doi:10.1006/jmbi.2000.4194 available online at http://www.idealibrary.com on
J. Mol. Biol. (2000) 304, 541±561
BII Nucleotides in the B and C Forms of
Natural-sequence Polymeric DNA: A New
Model for the C Form of DNA
Lorens van Dam* and Malcolm H. Levitt*
Department of Physical
Chemistry Arrhenius
Laboratory, Stockholm
University, S-106 91
Stockholm, Sweden
A combination of solid-state 31P and 13C NMR, X-ray diffraction, and
model building is used to show that the B and C forms of ®brous macromolecular DNA consist of two distinct nucleotide conformations, which
correspond closely to the BI and BII nucleotide conformations known
from oligonucleotide crystals. The proportion of the BII conformation is
higher in the C form than in the B form. We show structural models for
a 101 double helix involving BI nucleotides and a 91 double helix involving BII nucleotides. The 101 BI model is similar to a previous model of
B-form DNA, while the 91 BII model is novel. The BII model has a very
deep and narrow minor groove, a shallow and wide major groove,
and highly inclined bases. This work shows that the B to C transition in
®bers corresponds to BI to BII conformational changes of the individual
nucleotides.
# 2000 Academic Press
*Corresponding authors
Keywords: DNA structure; C-DNA; BII conformation;
model building
Introduction
An understanding of the conformational ¯exibility and polymorphism of DNA is necessary to
model its interactions with other molecules, such
as proteins and drug molecules. The earliest structural studies of DNA involved X-ray diffraction of
macromolecular ®bers, and identi®ed the A-form
and B-form double-helical structures (Watson &
Crick, 1953; Crick & Watson, 1954; Franklin &
Gosling, 1953). At low relative humidity and in the
absence of excess salt, a different ®ber diffraction
pattern was observed for LiDNA (Marvin et al.,
1958), and later for MgDNA (Skuratovskii &
Bartenev, 1979) and NaDNA (Rhodes et al., 1982).
It was postulated that this new type of diffraction
pattern was due to a different DNA structure,
called the C form. Various structural models of
C-form DNA have been suggested, most of which
are minor perturbations of the B-form structures
(Marvin et al., 1961; Arnott & Selsing, 1975;
Premilat & Albiser, 1984). Canonical A, B and
Abbreviations used: RH, relative humidity; CSA,
chemical shift anisotropy; odf, orientational distribution
function; IRLD, infra-red linear dichroism; MAS, magicangle-spinning.
E-mail addresses of the corresponding authors:
[email protected]; [email protected]
0022-2836/00/040541±21 $35.00/0
31
P solid-state NMR;
C-form DNA structures are displayed side-by-side
in many standard textbooks (e.g. Saenger, 1984;
Sarma, 1980). However, the C form has been
regarded as a ``laboratory curiosity'', with no biological signi®cance (Wolfe, 1993).
X-ray diffraction of oligonucleotide crystals, and
solution NMR, provide higher-quality structural
information, revealing great conformational diversity, such as the left-handed Z form in some
repeating oligonucleotide sequences (Wang et al.,
1979) and the BI and BII subconformations of
B-form-type structures (Fratini et al., 1982; Prive et
al., 1991; Grzeskowiak et al., 1991; Hartmann et al.,
1993; Schneider et al., 1997). However, the great
mass of structural information on oligonucleotides
has so far not revealed anything corresponding
clearly to the postulated structural models of
C-form DNA based on ®ber diffraction data.
The role of densely charged ions such as Li‡ and
Mg2‡ in promoting the B to C form transition and
inhibiting the B to A form transition remains unexplained. Furthermore, the standard B-form structural models from ®bers are inconsistent with the
heterogeneity of the oligonucleotide structures,
especially the existence of preferred BI and BII subconformations.
Recently, solid-state NMR has entered the scene
as a useful tool for investigating DNA structure.
This method has been used to investigate con# 2000 Academic Press
542
formational changes of DNA and intercalation of
drug molecules (Tang et al., 1990; Alam & Drobny,
1991 and references therein; Juang et al., 1995 and
references therein). DNA phosphate orientations
extracted from solid-state 31P NMR have showed
the existence of conformational polymorphs in A, B
and C-form ®bers (Song et al., 1997). The measured
phosphate orientations were in good agreement
with existing DNA models for A-form DNA, in
less good agreement for B-form DNA, and not at
all in agreement with currently existing models for
the C form of DNA (Song et al., 1997). Furthermore, anomalous 31P spectral lineshapes were
observed in that study, which were not totally
understood.
Here, we present further solid-state NMR data
on A, B and C-form DNA and show how this information may be combined with a small number of
X-ray constraints in order to resolve the mystery of
C-form DNA structure. Our results clearly show
that BI and BII conformations exist in natural
sequence DNA ®bers, and that the so-called B and
C forms in ®bers are distinguished only by the
relative proportions of these two conformations.
The existing models of C-form DNA, based solely
on X-ray ®ber diffraction, are incorrect, which
explains why no crystal structure has been identi®ed that clearly corresponds to existing models of
C-form DNA in ®bers. In reality, C-form DNA is
simply a BII-rich form of B-form DNA.
Furthermore, we are able to extract a 31P isotropic chemical shift difference of 1.8 ppm
between the two phosphate orientations found in
the B and C forms, the BII nucleotide phosphorus atom being less shielded than the BI
nucleotide phosphorus atom. This chemical shift
difference is consistent, both in sign and in size,
with the presence of both BI and BII nucleotides
in the DNA molecule, and explains the anomalous 31P spectral lineshapes.
We show structural models of the BII-rich structures of C-form DNA, based on the X-ray and
solid-state NMR constraints. These new structures
display considerable modi®cations with respect to
the canonical BI structures, such as a deep and
narrow minor groove, considerable inclination and
destacking of the bases, and the displacement of
the bases towards the major groove of the double
helix, causing it to become broad and very shallow.
This new DNA structural motif has obvious potential implications for protein-DNA interactions.
There is a considerable body of literature on the
conditions favoring the C form of DNA and on the
B to C form transition (e.g. see Fornells et al., 1983;
Pohl, 1976; Portugal & Subirana, 1985; Zimmerman
& Pheiffer, 1980; Bokma et al., 1987). Since C-form
DNA is now identi®ed as a BII-rich form of B-form
DNA, the accumulated knowledge on C-form
DNA may be mobilized towards understanding
the BI to BII transition, which could be of importance in protein-DNA interactions. We speculate
below that the promotion of the B to C form transition by Li‡ and Mg2‡, and the inhibition of the B
A BII Model for C-form DNA
to A form transition by the same ions, is associated
with the disruption of the phosphate hydration
shell.
Results
Samples
We obtained three samples of calf thymus DNA
®bers, called below A, B and C (see Materials and
Methods). Sample A is a NaDNA sample, prepared
at 66 % relative humidity (RH), conditions known
to favor the A form of DNA (Rupprecht &
Forslind, 1970b; Saenger, 1984). Sample B is a
LiDNA sample, prepared at 75 % RH with a large
excess of LiCl. These conditions are known to
favor the B-form of DNA (Rupprecht & Forslind,
1970; Saenger, 1984). Sample C is a LiDNA sample,
prepared at 66 % (NMR) or 75 % (X-ray) RH, with
no excess Li‡. These conditions are typical for the
preparation of C-form DNA (Rupprecht &
Forslind, 1970; Saenger, 1984).
All experimental techniques were performed on
pieces of the same samples, with precautions taken
for humidity control (see Materials and Methods).
X-ray diffraction
Figure 1 shows the ®ber X-ray diffraction patterns of the three samples. The NaDNA sample,
taken at 66 % relative humidity (RH), shows a
characteristic A-form pattern (Figure 1(a)). The
high-salt LiDNA sample at 75 % RH (sample B)
displays
a
characteristic
B-form
pattern
(Figure 1(b)). (Compare X-ray diffraction patterns
on page 47 of Saenger, 1984.)
The low-salt LiDNA sample at 75 % RH (sample
C) displays a typical C-form ®ber diffraction pattern (Figure 1(c)). The characteristic features are the
distinct doubling of the outermost azimuthal
Ê 1 the ®rst
re¯ections and the spot at x ˆ 0.10 A
layer-line (indicated by an arrow in Figure 1(c))
(Marvin et al., 1961; Saenger, 1984; Portugal &
Subirana, 1985).
The B-form pattern has been interpreted in terms
of a double helix with a winding angle of 36 per
base-pair, corresponding to exactly ten base-pairs
per turn (i.e. 101 symmetry), with a rise per residue
Ê (Crick & Watson,
along the helical axis of 3.38 A
1954; Arnott & Hukins, 1972).
The diffuse C-form pattern is harder to interpret,
and has been suggested to signify a non-integral
double helix with 9.33 base-pairs per turn, i.e. a 283
Ê (Marvin
helix, with a rise per residue of 3.32 A
et al., 1961; Arnott & Selsing, 1975; Leslie et al.,
1980). A 91 double helix, with winding angle 40.0 Ê has also
and an axial rise per residue of 3.32 A
been suggested (Marvin et al., 1961; Arnott &
Selsing, 1975; Leslie et al., 1980). A proposal for a
left-handed structure exists (Premilat & Albiser,
1984). If one excludes such unorthodox structures
(see Discussion), there is a broad consensus that
C-form DNA is a right-handed double helix with a
543
A BII Model for C-form DNA
larger winding angle and a smaller axial rise per
residue than B-form DNA.
Phosphate orientations from
Figure 1. X-ray diffraction pictures of the samples
used in this study. (a) Low-salt NaDNA sample at 66 %
relative humidity (sample A); (b) high-salt LiDNA
sample at 75 % relative humidity (sample B); (c) low-salt
LiDNA sample at 75 % relative humidity (sample C).
The arrow indicates the spot characteristic of the
C form.
31
P NMR
We performed rotor-synchronized two-dimensional magic-angle-spinning solid-state NMR (2Dsync-MAS) on the three DNA samples. The 2D 31P
spectra are shown in Figure 2, where the isotropic
resonances are marked by asterisks. There are
major differences between the spectra of samples A
and B, and smaller differences between the spectra
of samples B and C. Note that some of the lines are
visibly inhomogeneous with superposed positive
and negative contributions. This was seen also in
previous investigations (Tang et al., 1989; Song
et al., 1997). We return to this matter later on.
These 2D spectra arise from the modulations of
the 31P resonance frequency when the sample is
rotated with respect to the magnetic ®eld, because
of the chemical shift anisotropy (CSA) of the 31P
sites. The spectra may be analyzed to give the
orientation of the 31P chemical shielding anisotropy
tensor with respect to the rotor axis. Since the
DNA samples are macroscopically oriented and
have a known orientation with respect to the rotor
axis, the 2D spectra reveal the orientation of the
31
P CSA tensor with respect to the DNA ®ber
director. By assuming a certain orientation of the
31
P CSA principal axis system with respect to the
local symmetry of the PO4 unit, and by assuming
that the DNA helices are oriented along the ®ber
director, one may deduce the orientation of the
PO4 group with respect to the DNA helix.
The phosphate orientations are described most
conveniently by de®ning four three-dimensional
Cartesian axis systems, denoted P, M, H and D
in the following discussion. The P axis system is
chosen such that xP is the least shielded axis, yP is
the intermediately shielded axis, and zP is the most
shielded axis of the 31P chemical shift tensor. The
x-axis of the local molecular system M is perpendicular to the O10 -P-O20 plane and points to the
same side as O50 . The y-axis of the system M
bisects the O10 -P-O20 plane, and points from the
phosphorus atom towards the unsubstituted O10
and O20 . The z-axis of the system M is perpendicular to the xM and yM axes. O10 and O20 are the
unesteri®ed and charged phosphate group oxygen
atoms while O30 and O50 are those in the DNA
backbone chain. The axis systems P and M are
expected to coincide within around 10 . The
z-axis of the helix frame H is parallel with the helix
long axis, and the z-axis of the director frame D is
parallel with the average direction of the ®ber long
axes.
The relative orientation of these axis systems is
conveniently speci®ed by sets of Euler angles
PM ˆ {aPM, bPM, gPM}, MH ˆ {aMH, bMH, gMH} and
HD ˆ {aHD, bHD, gHD}, where the convention in
reference (Varshalovich et al., 1988) is used. The
molecular structural information is encoded in the
Euler angles MH (which gives the orientation of
544
A BII Model for C-form DNA
Figure 2. Rotor-synchronized 31P 2D spectra from: (a) the low-salt NaDNA sample (sample A) at 66 % RH; (b) the
high-salt LiDNA sample (sample B) at 75 % RH; and (c) the low-salt LiDNA sample (sample C) at 66 % RH. Asterisks
indicate the positions of the isotropic resonance lines.
the phosphate group with respect to the helix),
while the NMR experiment gives access to the
Euler angles aPD and bPD, describing the relative
orientation of the 31P CSA principal axis system
with respect to the ®ber director axis (the angle gPD
cannot be determined by this experiment). The
angles aPD and bPD, as determined by NMR, are
converted into phosphate orientational angles
MH, according to the procedure described in
Materials and Methods.
The 2D 31P spectra may be analyzed to obtain
the probability density of the Euler angles (aPD,
bPD). This is called the orientational distribution
function (odf) and may be represented as a 3D surface. Contour plots of these surfaces are shown for
the three samples in Figure 3. The peaks (light
regions) represent 31P chemical shift tensor orientation that occur commonly in these macromolecular DNA samples.
The A-form NaDNA sample (Figure 3(a)) shows
the largest intensity at (aPD,bPD) ˆ (65 72 ), with a
less intense peak at (0 ,60 ). The odf of the B-form
LiDNA sample, displayed in Figure 3(b),
shows two distinct peaks, one larger at
(aPD,bPD) ˆ (0 ,60 ), and one smaller at (aPD,bPD) ˆ
(0 ,22 ). The odf of the C-form LiDNA sample,
displayed in Figure 3(c), shows peaks at the same
places as the B-form sample, but with more intensity at the bPD ˆ 22 orientation. Similar results
were obtained by Song et al.(1997).
In the following discussion, we denote the three
odf peaks PA for the most intense peak in the
A-form odf (aPD,bPD) ˆ (65 ,172 ), Pl for the most
intense peak in the B and C-form odfs
(aPD,bPD) ˆ (0 ,60 ) and P2 for the less intense
peak in the B and C-form odfs (aPD,bPD) ˆ (0 ,22 ).
The relative proportions of the phosphate orientations in the three samples were deduced by integrating the distribution functions over suitable
angle ranges, and are given in Table 1.
These experimental results indicate the presence
of three distinct clusters of phosphate orientations
in the ®ber samples. In order to convert these
orientational distribution functions into molecular
structural information, the Euler angles (aPD,bPD)
must be converted into Euler angles (aMH,bMH)
describing the relative orientations of the phosphate groups with respect to the helix. By taking
into account the width of the odf peaks, the uncertainty in the orientation of the 31P CSA principal
axis systems, and the possible divergence of the
helix axis with respect to the director, we obtain
545
A BII Model for C-form DNA
the experimental determination of (aMH,bMH).
Replacement of aMH by aMH or 180 aMH, and
replacement of bMH by 180 bMH, in all possible
combinations (eight in total), leads to indistinguishable solid-state NMR results.
If desired, the Euler angles aMH and bMH may be
related to the commonly used phosphate conformational angles OO and OPO (Pohle et al., 1984).
The angle OO describes the angle between zM and
the helix axis, while OPO describes the angle
between yM and the helix axis. The angle parameters are related through cos(OO) ˆ cos(bMH)
and cos(OPO) ˆ sin(bMH)sin(aMH).
PA : …OPO ; OO † ˆ …30 12 ; 72 8 †
P1 : …OPO ; OO † ˆ …89 11 ; 60 9 †
…2†
P2 : …OPO ; OO † ˆ …90 7 ; 23 8 †
The angles OPO and OO are easier to visualize
than the Euler angles aMH and bMH, and give a
clearer indication of the error margins in the phosphate orientations, which can be seen to be
approximately the same for PA, P1 and P2. In our
analysis, we use the Euler angles, because of their
more convenient mathematical properties.
A representation of the PA, P1 and P2 orientations
is shown in Figure 4. These orientations differ
slightly from those given by Song et al. (1997).
Note that the orientations Pl and P2 both have the
O10 -P-O20 bisector perpendicular to the helix axis,
and that the O10 -P-O20 plane is almost parallel
with the helix axis in the P2 orientation.
31
Figure 3. Contour plots of the phosphate orientational
distribution p(aPD, bPD) for the: (a) low-salt A-form
NaDNA sample (sample A); (b) high-salt B-form LiDNA
sample (sample B); and (c) low-salt C-form LiDNA
sample (sample C).
the following phosphate orientations and their con®dence limits:
PA : …aMH ; bMH † ˆ …65 9 ; 72 8 †
P1 : …aMH ; bMH † ˆ …0 13 ; 60 9 †
…1†
P2 : …aMH ; bMH † ˆ …0 30 ; 23 8 †
When considering the phosphate orientations, it
is necessary to take into account the ambiguity in
P isotropic chemical shifts
A detailed examination of the 2D 31P lineshapes
(see Materials and Methods) indicates that the P1
and P2 orientations are associated with slightly
different isotropic chemical shifts. The 31P isotropic
chemical shift for phosphate groups with the P2
orientation is 1.8 ppm higher (i.e. less shielded)
than that for phosphate groups with the P1 orientation. As discussed below, the 1.8 ppm difference
between the 31P isotropic chemical shifts of the P1
and P2 orientations provides useful information on
the backbone torsion angles a and z.
13
C NMR
Figure 5 shows the 13C CPMAS spectra for the
three samples acquired under 1H decoupling at a
spinning frequency of 2.6 kHz. We used the 13C
resonance of the thymidine CH3 group as chemical
shift reference at d ˆ 14.6 ppm (Leupin et al., 1987).
The NaDNA A-form spectrum ((a)) is signi®cantly
different from that of the LiDNA B-form ((b)) and
C-form ((c)) spectra, while the latter two are almost
identical, disregarding the two resonances at 49
and 61 ppm (indicated by asterisks), which stem
from the polypropylene foil used to wrap the
samples (this was veri®ed by adding foil, whereupon these signals grew in intensity). The most
546
A BII Model for C-form DNA
Table 1. Relative populations of the phosphate orientations in the samples used
DNA sample
A
B
C
PA (%)
P1 (%)
P2 (%)
P2/P1
50
3
3
21
59
51
20
36
45
0.61
0.88
The relative populations were found by evaluation of the integral
Z a2 Z b2
p…aPD ; bPD † sin bPD dbPD daPD ;
a1
b1
with p(aPD,bPD) the orientational distribution functions as displayed in Figure 3. The integration limits were chosen as follows: PA,
(a1 ˆ 40 , a2 ˆ 90 , b1 ˆ 50 , b2 ˆ 90 ); P1, (a1 ˆ 0 , a2 ˆ 30 , b1 ˆ 38 , b2 ˆ 80 ); and P2, (a1 ˆ 0 , a2 ˆ 90 , b1 ˆ 0 , b2 ˆ 38 ).
noticeable variations occur in the 60-80 ppm
region, where resonances are found at 79 and 69
ppm for the B and C forms, while resonances are
found at 71 and 64 ppm for the A form. This is in
accordance with the isotropic chemical shifts for
the C30 (higher shift) and C50 (lower shift) atoms of
these different DNA forms as reported by Santos
et al. (1989), who saw these differences in the 13C
spectra for the A and C forms of DNA. The other
sugar resonances are located at 39 ppm (C20 ) and
86 ppm (C10 and C40 ), while resonances at values
higher than 90 ppm are from aromatic 13C sites,
and belong to the bases.
The sugar 13C chemical shifts of the B and Cform sample agree well with those found for Bform oligonucleotides in solution state NMR
(Leupin et al., 1987), which are known to occur in
the C20 -endo puckering mode. The 5-10 ppm shift
for the C30 and C50 resonances towards lower
values of chemical shift d for the A-form sample
was seen by Santos et al. (1989). We take the similarity between B and C-form 13C spectra as evidence that the sugar puckering in nucleotides
associated with the P1 and P2 phosphate orientations are both in the C20 -endo region.
Structural Model Building
As discussed by Song et al. (1997), and treated
further below, the PA phosphate orientation
extracted by 31P solid-state NMR agrees well with
that found in A-form DNA crystal structures of oligonucleotides and A-form models from ®ber diffraction. The P1 and especially the P2 phosphate
orientations, however, are not in agreement with
those from previous models for the B and C forms
DNA based on ®ber X-ray diffraction. In particular,
the P2 phosphate orientation has not been found
before in models for either B or C-form DNA. We
therefore built models to combine our information
on the ®t phosphate orientations with the information on the helix parameters available from the
X-ray diffraction patterns.
The conformation of a nucleotide in DNA is
fully speci®ed by using seven torsional angles (a,
b, g, d, e, z, and w), under the assumption that the
bond lengths and three-atom bond angles are
known. We use the standard labeling of the atoms
and the torsional angles (see Figures 2 and 3 of
Saenger, 1984).
In order to simplify the discussion, we use
the following nomenclature: cis ˆ 0(30) (c),
gauche‡ ˆ 60(30) (g‡), anticlinal‡ ˆ 120(30) (a‡), trans ˆ 180(30) (t), anticlinal ˆ 240(30) (a ) and gauche ˆ 300(30) (g ) for the ranges of
the torsional angles. The notation c/g‡ is used to
designate a torsional angle on the border of cis and
gauche‡, while the symbol [c,g‡] is used to denote
the combined ranges of cis and gauche‡ ( 30 to
90 ). We use for the twist, h for the helical rise
per residue, p for the pitch, y for the tip angle, and
Z for the inclination, as recommended by the Cambridge DNA Nomenclature Accord (Diekmann,
1989).
X-ray ®ber diffraction and solid-state NMR are
complementary methods. X-ray ®ber diffraction
provides high-quality information as to the separation of the bases (the helical rise per residue) and
pitch of the helix, while solid-state NMR provides
detailed information on the orientation of the phosphate groups, and qualitative information on the
torsional angles a, d and z, through the 31P and 13C
chemical shifts. We constructed structural models
of DNA double helices as found in ®bers combining these complementary pieces of information.
A computer program was written that takes the
aMH and bMH angles from our 31P NMR measurements, and combines this information with constraints on the winding angle and the helical rise
per residue from ®ber X-ray diffraction. The DNA
models are restricted to be right-handed, antiparallel, double helices with virtually planar WatsonCrick pairing base-pairs and a (pseudo) dyad axis
perpendicular to the helix axis along the x-axis of
the base-pair (Diekmann, 1989) (the dyad axis travels along with the helical twist). Restrictions were
placed on the inclination and the tilt of the bases,
and atoms more than three bonds away from each
other were not allowed within van der Waals distance. Alternating m5CG base-pairs were used, the
sequence thus being (m5CG)n, where 5-methylcytosine (m5C) was used as a pseudo base to account
for the bulky methyl group of thymidine.
A BII Model for C-form DNA
547
Figure 4. A representation of the
phosphate orientations: (a) PA
(aMH,bMH) ˆ (65 ,72 );
(b)
P1
(aMH,bMH) ˆ (0 ,60 ); and (c) P2
(aMH,bMH) ˆ (0 ,22 ). The local
molecular symmetry axis system M
is shown. The O10 -P-O20 plane is
indicated by a wedge.
The P1 phosphate orientation
The P1 phosphate orientation (aMH,bMH) ˆ
(0 13 , 60 9 ) is the dominant one in B-form
DNA according to our measurements (see Table 1).
It is likely that at higher relative humidity, which
is known to favor the B form of DNA, the P1 conformation would be even more dominating. We
548
A BII Model for C-form DNA
Figure 5. 13C CPMAS spectra for:
(a) low-salt A-form NaDNA sample
(sample A); (b) high-salt B-form
LiDNA sample (sample B); and (c)
low-salt C-form LiDNA sample
(sample C). Asterisks, at 48 and 60
ppm, indicate the peaks generated
by the polypropylene foil in which
the samples were wrapped (see
Materials and Methods).
therefore construct a B-form DNA model consisting only of P1-type phosphate groups and with the
Ê , ˆ 36.0 B-form helix parameters h ˆ 3.38 A
(corresponding to 101 symmetry).
The angles (aMH,bMH) carry an ambiguity in
their sign, as described above. On basis of the fair
agreement of our P1 phosphate orientation using
the interpretation (aMH,bMH) ˆ (0 13 , 60 9 )
with that of previous models for B-form DNA and
with B-form DNA oligonucleotide phosphate
orientations (Song et al., 1997), we used this
interpretation only when building B-form structural models.
We scanned through conformational space in the
ranges 270 4 a 4 360 [g ,c], 120 4 d 4 160 [a‡,t], 150 4 e 4 270 [t,a ], 150 4 z 4 270 [t,a ], which covers all of the previously suggested
B forms and extends over the full BI and BII
nucleotide ranges, excepting extremes (see
Schneider et al., 1997). The range for d is the
full C20 -endo range (Saenger, 1984), in agreement
with the 13C chemical shift data. We used
(aMH,bMH) ˆ (0 13 , 60 9 ) as determined by
experiment, allowing the phosphate angles to vary
within their error limits (see Materials and
Ê
Methods). Using h ˆ 3.38 0.1 A
and ˆ
36 1 , 14 structures could conform to the
constraints (see Materials and Methods). The structures that emerged had the following ranges of
torsional angles: a ˆ 290-318 (g ), d ˆ 122-136 (a‡), e ˆ 224-238 (a ), z ˆ 224-246 (a ) and
w ˆ 226-250 (a ). In Figure 6, 12 base-pairs of the
best structure, judged by h and , is shown. It has
the torsional angle set: a ˆ 316 , b ˆ 126 , g ˆ 50 ,
d ˆ 126 , e ˆ 226 , z ˆ 224 and w ˆ 229 . (The
coordinates for this 101 BI double helix are available as Supplementary Material).
The structure is similar to the B-form DNA
model described by Premilat & Albiser (1983). It is
Ê as opposed to 9.3 A
Ê , but
slightly wider, 9.5 A
otherwise has practically the same inclination and
groove widths as the Premilat and Albiser B-form
549
A BII Model for C-form DNA
Figure 6. A model for B-form
DNA consisting purely of BI-type
nucleotides. The symmetry is 101
and the phosphate angles are
aMH ˆ 13 , bMH ˆ 51 . The basesequence is (m5CG)n. (a) Stick
model of 12 base-pairs. (b) Space®lling model of 12 base-pairs.
(c) Top view of two adjacent basepairs. The intersection of the
(pseudo C10) helical symmetry axis
with the plane is shown by a circle.
model (see Table 3). The nucleotide torsional angle
set in our model is close to that of the nucleotide in
Premilat and Albiser's B form, but with the noticeable difference that the e angle is in the a range,
although close to the t range, while the Premilat
and Albiser' B-form nucleotide has e in the t range
but close to the a range. The model is also quite
close to the B-form model described by
Chandrasekaran & Arnott (1996), but clearly inconsistent with the older B-form model described by
Arnott & Hukins (1972).
accounts for 45 % of the phosphate groups, has
not been described in DNA models from ®ber Xray diffraction. In order to propose a structural
model associated with this orientation, we require
estimates for the helical rise per residue, h, and the
winding angle, , for the nucleotides.
Previous structural models of C-form DNA have
interpreted the X-ray diffraction pattern of this
form in terms of a uniform conformation with
Ê and ˆ 38.6 (283
helical parameters h ˆ 3.32 A
symmetry) or possibly ˆ 40.0 (91 symmetry)
(Marvin et al., 1961; Arnott & Selsing, 1975). However, it is now known that the C form contains two
distinct phosphate orientations, presumably associated with different nucleotide conformations. Furthermore, the sample used in the solid-state NMR
studies may have a P1/P2 ratio different from
The P2 phosphate orientation
The P2 phosphate orientation, (aMH,bMH) ˆ
(0 30 ,23 8 ), found in the odf of B-form DNA
and especially in the odf of C-form DNA, where it
Table 2. Torsional angles for ®brous DNA models and oligonucleotide crystal averages, grouped into categories
DNA form
a
b
g
‡
d
‡
e
z
w
Reference
Arnott & Hukins (1972)
Premilat & Albiser
(1986)
Chandrasekaran et al.
(1989)
Schneider et al. (1997)
Arnott & Hukins (1972)
Premilat & Albiser
(1983)
Chandrasekaran &
Arnott (1996)
Schneider et al. (1997)
Schneider et al. (1997)
Marvin et al. (1961)
Arnott & Selsing (1975)
This work
This work
Arnott A
g
t
g
g
t
g
t
Premilat A
g
t
g‡
g‡
a
g
t
Chandrasekaran A
Oligo A
Arnott B
g
g
g
t
t
a
g‡
g‡
g‡
g‡
g‡
t
t/a
[t,a ]
t
g
g
a
t
t
a
Premilat B
g /c
a‡/t
g‡
a‡
t
a
a
Chandrasekaran B
Oligo BI
Oligo BII
Marvin C
Arnott C
BI 101
BII 91
g /c
g
g
g
g
g
[g ,c]
a‡
[a‡,t]
[a‡,t]
a‡
t
a‡
a‡
c/g‡
g‡
g‡
g‡
g‡
g‡
g‡
a‡
a‡
a‡
a‡
t
a‡
[a‡,t]
a
t
a
t/a
t
a
[t,a ]
t
[a ,g ]
t
t/a
a
a
t
a
a
a
a
a
a
[a ,g ]
Table 3. Phosphate group angles, torsional angles, helical parameters and groove widths for DNA models from ®ber X-ray diffraction, and mean values for the torsional
angles of oligonucleotide X-ray crystallography
DNA form
aMH
bMH
a
b
g
d
e
z
w
r(P)a
h
Mgb
mgb
Zc
yd
P-Pe
Arnott A
Premilat A
Chandrasekaran A
Oligo A mean
Arnott B
Premilat B
Chandrasekaran B
Oligo BI mean
Oligo BII mean
Marvin C
Amott C
BI 101 modelf
BII 91 modelg
BII 91 statistics of
24 best structuresh
74
52
61
41
23
19
13
31
13
0
304
430i
77
65
61
77
57
42
44
73
51
23
154
431i
275
300
308
293
313
329
330
298
298
315
321
316
330
330
16
208
164
175
174
214
149
136
176
146
143
200
126
114
122
13
46
51
42
56
36
37
31
48
48
48
37
50
49
50
8
85
81
79
81
156
132
143
128
144
141
157
126
138
145
9
178
224
212
203
155
203
219
184
246
211
161
226
228
221
13
311
273
285
289
265
226
199
265
174
212
254
224
180
181
10
206
195
203
199
262
240
262
250
271
263
263
229
254
266
16
8.9
8.9
8.6
8.9
9.3
9.2
9.05
8.7
9.5
8.4
8.7
0.4
2.56
2.82
2.55
3.38
3.39
3.38
3.32
3.31
3.38
3.32
3.32
0.02j
32.7
32.7
32.7
36.0
36.0
36.0
38.6
38.6
36.0
40.0
40.0
0.2j
8.2
10.7
8.0
17.3
17.3
17.2
16.1
14.6
17.1
16.0
15.0
1.2
16.7
16.6
16.8
11.7
12.5
11.7
11.7
10.7
13.8
9.9
11.4
1.5
18
21
23
6
1
4
5
8
3
17
14
4
1
6
2
1
4
7
6
2
3
4
4
2
5.6
5.8
5.5
6.5
6.7
6.6
6.8
6.6
6.7
6.6
6.8
0.2
Ê ngstroÈm (A
Ê ) for distances. References are as in Table 2.
Units are degrees ( ) for angles and A
a
Radial coordinate of the phosphorus atom.
b
Major (Mg) and minor (mg) groove widths measured as the distance between phosphorus atoms on different strands, spanning the groove. The phosphorus atoms are selected by drawing
lines between neighboring phosphorus atoms on the same strand, and then selecting pairs of phosphorus atoms on different strands that lie closest to a line that is perpendicular to the intrastrand P-P lines.
c
Measured as the angle between the C6 to N3 vector in cytosine and the projection of it onto the plane perpendicular to the helix axis.
d
Measured as the angle between the C2 to C4 vector in cytosine and the projection of it onto the plane perpendicular to the helix axis.
e
Distance between adjacent phosphorus atoms on the same strand.
f
Model as in Figure 6.
g
Model as in Figures 7 and 8.
h
Mean value (top row) and standard deviation (bottom row) of 24 best 91 BII structures (see the text).
i
Ranges of aMH and bMH, respectively.
j
Limits on h and used for selection of the 24 best structures (see the text).
551
A BII Model for C-form DNA
those giving rise to the X-ray diffraction patterns
on which the previous structural models are based.
In this work, we assume that the nucleotide corresponding to the P2 phosphate orientation has a
Ê and a winding
helical rise per residue of h ˆ 3.32 A
angle of ˆ 40 . This winding angle is at the high
end of estimated winding angles for the C form: in
this way we try to take into account the fact that
the C form appears to be a mixture of a low-winding-angle BI form and a second conformation that
we wish to determine. In any case, the small difference between 283 symmetry, as both right-handed
C-form DNA models have (Marvin et al., 1961;
Arnott & Selsing, 1975), and a 91 helical symmetry
produces no signi®cant difference to the outcome
of the modeling.
In addition to the X-ray constraints, we use the
13
C chemical shift data, which indicates a C20 -endo
sugar conformation.
Due to the symmetries involved, as mentioned
above, the 31P 2D spectra may be interpreted in
terms of the following four possible ranges for the
P2 phosphate orientation: (aMH,bMH) ˆ (030 ,
(180 30 , 23 8 ), (0 30 , 157 8 ) and
(180 30 , 157 8 ). All these (aMH,bMH) combinations were tried in the modeling, which
extended over the full conformational space within
the C20 -endo range, i.e. ranges 0 4 a 4 360 ,
120 4 d 4 160 , 0 4 e 4 360 and 0 4 z
4 360 . Out of approximately 1010 modeled
nucleotides with the observed P2 phosphate orientation, only about 200 nucleotide conformations
were consistent with the assumed axial rise
Ê , the assumed winding angle
h ˆ 3.320.1 A
ˆ 401 , and the C20 -endo sugar pucker. These
structures fell into two groups, which may be
denoted by the torsional angle ranges (a,b,g,d,e,z,
and w) of the nucleotide:
…‰g ; cŠa‡ g‡ ‰a‡ ; tŠ‰t; a Št‰a ; g Š†
…3a†
…g‡ a t‰a‡ ; tŠa t‰a ; g Š†
…3b†
and:
Both these angle combinations are consistent with
the P2 31P isotropic chemical shift, which is 1.8
ppm higher (more deshielded) than that of the P1
phosphate
group.
Theoretical
calculations
(Gorenstein & Kar, 1975; Gorenstein, 1983) have
shown that the angle set az ˆ gt, regardless of the
sign of the a angle, should resonate 1-2 ppm higher
than the BI angle set az ˆ g [g ,a ]. This property
has been used in solution NMR to identify BII
nucleotides, which have the angle set az ˆ g t
(Gorenstein et al., 1988; Gorenstein, 1992).
Although the conformations (3a) and (3b) are
both consistent with the measured P2 phosphate
orientation, the 31P isotropic chemical shift and the
13
C chemical shifts, the conformation (3b) may be
excluded on energetic grounds. Theoretical energy
calculations on nucleotides and nucleosides have
shown that b torsion angles in the range [a‡,t] and
g torsion angles in the [g‡,c] range are energetically
favorable. Indeed, all known right-handed nucleotides adopt bg torsional angles in this range
(Schneider et al., 1997). The bg ˆ a t conformation
of (3b), on the other hand, is highly unfavorable
energetically (Saran et al., 1972).
Our conclusion is that (3a) is the correct structure for the nucleotide associated with the P2
phosphate orientation. The conformation (3a) is
very close to the typical BII conformation found in
oligonucleotides (Hartmann et al., 1993; Schneider
et al., 1997). The BII nucleotide conformation is
often distinguished from the BI nucleotide conformation via the value of e-z, which is around ‡90 for BII nucleotides, and around 90 for BI nucleotides (Fratini et al., 1982; Hartmann et al., 1993;
Bertrand et al., 1998). For our structure (3a), we
®nd 2 4 (e-z) 4 74 for (aMH,bMH) ˆ (0 30 ,
23 8 ). This range of e-z is consistent with the e-z
vales for BII nucleotides that have been found in
oligonucleotide crystals (Hartmann et al., 1993),
although theoretical energy calculations support a
value of e-z between 100 and 150 (Bertrand et al.,
1998). Energy calculations show the BII nucleotide
to have a stable twist of ˆ 40 (Bertrand et al.,
1998), as does our 91 double helix.
A superposition of the 24 best structures of
type (3a), judged by their winding angles
(
ˆ 400.2 ) and axial rise per residue
Ê ), is shown in Figure 7(b). All these
(h ˆ 3.320.2 A
structures are of the BII type, with a torsion angle
set (a, b, g, d, e, z, w) ˆ ([g ,c], a‡, g‡, [a‡,t], [t,a ], t,
[a ,g ]), and their corresponding 91 double helices
have similar overall features. The average torsional
angles and helical parameters for these 24 structures, together with their standard deviations, are
displayed in Table 3.
A pure BII helix of symmetry 91 with pitch
Ê for aMH ˆ 0 and bMH ˆ 23 is depicted in
3.32 A
Figures 7(a) and 8. This particular structure has
torsional angles a, b, g, d, e, z, and w close to the
average of the structures in group (3a) and the aMH
and bMH angles at the center of the orientational
distribution for the phosphate group. The torsional
angles are a ˆ 330 , b ˆ 114 , g ˆ 49 , d ˆ 138 ,
z ˆ 180 , and w ˆ 254 . The structure displays a
strikingly deep and narrow minor groove, and a
very broad and shallow major groove, as is clearly
seen in the space-®lling model shown in Figure 8.
Ê wide and the major
The minor groove is 10 A
Ê wide, as estimated from P-P
groove is 16 A
distances (see the legend to Table 3 for details).
Adjacent phosphorus atoms on the same strand
Ê apart, the radial coordinate for the P
are 6.6 A
Ê , i.e. about 1 A
Ê smaller than for BI
atom being 8.4 A
DNA. The helical pseudo C9 symmetry axis lies far
into the minor groove of the structure, as clearly
seen in the top view, Figure 7(c). The base plane
normals are inclined by around 17 from the helix
axis, and there is a small tip of 4 . The closest
distance between unesteri®ed oxygen atoms of
Ê , which is over the minor
phosphate groups is 8.7 A
552
A BII Model for C-form DNA
Figure 7. A 91 DNA model consisting of purely BII-type nucleotides with aMH ˆ 0 , bMH ˆ 23 . The base sequence
is (m5CG)n. (a) Stick model of 12 base-pairs; (b) superposition of the 24 best 91 structures; and (c) top view of two
adjacent base-pairs. The intersection of the (pseudo C9) helical symmetry axis with the plane is shown by a circle.
groove. (The coordinates for this 91 BII doublehelix are available as Supplementary Material.)
The combined X-ray diffraction and solid-state
NMR constraints lead to the conclusion that the
typical phosphate orientations P1 and P2 found in
B and C-form DNA correspond closely to the
nucleotide conformations BI and BII found in oligonucleotide crystals. Note that the modeling procedure does not assume a correspondence between
the phosphate orientations P1/P2 and the nucleotide conformations BI/BII from the beginning. The
modeling procedure itself excludes all other possibilities, with the exception of one postulated form,
which could be ruled out on energetic grounds.
This is strong evidence that our structural models
are essentially unique, except for possible ®ne
adjustments.
Discussion
The torsion angles and structural parameters for
the new BI 101 double helix and the new BII 91
double helix are compared with previously
suggested models of A, B and C-form DNA in
Tables 1 and 2. All of these structures are righthanded, antiparallel double-stranded helices with
Watson-Crick base-pairs. We exclude the
suggested left-handed C forms (Premilat & Albiser,
1984), since these are unlikely to be compatible
with the non-cooperativity of the B to C transition
as evidenced by mechanochemical experiments
(Rupprecht et al., 1994), circular dichroism (Bokma
et al., 1987) and ®ber X-ray diffraction
(Skuratovskii & Bartenev, 1979).
The Tables include the torsional angle ranges for
A, BI and BII nucleotides obtained from compilation of the nucleotide conformations in 34 B-form
and 30 A-form oligonucleotides, as determined by
single-crystal X-ray diffraction (Schneider et al.,
1997).
The new ®ber BI and BII structures are in good
agreement with observed structural motifs for oligonucleotides. The new BI structure is in reasonable agreement with the B-form models described
by Premilat & Albiser (1983) and by
Chandrasekaran & Arnott (1996). There is, how-
553
A BII Model for C-form DNA
Figure 8. Stereo view of a space-®lling model of the
91 BII double helix (the same as in Figure 7) showing
the shallow major goove and the deep minor groove.
ever, little agreement between the new BII structure and previous models of C-form DNA, except
for the C form structure described by Marvin et al.
(1961), which is quite close. We conclude that the
®ber diffraction pattern of C-form DNA, which is
poor in information due to the bad crystallinity of
the C form, has previously been misinterpreted,
presumably because of the existence of two conformational populations in the samples
This point is emphasized by Figure 9, which
shows the ranges of (aMH,bMH) for the PA, P1 and
P2 phosphate orientations, as well as those found
in DNA structural models. There is good mutual
agreement between the three ®ber X-ray diffraction
A-form models (Arnott & Hukins, 1972; Premilat &
Albiser, 1986; Chandrasekaran et al., 1989), and
with the PA phosphate orientation. For the B and
C-form models from X-ray diffraction (Arnott &
Hukins, 1972; Premilat & Albiser, 1983;
Chandrasekaran & Arnott, 1996; Marvin et al.,
1961; Arnott & Selsing, 1975) on the other hand,
there is little mutual agreement in the phosphate
orientations. However, some of the B and C-form
phosphate orientations are fairly close to those of
the P1 and P2 phosphate from solid-state NMR. A
selection of phosphate orientations that have been
termed BII in oligonucleotide crystal studies
(Fratini et al., 1982; Prive et al., 1991; Grzeskowiak
et al., 1991) are shown in Figure 9. They display a
wide scatter, with some clustering around the P2
phosphate orientation.
The existence of BII nucleotides in C-form DNA
was suggested before, in conjunction with static
31
P NMR of oriented ®bers (Shindo et al., 1984;
Shindo, 1985). However, these results were interpreted in terms of three phosphate orientations
(called BI, BII and C), which we now know to be
incorrect.
Figure 9. The aMH and bMH angles for various DNA
models (symbols) and the aMH and bMH angle ranges
found in this investigation; PA, P1 and P2 (boxes). Diamonds are for A-form DNA models, circles for B-form
DNA models, and squares for C-form DNA models. See
Table 3 for references. Crosses are for phosphate orientations found in BII nucleotides in oligonucleotide crystals. The crosses at (aMH,bMH) ˆ (18 ,31 ) and (26 ,48 )
correspond to the G10-C11 and G22-C23 steps described
by Fratini et al. (1982), respectively. The crosses at
(aMH,bMH) ˆ (13 ,22 ) and (35 ,14 ) correspond to the
C12-A13 and T8-G9 steps of the CG octamer described
by Prive et al. (1991), respectively. The cross at
(aMH,bMH) ˆ (17 ,38 ) corresponds to the T4-C5 step
described by Grzeskowiak et al. (1991).
The BI/BII helix and the C form of DNA
The pure BII helix shown in Figures 7 and 8
is an idealization. In reality, our C-form sample
contains around 51 % BI and 45 % BII nucleotides.
Oligonucleotide structures have shown that BII
conformations are most often observed at pyrimidine-purine (Y-R) steps and rarely at pyrimidinepyrimidine (Y-Y) steps (Winger et al., 1998;
Bertrand et al., 1998), indicating a base-sequence
depencence of BII occurence. At the moment, it is
not known whether the BI and BII nucleotide conformations in our DNA ®bers occur in a contiguous sequence or in a pseudo-random intimate
mixture. These questions may be addressed by
further solid-state NMR experiments exploiting
through-space 31P-31P couplings, which are
planned.
An intimate mixture of BI and BII is common in
crystals (Fratini et al., 1982; Grzeskowiak et al.,
1991; Prive et al., 1991). A mixed BI/BII model is
supported by the non-cooperativity of the B to C
form transition, as studied by mechanochemical
experiments (Rupprecht et al., 1994), circular
dichroism (Bokma et al., 1987) and ®ber X-ray diffraction (Skuratovskii & Bartenev, 1979). An intimate mixture of BI and BII nucleotides may
explain the infra-red linear dichroism (IRLD)
554
results reported by Pohle et al. (1984), who determined C-form phosphate orientations that are very
different from our ®ndings. These IRLD measurements were ®t to a single phosphate orientation,
which the solid-state NMR data show to be an
oversimpli®cation. More recent IR absorption
investigations have demonstrated the existence of
BII substates in ®brous B-form DNA (Pichler et al.,
1999, 2000). Intimate mixing of BI and BII nucleotides might explain the lack of long-range order in
the DNA ®bers and therefore the diffuse X-ray diffraction features.
Our model of the 91 BII helix is to be taken as an
extreme case, in which nine contiguous BII nucleotides make up an entire turn of the helix. It is unlikely that a full helix turn exists in our C-form
sample, although occasional contiguous sequences
of three or four BII nucleotides are to be expected
on statistical grounds, assuming that the incidence
of these conformations is random and uncorrelated. The approximate validity of a pseudorandom BI/BII conformational model is supported
by the observed non-cooperativity of the B to C
transition.
Unfortunately, it is not feasible at this time to
reconstruct the X-ray diffraction pattern using the
new BI/BII model. One would need information
about the distribution of the two conformations
and about the packing of the helices. The diffuse
X-ray pattern suggests an intimate mixture of conformations with low repetitive order. This further
complicates the matter, since the unit cell is not
well de®ned.
The BI to BII transition
This work shows that the B to C form transition
in ®brous DNA may now be interpreted in terms
of BI to BII conformational changes. The numerous
studies on the B to C-form transition have become
relevant for the understanding of the BI/BII conformational equilibrium. In particular, it is now
clear that metal ions of high charge density, i.e. Li‡
and Mg2‡, favor the BI to BII conversion, and
hence the transition of DNA from the B form to the
C form at low relative humidity.
When complexed with less densely charged
counterions, as Na‡, K‡ and Cs‡, DNA takes on
the A-form at low water content (Rupprecht
et al., 1994; Schultz et al., 1992; Weidlich et al.,
1988). The stability of the A form of DNA at
low relative humidity has been attributed to the
existence of a network of water molecules, making it possible for the phosphate groups to share
water molecules for hydration (Saenger, 1984;
Saenger et al., 1986 and references therein). A
molecular dynamics investigation of Li, Na and
CsDNA showed that the Li ions can bind very
tightly to the unesteri®ed phosphate oxygen
atoms with no water mediation but with a joint
hydration shell around the Li-phosphate complex
(Lyubartsev & Laaksonen, 1998). This conclusion
is supported by NMR diffusion studies, which
A BII Model for C-form DNA
determined very slow diffusion for Li‡ as compared to Cs‡ (van Dam et al., 1998). A change
in phosphate hydration pattern upon the BI to
BII transition has been implicated in several molecular dynamics studies (Winger et al., 1998;
Pichler et al., 2000). These pieces of evidence
suggest that Li‡ and Mg2‡ inhibit the formation
of the A form at low humidity by breaking up
the hydration shells of the phosphate groups
and preventing the formation of A-form
hydration bridges. It is possible that charge neutralization of the phosphate group upon complexation with a densely charged cation induces
or stabilizes the BII conformation. Charge neutralization of the nucleotide has a large effect on
its conformation (Gurlie & Zakrzewska, 1998;
Gurlie et al., 1999), although some evidence
speaks against BII conformations being favored
by this effect (Tisne et al., 1999).
Possible biological implications
The present work has identi®ed the C form of
DNA as a BII-rich B form, and indicates that BII
nucleotides are stabilized by complexation of DNA
phosphate groups with densely charged cations.
The DNA used in the present study is polymeric
(approximately 75 kb) and of natural sequence. In
C-form DNA, around 45 % of the nucleotides are in
the BII conformation, which may be contrasted to
the mere occasional presence of BII nucleotides in
oligonucleotide crystals. It is reassuring that BII
nucleotides have now been clearly identi®ed both
in oligonucleotide crystals and in DNA ®bers of
natural length and base sequence.
Although Li‡ is not abundant in biological systems, the even more densely charged Mg2‡ does
occur physiologically in millimolar concentrations.
Solid-state NMR has shown that C-form MgDNA
displays a set of phosphate orientations similar to
that for C-form LiDNA (Song et al., 1997). This
suggests that Mg2‡ may disrupt the hydration of
the DNA phosphate groups and favor the BI to BII
transition. A molecular dynamics simulation of
DNA in the presence of Mg2‡ showed no correlation between Mg2‡ binding and BII conformations (Winger et al., 1998). However, this
simulation was carried out in large excess of water,
158 water molecules per nucleotide, whereas our
experiments at 66 to 75 % relative humidity correspond to around ten water molecules per nucleotide (Lee et al., 1987), i.e. a completed, or nearly
completed, inner DNA hydration shell but no
outer DNA hydration shell (Saenger, 1984).
C-form DNA is found under low-humidity conditions, and presumably the BI-BII interconversion
is sensitive to hydration conditions. Highly compacted low-humidity conditions for DNA may be
found in biological entities such as nucleosomes,
sperm and viral heads. Local dehydration occurs
upon protein binding to DNA, through exclusion
of water molecules (LundbaÈck & HaÈrd, 1996; HaÈrd
& LundbaÈck, 1996).
555
A BII Model for C-form DNA
It has been observed that the C form of DNA
®bers is favored at low humidity by the presence
of certain organic amines and polyamines
(Portugal & Subirana, 1985; Suwalsky et al., 1969).
This suggests that positively charged amino acids
(and by implication, certain protein side-chains)
may stabilize BII nucleotides. We are currently
investigating some amine-DNA and amino acidDNA complexes by solid-state NMR.
The BII structural model shown in Figures 7 and
8 has potential relevance for protein-DNA interactions and for the mechanism of DNA bending.
The model displays an overwinding of the helix, a
narrow and deep minor groove, and a wide and
shallow major groove, which are all features commonly found in protein-DNA complexes (Travers,
1993). The displacement of the bases into the major
groove may be a relevant property, since it may
facilitate external access to the base sequence. Furthermore, the BII model shows high base-pair inclinations, suggesting that BI-BII steps cause the
double helix to bend. Helix curvature associated
with BII conformations has been observed by solution NMR in a DNA hexadecamer related to the
HIV-1 kB binding site (Tisne et al., 1998).
Conclusions
31
P solid-state NMR shows that the same two
distinct phosphate orientations exist in the B and
the C forms of DNA ®bers, with different relative
populations for the two forms. We have built structural models for these two forms, using helical
parameters derived from X-ray diffraction and
additional constraints from 31P and 13C NMR. The
major form in both B and C-form DNA is found to
closely resemble known structural models of
B-form DNA, with a conformation close to that of
the BI nucleotides found in oligonucleotide crystals. The minor form in both B and C-form DNA is
close to the BII nucleotide conformation also found
in oligonucleotide crystals. This nucleotide can be
used to build an idealized BII double helix with a
winding angle of 40 and a helical rise per residue
Ê.
of 3.32 A
This 91 BII double helix has no resemblance to
previously proposed DNA models but is consistent
with observations of BII nucleotide conformations
in oligonucleotides, and recent observations of a
BII substate by infrared spectroscopy of B-form
DNA ®bers.
In conclusion, we have shown that previous
structural models of C-form DNA, based purely on
X-ray ®ber diffraction data, are incorrect. By combining X-ray and solid-state NMR information, a
more complex picture emerges, in which both BI
and BII conformations are involved. An idealized
structural model, based on repeating BII nucleotides, displays interesting features, such as the displacement of the bases to the outside of the helix in
the major groove, and the development of a very
deep minor groove. These features may have
relevance to DNA bending and to protein-DNA
interactions.
Materials and Methods
Sample preparation
Three samples, labeled A, B, and,C, were prepared
from highly polymeric DNA through the wet-spinning
method (Rupprecht, 1963, 1970).
Sample A, prepared from salmon testes DNA (Fluka),
was spun in an ethanol/water bath containing NaCl and
subsequently bathed in an ethanol/water (80:20, v/v)
solution containing 0.1 mM NaCl, the contents of the
bath being changed three times.
Samples B and C, prepared from ultra-pure calf thymus DNA (D. Lando, Belorussian Academy of Sciences,
Minsk), were spun in ethanol/water baths containing
LiCl and subsequently bathed in an ethanol/water
(80:20, v/v) solution containing 220 mM LiCI for sample
B, and 30 mM LiCl for sample C. The contents of the
baths were changed three times.
After the bathing, the samples A, B, and C were
slightly dried at ‡5 C, subsequently released from the
spinning cylinders, and then equilibrated over a saturated NaCl solution, providing 75 % relative humidity,
so that the ®bers merged into a ®lm. After this, the
samples A, B, and C were folded into NMR samples of
approximate dimension 14 mm 2.5 mm 2.5 mm,
keeping the ®ber direction parallel for all sheets.
Humidity control
The water content of the samples was adjusted by
equilibration in desiccators containing appropriate
saturated salt solutions. The salt solutions used were:
NaNO2 (66 % relative humidity) and NaCl (75 % relative
humidity) (O'Brien, 1948; Spencer, 1926).
X-ray diffraction
X-ray diffraction patterns were taken from pieces of
the same samples as used for the NMR studies. CuKa
Ê wavelength). The specimen to
rays were used (1.542 A
®lm distance was 50 mm. Typical exposure times were
around four hours, during which the sample was kept at
constant relative humidity in a sealed box containing the
appropriate salt solution.
NMR equipment
A Varian In®nity NMR system was used with magnetic ®eld of 4.7 T, giving a 1H Larmor frequency of
200.20 MHz, 81.04 MHz for 31P and 50.34 MHz for
13
C (see Levitt (1997) for the negative signs). The rather
low magnetic ®eld made it easier to work with these
lossy dielectric samples. A standard three-channel 6 mm
magic-angle-spinning (MAS) Apex-II probe from Varian
Instruments was employed. All experiments were performed at ambient temperature. The DNA samples were
wrapped in polypropylene foil (Freshcling) to retain
humidity, and mounted in the ZrO2 rotor so that the
®ber axis was perpendicular to the magic-angle-spinning
axis. Al2O3 powder was used to ®ll out the remaining
space between the sample and the rotor walls, to accomplish mechanical balance when spinning.
556
31
A BII Model for C-form DNA
daniso ˆ dzz
P NMR
Rotor-synchronized 31P 2D MAS spectra at a spinning
frequency of 2000 1 HZ kHz were recorded using a
standard pulse-sequence (Harbison & Spiess, 1986), with
a cross-polarization interval of 1 ms from 31P to 1H and a
1
H decoupling nutation frequency of around 50 kHz. The
radiofrequency pulse-sequence was synchronized with
the sample rotation by causing the pulse programmer to
wait for the optical tachometer signal. The synchronization interval tl was incremented in 16 equal steps spanning one rotor period. The complex 2D data sets s(tl,t2)
were Fourier transformed and phase-corrected to ®rst
order in both dimensions to produce the 2D spectra
S(ol,o2).
The peaks were integrated to obtain a 2D set of amplitudes a(k1,k2), where k1 and k2 are integer indices in the
o1 and o2 spectral dimensions, respectively. Due to the
perpendicular orientation of the DNA ®ber director with
respect to the spinning axis, spectral intensity is expected
to show up only at every other o1 slice (Schmidt-Rohr &
Spiess, 1994). This was indeed found to be the case, and
therefore only k1 ˆ even slices are shown in Figure 2.
The isotropic shift has the index (k1,k2) ˆ (0,0), and is
marked with an asterisk in Figure 2. The k1 index runs
from 6, the frontmost slice, to ‡6, the backmost slice,
in all 2D spectra in Figure 2. Sidebands to the left of the
isotropic peak have a negative sign of the k2 index, and
sidebands to the right of the isotropic peak have a positive sign of the k2 index (see Song et al. (1997) for more
information on the indexing). The orientational distribution function p(aPD,bPD) of the 31P CSA tensor with
respect to the ®ber director was obtained from a(k1,k2)
using an analysis method similar to that described by
Harbison et al. (1987). This consists of setting up simulations with distribution function components proportional to spherical harmonics, YLq(bPD,aPD), with L ˆ 0,
2, 4, . . . , Lmax and q ˆ L, L ‡ 1, . . . , L. These simulated
sub-spectra are superposed with variable coef®cients, in
order to obtain the best ®t to the experimental spectrum.
Only even ranks L are required, since the DNA molecules are bipolar. The ®tting procedure started with an
analysis using Lmax ˆ 2. The result of that procedure was
used as a starting point for a ®t with Lmax ˆ 4, and so
on, up to the ®nal ®t, which used Lmax ˆ 10. At each
step, the minimization function consisted of the meansquare deviation between experimental and simulated
sideband amplitudes, plus a penalty for negative probabilities, which was evaluated at a ®ne grid of points
spanning the {aPD, bPD} surface. This ®tting procedure
was found to be more stable and reliable than the unconstrained least-squares ®t described by Harbison & Spiess
(1986) and by Schmidt-Rohr & Spiess (1994).
The simulations require knowledge of the principal
values of the 31P CSA tensor. We estimate these principal
values by measurements on ``powdered'' LiDNA and
NaDNA ®bers, obtaining (daniso ˆ 92.5 ppm, Z ˆ 0.64)
for the LiDNA samples, and (daniso ˆ 98/.8 ppm,
Z ˆ 0.65) for the NaDNA sample. We checked that interchanging these values made little difference to the
results. The CSA values are speci®ed here using the
deshielding convention, with:
Z ˆ …dyy
diso
dxx †=daniso
The 31P isotropic chemical shifts for the P1 and P2 orientations were obtained by analyzing the spectrum from the
C-form sample (sample C) in more detail, as illustrated in
Figure 10(a), which shows an expansion of the peaks with
indices (k1, k2) ˆ (0, 1), (0,0), (0,1). The peaks have a
clearly non-Lorentzian form. An unconstrained ®t of the
®ve most intense peaks (k1k2) ˆ (0, 2), (0, 1), (0,0), (0,1)
and (0,2) to ten Lorentzians showed that two partly overlapping Lorentzians for every peak was likely to be a
good interpretation. The free ®tting resulted in the pairwise Lorentzian lines being 143, 144, 144, 137 and 144 Hz
apart, corresponding to 1.8 ppm, with linewidths ranging
from 130-170 Hz for the less shielded line and 145-185 Hz
for the more shielded. The MAS sidebands were within
5 Hz from the positions expected. This implies the existence of two different isotropic 31P chemical shifts in the Cform sample, 1.8 ppm apart. Figure 10(b) shows the ®t to
the (k1k2) ˆ (0, 1), (0,0), (0,1) peak, and Figure 10(c) the
residual intensity when the ®tted spectrum is subtracted
from the experimental.
Once the isotropic shift difference is known, it is possible to ®t the entire 2D spectrum to two partly overlapping Lorentzians per sideband using only the
amplitudes of the peaks as variables. This ®t was consistent throughout the 2D spectrum. It explains the occurrence of peaks with superposed positive and negative
components, as illustrated in Figure 10(d)-(f), where the
experimental (k1,k2) ˆ ( 2, 2), ( 2, 3) and ( 2, 4)
peaks and the ®tted Lorentzians for the C-form sample
are shown. This interpretation was already suspected by
Harbison and co-workers (Tang et al., 1989).
This ®tting procedure produced two 2D data sets
a1(k1,k2) and a2(k1,k2), which were used to extract orientational distribution functions as before. The resulting odfs
from an analysis with Lmax ˆ 10 are shown in Figure 11.
Although the odf lineshapes are somewhat distorted as
compared to those in Figure 3, it is clear that each isotropic chemical shift corresponds to one phosphate orientation, with the more shielded peak corresponding to the
P1 orientation in the phosphate odf, and the less shielded
corresponding to the P2 orientation. From the ®tted
intensities to the central k1 ˆ 0 slice, we ®nd that in our
C-form sample, 70 % of the phosphate groups are in
the P1 orientation and 30 % are in the P2 orientation.
These results are in only rough agreement with the integrals of the orientational distribution function (Table 1),
for reasons that are not understood at this point.
13
C NMR
13
C NMR MAS spectra were recorded using crosspolarization from 1H to 13C with a contact time of 1 ms,
followed by data acquisition under strong 1H decoupling
with a nutation frequency around 50 kHz. The spinning
frequency was 2.6 kHz.
Phosphate orientations
jdzz
diso j > jdyy
diso j5jdxx
diso ˆ …dxx ‡ dyy ‡ dzz †=3
diso j
The measured values of (aPD,bPD) must be converted
into the structurally signi®cant angles (aMH,bMH), where
P is the 31P CSA principal axis system, M is the local
molecular frame of the phosphate group (see Figure 4),
H is the helix frame, and D the director frame. This
557
A BII Model for C-form DNA
Figure 10. (a) The three most intense peaks, k2 ˆ 1, 0, 1, of the center slice, k1 ˆ 0 of the 31P rotor-synchronized
MAS spectrum for C-form LiDNA. (b) The unconstrained deconvolution (see the text) of (a) into two sets of Lorentzians. (c) The unconstrained deconvolution (see the text) of (a) into two sets of Lorentzians. (c) The residual intensity
when (b) is subtracted from (a). (d) Three peaks, k2 ˆ 4, 3, 2, of the k1 ˆ 2 slice of the same 2D spectrum as in
(a). (e) Constrained deconvolution (see the text) into two sets of Lorentzians. (f) The residual intensity when (e) is
subtracted from (d).
requires assuming certain sets of Euler angles
PM ˆ {aPM,bPM,gPM} and HD ˆ {aHD,bHD,gHD}, relating
the CSA principal axis frame to the molecular frame, and
the helical frame to the director frame, respectively.
Single crystal NMR studies of the model compound barium diethyl phosphate have shown the following
relation between the P and M axis systems: aPM ˆ 90.3 ,
bPM ˆ 8.9 and gPM ˆ 97.0 (Herzfeld et al., 1978). This
represents an offset from perfect coincidence of the P
and M frames by 7-13 . Similar PM angles have been
found in other diesteri®ed phosphate model compounds
(Kohler & Klein, 1977), as well as from theoretical studies
(Ribas Prado et al., 1979). We must also assume a spread
of the Euler angles HD describing the orientations of
DNA helix axes with respect to the ®ber director. In previous X-ray diffraction measurements, the spread
between the H and D frames was seen to have a standard deviation of around 2 in these kind of samples
(Rupprecht, 1970). This small deviation is supported by
the relatively narrow peaks in the determined distribution p(aMD,bMD), the width being roughly the same for
all three peaks PA, P1 and P2, see equation (2). In practice, we neglect the slight deviation between the H and
D frames.
We generated estimates for the angles (aPM,bPM) for
the PA, P1, and P2 orientations by (i) generating an
ensemble of CSA principal axes using the broadened dis-
tributions of (aPD,bPD) measured in the experimental odf
plots shown in Figure 3, where we took into account the
tendency for the odf peak to merge with its ``mirror
image'' about aPD ˆ 0 , as described by Schmidt-Rohr &
Spiess (1994), and (ii) generating an ensemble of local
molecular axis systems M with a standard deviation of
10 from the principal axis system P, in order to take
into account the uncertainty in the CSA principal axes.
The estimated standard deviation in the orientations
(aPD,bPD) were taken to be (5 ,5 ) for the PA peak,
(10 ,5 ) for the P1 peak, and (20 ,5 ) for the P2 peak. A
large set of angles (aMH,bMH) was generated from these
ensembles, and the results analyzed statistically, to
obtain the phosphate orientational angles and their standard deviations, given in equation (1).
Model building
The modeling computer program scans through a
speci®ed part of conformational space for models of
DNA consistent with certain criteria. The modeling is
completely unbiased and takes no account of the energies of the different conformations. Bond lengths and
three-atom bond angles for the backbone phosphate and
sugar units were set to average values as described
(Saenger, 1984, page 70 C20 -endo deoxyribose sugar, and
page 86 phosphate). The bond lengths and angles for the
558
A BII Model for C-form DNA
The modeling procedure was checked by feeding it
with the (aMH,bMH) angle pairs corresponding to the existing models of B and C-form DNA (see Table 3). All the
three existing B-form models and the two right-handed
C-form models were successfully reproduced by the modeling procedure to within 10 in torsional angles.
We estimate that around 1010 possible nucleotide conformations were checked in the modeling for the C form.
Details on the modeling procedure are available from
the authors upon request.
Acknowledgments
We thank Drs Allan Rupprecht and Nikolay Korolev
for making the DNA samples and for acquiring the
X-ray diffraction pictures. We thank Dr Latha Kadalayil
for discussions, and Dr Nikolay Korolev and Lic. Arvid
Nilsson for help. This work was sponsored by the Swedish Natural Science Research Council and the GoÈran
Gustafsson Foundation for Research in the Natural
Sciences and Medicine.
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Figure 11. Contour plots of the phosphate orientational distribution functions for the (a) more shielded
(lower d) peak and (b) less shielded (higher d) peak of
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0.1 A
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Edited by A. Klug
(Received 2 May 2000; received in revised form 12
September 2000; accepted 29 September 2000)
http://www.academicpress.com/jmb
Supplementary material comprising atomic coordinates for the DNA models is available from JMB
IDEAL.
A BII Model for C-form DNA
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