1.65
-1.65
-2.46
3.83
6.13
10.63
4.94
5.23
8.01
8.01
5.23
4.94
10.63
8.89
13.13
8.24
6.03
6.03
8.24
13.13
8.89
3.94
4.18
4.15
3.71
3.71
4.15
4.18
3.94
4.00
31.3
3.20
7.20
9.07
4.4
32.7
2.56
22.0
6.0
4.1(c)
14.0(a) 16.2(a)
34.0(a) 3.10(b)
3.5(a)
9.0(b)
8.6(d)
31.6(c) 2.84(a)
The parameters are those defined by Dickerson and collegues
(11,35-37) where the helix twist measures the angle at which the
Cl'-Cl1 vectors of two successive base pairs intersect when
viewed down the helix axis, base pair roll and tilt describe
rotations of the best plane through a base pair about the long
and the short axis of the base pair, respectively. The propellor
twist is the angle of rotation about the C6(pyrimidine)-C8(purine)
vector between the bases of a pair a'nd D measures the perpendicular displacement of this vector from the helix axis. The
corresponding parameters for A-DNA as defined by fiber diffraction
methods are taken from Arnott and colleages (9). X-ray max. and
min. give the largest and smallest mean values for each parameter
as observed by single crystal analysis of oligonucleotides (2):
(a) d( i CCGG) (15,16), (b) d(CCCCGGGG) (18), (c) d(GGGGCCCC) (19),
(d) d(GGCCGGCC) (17).
stretch leads into a poly(G) stretch. At first glance, this reduction in local helix twist angle might be regarded as a proper
Calladine strategy (10,11) for avoiding an inter-strand minor
groove clash between the guanine bases at a pyrimidine-purine
step.
In addition to the mean helix twist angle other parameters
have adopted values outside the range previously observed in
crystalline A-DNA fragments (2) and different from the fiber diffraction model of A-DNA (9). The mean rise per base pair is 3.2 A
and thus larger than expected for A-DNA, the average base pair
tilt against the helix axis is only 7.2° and, most significantly,
the major groove width measures 9.8 A (P-P separation minus 5.8 A
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3.
U
8
12
16
20
Base pair tilt Is)
Figure 2. Dependence of major groove width and average rise per
base pair on the mean base pair tilt in oligonucleotide crystal
structures in the A and B-DNA conformation. Data are taken from
(2). The solid symbols represent the fiber diffraction models of
B-DNA (top left) and A-DNA (bottom right), symbols for a DNA/RNA
hybrid (22) have been placed in parentheses and symbols for
d(GCCCGGGC) are shown enlarged. The linear correlation coefficient
with the mean base pair tilt is -0.983 for the major groove width
and -0.976 for the mean rise per base pair.
to account for the van der Waals radii of the two phosphate
groups). Base pair tilt angle, rise per base pair and major
groove separation are highly correlated as shown in Figure 2
which may also serve to place the global structure of d(GCCCGGGC)
in the context of known DNA conformations.
The polymorphism of A-DNA structure is most dramatically demonstrated by a comparison of the major groove widths of crystalline d(GCCCGGGC) and a model of the same sequence based on idealized coordinates from fiber diffraction studies of random-sequence
DNA in the A conformation (7-9, see Figure 3 ) . There is much difference in the accessibility of bases in the major groove which
may be important concerning a possible role of A-DNA in living
cells (20,39). The low-resolution structure of a 9 base-pair DNA
fragment covering part of the binding site for Xenopus laevis
transcription factor IIIA (20) also shows A-form DNA with a very
wide major groove.
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(a)
(b)
Figure 3. Space filling drawing of (a) the crystal structure of
d(GCCCGGGC) and (b) an A-DNA model for the same sequence based on
idealized coordinates from fiber diffraction (7-9) in an orientation which emphasizes the difference in major groove separation.
Phosphorous atoms have been drawn in black to mark the sugarphosphate backbone.
Base pair geometry and stacking
Four individual base pairs and four base pair steps are
found in the symmetric octamer double helix. All base pairs display the usual positive propellor twist. The roll angles (about
the long axis Of a base pair) for individual base pair steps in
d(GCCCGGGC) are 6.3" (G1-C2/C16-G15), 9.0° (C2-C3/G15-G14), 3.1*
(C3-C4/G14-G13) and -0.1* (C4-G5/G13-C12) . This behaviour is in
contrast to the Calladine/Dickerson predictions (10,11) which
would call for a maximum roll angle at the central C-G base pair
step to relieve a possible minor-groove purine-purine clash.
The local helical conformation of DNA is dictated by the sequence-dependent stacking of base pairs. In the d(GCCCGGGC) helix
stacking involving the guanine bases is strongly favoured over
stacking with cytosines (Figure 4 ) . From the terminal base pair
step into the center of the helix we observe increasing sliding
out of the stack of base pairs, predominantly along their long
axes, resulting in perfect inter-strand stacking between G5 and
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C16...G1
G15...C2
C16...G1
G15...C2
G15...C2
G14...C3
G15...C2
G14...C3
G14...C3
G13...C4
G14...C3
G13...C4
G13...C4
C12...G5
Figure 4. The four different base pair steps of d(GCCCGGGC)
viewed down the helix axis. The upper nucleoside pair and the
phosphate units are shown with solid bonds. Note the C3'-exo
sugar pucker of C16 (top) and the pronounced interstrand stacking
between G5 and G13 (bottom) which is made possible by a reduction
in local helical twist and a fully extended sugar-phosphate backbone.
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Table 3
Stereochemistry of the sugar-phosphate backbone.
a
Gl
C2
C3
C4
G5
G6
G7
C8
0
-84 .6
162.4
-60 .7
174.5
-82 .2 -173.7
-170 .5 -163.9
-78 .4 -178.0
-69 .1
173.6
-49 .7
166.8
7
6
e
47.6
69.2
42.0
54.0
142.8
57.0
61.6
55.8
78.5
71.3
80.9
81.0
67.6
80.1
78.8
145.8
-161.4
-159.6
168.3
-178.2
-154.9
-148.3
-161.9
r
-55.7
-63.4
-46.0
-69.3
-72.6
-74.5
-66.9
X
-158.7
-161.9
-159.0
-160.4
-177.3
-173.4
-165.5
-119.9
T
M
39.1
43.9
45.0
35.1
47.8
41.8
43.2
42.8
P
8.6
21.9
12.8
22.3
16.0
18.9
15.3
186.5
pucker
C3 '-endo
C3 1-endo
C3 '-endo
C3 '-endo
C3 '-endo
C3 '-endo
C3 '-endo
C3 1 -exo
Backbone torsion angles are defined as P a 05' 0 C5' 7 C4' 5 C3' £ 03' f P
and the glycoayl angle is C2_tU X Cl '_O4"'~rpyrTmldTne«T~an3 C4 H9 X cl'~o"4'
(purines). The p«eudorotation magnitude (TM> and phase (P) are gTven according to (41).
G13 at the central C-G step. In order to maximize this stacking
interaction the roll angle between the central base pairs has to
be close to zero. A buckling of the central base pairs with an
angle of -4.6* contributes to a parallel orientation of the guanine planes. Since, in addition, the propellor twist of the C4G13 base pair (and the symmetry-related G5-C12) is rather small
(6.0°), the sliding of base pairs positions the six-membered
rings of the guanine bases almost perfectly flat atop each other.
The extended backbone at C4-G5, necessary to permit inter-strand
purine-purine stacking, causes the reduction in local helix twist
angle (see above).
The single parameter that determines the local structure of
d(GCCCGGGC) is the cross-strand stacking of guanine bases at the
central C-G step. Analogous behaviour has been described for the
helical structure of the related A-DNA octamers d(CCCCGGGG) (18)
and d(GGGCGCCC) (40). Calladine and Drew (40a) have early on recognized the importance of pyriraidine-purine (and here especially
C-G) steps for DNA helix structure. They have proposed two distinctly different stacking geometries: pyrimidine-purine stacking
on the same strand in B-DNA and cross-strand stacking of purines,
as observed in this structure, in A-DNA. Since preceding dinucleotide steps in d(GCCCGGGC) have no preference for either mode,
the other base pairs follow the motion of the central guanines
forced by "the passive, elastic stiffness of the backbone" (40a),
as evidenced by the increase of the base pair slide (1.1 - 1.6 1.7 - 2.3 A) from end to center of the helix. In this sense the
slide is in fact a cooperative parameter capable of being transmitted along the DNA helix.
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Sugar-phosphate backbone
In the refinement of the octamer structure no restraints
have been placed on sugar pucker and backbone torsion angles. Yet
seven out of the eight deoxyribose units have adopted the usual
C3'-endo conformation (Table 3 ) . The degree of puckering as measured by the pseudorotation magnitude 7"M (41) is almost uniform
over the entire strand. Only the 3'-terminal sugar has adopted a
pucker in the opposite domain (C3'-exo) which remains without major consequences for the helix structure. A similar observation
has been made for d( CCGG) (15,16). It may be remarked once again
that the A-DNA conformation appears to allow less variability in
sugar pucker than the B form.
With the exception of nucleotide unit G5 and the C3'-exo
sugar pucker of C8 all backbone torsion angles fall into the
range expected for A-DNA (2). To permit the above discussed
cross-strand stacking of guanines in the center of the helix
torsion angles a and 7 have to change from their standard values
(-)gauche and (+)gauche to trans, i.e. the backbone has to be
fully extended. As a consequence, the P-P separation between the
nucleotide units 5 and 6 is increased to 6.8 A over an average
of 6.0(3) A for the remaining intra-strand phosphate-phosphate
steps. Again, this phenomenon has been noted before for C-G base
pair steps in A-DNA (18,40). Inasmuch as it suggests a continuum
of possible conformations, the term "elastic stiffness" then
appears to be no adequate decription of the DNA backbone since
the latter is capable of adopting distinct conformational states
within one helix type.
Concerning a possible biological function it should be
pointed out that here sequence information is reflected in the
conformation of the sugar-phosphate backbone. An information
readout, thus, would not necessarily have to involve direct
contact (of a protein, e.g.) with the bases but could occur by
interaction with the phosphate groups.
Thermal parameters
Atomic temperature factors of linked atoms are restrained
during least-squares refinement to prevent unreasonable differences of thermal motion (33). As often observed in oligonucleotide crystal structures, in d(GCCCGGGC) the mean isotropic
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o
Figure 5. Hydration of the d(GCCCGGGC) double helix. The view is
into the major groove as in Figure lc. Water molecules appear as
open spheres.
temperature factors are lowest for atoms in the bases (21.5 A 2 )
and highest in the phosphate groups (27.4 A 2 ) with sugars in
between (24.8 A 2 ) . There is a tendency of atoms in central bases and sugars to adopt lower B values than at the ends of the
helix but not so for the phosphate groups.
For the solvent structure no partial occupancies were
allowed. The temperature factors thus take up the effect of
incomplete occupancy of water sites and do not strictly reflect thermal motion. Temperature factors of water oxygen
atoms range from 27 to 60 A 2 .
Hydration
The interaction with water and dissolved ions plays an important role in the structure of biological raacromolecules (42).
Mechanisms for the stabilization of B-DNA by the minor groove
"spine of hydration" (43) and of A-DNA by "economics of hydration"
(44) have been proposed. The influence of cations which have to
be present to neutralize the phosphate charges of DNA, however,
must not be overlooked. At the present resolution the identification of metal ions in the solvent sphere of d(GCCCGGGC) proved
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impossible. The discussion of hydration of the octamer will
therefore be restricted to a few general comments. A detailed
analysis of the solvent structure will have to await the analysis
of higher-resolution X-ray diffraction data which have recently
been collected at the DESY synchrotron source in Hamburg.
Of the 68 water molecules identified per double helix all
but 8 are in hydrogen-bonding distance (less than 3.5 A) to polar
DNA atoms. The present crystal structure analysis has thus revealed little more than the primary hydration shell of the octamer. Since 10 water molecules can make hydrogen bonds to symmetryrelated helices in the crystal lattice and thus contribute twice
to the hydration of one helix, the inner hydration shell contains
at least 70 water molecules. The wide and deep major groove of
d(GCCCGGGC) and the phosphate backbone are the primary hydration
sites (Figure 5 ) . The phosphate groups are individually hydrated?
no water molecules are found that bridge between adjacent phosphate groups by hydrogen-bonding as might have been predicted
(44) although, with the mentioned exception of phosphate groups 5
and 6, all intra-strand phosphate-phosphate distances are in the
normal A-DNA range. Binding of water molecules in the minor
groove of the helix is partially hindered by lattice contacts.
Therefore this crystal form is not a good model to study minorgroove hydration in A-DNA.
Crystal packing
The formation of the d(GCCCGGGC) double helix by a crystallographic dyad axis will not be considered here. Contacts in the
crystal lattice between octamer helices involve van der Waals
interactions and hydrogen bonds. Little can be learned about
charge-charge interactions since dissolved cations could not be
located.
Inter-helix contacts include four possible hydrogen bonds:
N2 of residue G5 to 04" of Gl (3.0 A ) , N2 of G6 to N3 of Gl
(3.2 A) and the analogous contacts with the equivalent strand.
Using a cutoff distance of 4.0 A for van der Waals interactions,
there are 122 non-bonded contacts between octamer helices, 24 of
which involve atom pairs closer than 3.5 A. In addition, there
are 10 water molecules which might bridge between helices by
hydrogen bonding.
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Figure 6. Packing of the terminal three base pairs of two symmetry-related molecules (narrow lines) against the flat minor
groove of the helix in d(GCCCGGGC) crystals.
Most intermolecular contacts arise from a packing of the
terminal base pairs of two symmetry-related helices into the flat
minor groove of the d(GCCCGGGC) double helix (Figure 6 ) . There is
a very intimate inter-helical fit which undoubtedly greatly stabilizes the crystal lattice. A similar packing scheme has been
observed in most A-DNA crystal structures analysed so far (12-25).
Relevance to DNA solution structure and biological significance
It is possible to determine the global conformation of oligonucleotides in solution by NMR methods (45,46). By this method
and by Raman spectroscopy a number of oligonucleotides that form
A-DNA in the crystal were shown to adopt the B conformation in
solution (47-49) thus questioning the relevance of crystalline ADNA to the structure of DNA in aqueous solution. It should be
kept in mind, however, that by NMR spectroscopy the overall conformation of a chain-like molecule like DNA has to be deduced
from short-range inter-proton distances whose accurate determination is still a matter of debate (50) while other spectroscopic
methods may not identify an oligonucleotide as d(GCCCGGGC), showing almost B-DNA like base pair tilt and rise, as A-DNA.
Based on its sequence the octamer may be expected to display
structural features reminiscent of the homopolyraer poly(dG).
poly(dC). Fibers of this polymer have been reported to retain an
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A-DNA like conformation even at high relative humidity (51). Solution X-ray scattering experiments have yielded a radius of gyration closer to double-helical RNA than to DNA (52). The helical
repeat of poly(dG).poly(dC) in solution is 10.7 base pairs per
turn (53) and thus nearly identical with the repeat of mixed-sequence DNA (53,54) but markedly different from the 10 base pairs
per turn of fiber diffraction B-DNA (7-9). These facts taken together suggest that in solution conformational differences between helix types are more subtle than in our models and intermediate forms between A and B-DNA exist of which poly(dG).poly(dc)
may be one. It thus seems natural that d(GCCCGGGC) forms an A-DNA
helix with certain structural characteristics such as major
groove width, base pair tilt and rise resembling B-DNA.
The biological significance of A-DNA has been discussed recently (20,39). With this respect the possibility of A-DNA to
open its major groove as wide as in d(GCCCGGGC) may be important,
e.g. for protein-DNA interaction and information readout. The
characteristic cross-strand guanine-guanine stacking at the central C-G step of the octamer has been shown to be reflected in
the conformation of the sugar-phosphate backbone. An information
readout from DNA thus must not necessarily involve direct contact
with the bases in the helical grooves but can be achieved by
sensing backbone conformation in some cases. This may be illustrated by an admittedly speculative example. The enzyme DNase I
has been shown to cleave phosphodiester bonds with rates depending on DNA sequence and local structure (55-58). While poly(dG).
poly(dc) like sequences are generally cut less frequently than
mixed-sequence DNA (58) cleavage rates at all three CCC V G sites
in a 160 base pair DNA fragment were significantly above those of
neighbouring dlnucleotides (57). In'd(GCCCGGGC) this cleavage
site shows a unique all-trans backbone conformation and a phosphate-phosphate separation increased by nearly 1 A. If oligo(dG).
oligo(dC) stretches in a mixed-sequence environment indeed adopt
an A-DNA like conformation this may explain the generally reduced
cleavage rates within these sequences. The enhanced cutting frequency at the C-G step may then be attributed to the higher exposure to nucleophilic attack of the guanine phosphate group in the
extended backbone segment.
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Nucleic Acids Research
ACKNOWLEDGEMENTS
The authors thank Drs. E. Westhof, J.L. Sussman and R.E.
Dickerson for computer programs and W. Saenger for reading the
manuscript and helpful discussions. U.H. and H.L. acknowledge
financial support from the Deutsche Forschungsgemeinschaft and
the StSndige Koramission ftlr Forschung und wisaenschaftlichen
Nachwuchs der Freien Universitat Berlin and M. Steifa for invaluable technical assistance. A stipend from the Studienstiftung
des Deutschen Volkes to H.L. and help in DNA synthesis by W.
Heikens and M. Becker are also gratefully acknowledged. We wish
express our gratitude to a referee for valuable suggestions.
*To whom correspondence should be addressed
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