Volume 15 Number 14 1987
Nucleic Acids Research
Two-dimensional NMR investigation of a bent DNA fragment: assignment of the proton
resonances and preliminary structure analysis
Agustin Kintanar1, Rachel E.Klevit2 and Brian R.Reid 12
Departments of 'Chemistry and 2Biochemistry, University of Washington, Seattle, WA 98195, USA
Received March 9, 1987; Revised and Accepted June 10, 1987
ABSTRACT
The resonances of all the non-exchangeable protons (except 5'H and 5"H) of
d(CGAAAAATCGG) + d(CCGATTTTTCG), a putatively bent DNA duplex, have been
assigned using 'H two-dimensional nuclear magnetic resonance methods. The nuclear
Overhauser effect data indicate an overall B-form structure for this double-helical DNA
undecamer. However, several features of the NMR data such as some unusually weak
C8/C6 proton to Cl' proton NOE cross-peaks, the presence of relatively intense C2H to
Cl'H NOE cross-peaks, and unusual chemical shifts of some 2", 2', and 1' protons suggest
a substantial perturbation of the helix structure at the junctions and along the length of
the tract of A residues. These structural deviations are considered in terms of models of
DNA bending.
INTRODUCTION
In the last few years it has become apparent that the helix axis of certain doublehelical DNA sequences is bent in solution. The first evidence for DNA bending came from
the anomalously slow mobility of some restriction fragments from kinetoplast minicircles
of tropical parasites (1-3) and from bacteriophage A DNA (4) upon polyacrylamide gel
electrophoresis. A bent piece of DNA migrates slowly through a polyacrylamide gel due
to
its decreased ability to snake through the pores of the gel. In an elegant study, Wu and
Crothers (5) showed that the locus of the aberrant electrophoretic behavior in kinetoplast
DNA lay in a sequence containing recurring short stretches of 5 or 6 A residues with a
periodicity of about 10 base pairs. They proposed a model in which each oligo-A tract
induces a small kink or local helical distortion, and suggested that these defects are propagated in phase with the helical repeat (10-11 base pairs) resulting in a stable macroscopic
bend.
Recently, Hagerman (6) and Koo et al. (7) carried out a systematic study of synthetic
model DNA duplexes to determine which sequences exhibit bending. Both groups confirmed that continuous runs of A residues are required for the phenomenon; interrupted
runs (with a C, G or T in the middle) or runs of G residues in phase with the helical repeat
do not exhibit anomalously slow gel electrophoretic mobilities. Koo et al. found that a run
of 5 or 6 A residues was required for maximal bending, with 4 contiguous A's exhibiting a
—^——^—^__^^-____^_^_^___^__^^—^^—^_^-^^-^——_^__^_^_
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© IR L Press Limited, Oxford, England.
Nucleic Acids Research
smaller, but detectable, retardation. They concluded that accurate phasing of the 3' ends
•* •>
of the oligo-A stretches was more important than phasing of the 5' ends, suggesting that
bending is localized primarily at the 3' junction Hagerman's study indicated that three
_^
contiguous A residues was sufficient for virtually maximal bending; however. Hagerman's
A
sequences were of the type (GGAAATTTCC)n whereas the Koo et al. sequences contained
no internal palindromic symmetry within the 10 base pair repeat unit. Superimposing the
*"
^ .
additional two-fold symmetry creates 5'-AAAT-3' junctions in both strands. These workers
also investigated the role of bases flanking the A tracts and found that a T residue at the 3'
end produced maximal bending, but otherwise the identity of the flanking base mattered
little.
Numerous models have been proposed to account for the molecular basis of the observed sequence-directed bending. The models may be grouped into two classes as discussed by Koo et al. (7). The first class assumes that the runs of A residues retain a
general B-DNA structure and proposes that bending is induced by interactions of neighbor and next-nearest neighbor nucleotides. The second class of models assumes a different
right-handed helical structure for the A tract and proposes that bending occurs at the
junctions of the A tracts and adjacent B-DNA. Clearly, the understanding of DNA bending would be greatly enhanced if a detailed molecular structure of the junctions and of the
*<
v*
A
•*
y .,
oligo-A tract were available.
Two-dimensional ' H NMR has the potential for elucidating the structure of biological macromolecules in solution (8). The use of NOESY, COSY and RELAY 2D NMR
experiments facilitates the assignment of the cofolex 'H NMR spectra of proteins or
DNA molecules. Currently, it is possible to assign the spectrum of a non-symmetric DNA
duplex up to about 25 base pairs long. The NOESY experiment is based on the throughspace dipolar coupling between protons. With care, the resulting data can be converted
into distances between pairs of H-atoms <4.5 A apart. Provided there are enough measured distances, in theory one should be able to determine 06 initio the structure of the
molecule using distance geometry methods (9,10). Recently, the structures of several unusual DNA oligonucleotides were determined using two-dimensional NMR and distance
geometry methods. These include a hairpin (11), a duplex with a base pair mismatch (12),
and a duplex with an extrahelical residue (13).
We have thus undertaken a two-dimensional 'H NMR study of d(CGAAAAATCGG)
+ d(CCGATTTTTCG) with the aim of determining the detailed structure in solution in
order to elucidate the molecular basis of DNA bending. This sequence contains a tract
of 5 A residues followed by a T nucleotide: such a —GAAAAAT— sequence has been
shown to be the basis of the bending phenomenon. Here, we report the assignment of all
non-exchangeable proton resonances (except 5' and 5" protons) in this molecule. Several
interesting and unusual features of the 2D NMR spectra allow us to make some qualitative
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^
»_«
"*•
Nucleic Acids Research
comments about the structure in terms of the proposed models for the origin of DNA
bending. This work represents the first step towards determining the three-dimensional
structure of bent DNA by NMR and distance geometry methods.
MATERIALS ANT)
Sample Preparation. The DNA 11-mer d(CGAAAAATCGG) and its complement
d(CCGATTTTTCG) were synthesized using solid-phase phosphite triester techniques on
an Applied Biosystems 380A DNA synthesizer. Each strand was synthesized on a 10 /jmole
scale, yielding over 500 A260 units of crude material.
The individual strands of DNA in 0.5 M NaCl were precipitated with ethanol, redissolved in 1.5 ml H2O and chromatographed on a superfine Sephadex G-25 column (120
cm x 2 cm) in distilled H2O. Fractions of 1.5 ml were collected at a flow rate of 20 ml/hr.
The elution profile was monitored at 260 nm and aliquots of selected fractions (ca. 0.05
A26o) were 5' end-labelled with "7-32P-ATP (New England Nuclear) using polynucleotide
kinase (Sigma Chemical), then electrophoresed on a 20% polyacrylamide denaturing gel
(8M urea). Autoradiograms of these gels revealed good separation of the desired major
product, from the shorter failure sequences. Fractions containing only the full-length 11mer were pooled, resulting in the recovery of ~400 A260 units of each purified DNA strand
(-75% yield).
Equimolar amounts of each strand were mixed in 5 ml of 10 mM phosphate buffer
(pH 7) and annealed together by heating to ~75 °C and cooling slowly. The duplex
DNA was applied to a hydroxyapatite column (15 cm x 2.5 cm) and eluted at room
temperature with a gradient of 10mM-400mM phosphate buffer (pH 7) to remove singlestranded material. The duplex DNA (~20 mg) was then lyophilized, redissolved in 1.5
ml H2O and desalted on a superfine Sephadex G-15 column (6C cm x 2 cm) eluted with
distilled H2O. The desalted duplex DNA was lyophilized and redissolved in 0.4 ml buffer
containing 20 mM sodium phosphate (pH 7), 100 mM sodium chloride, 0.1 mM EDTA.
The sample was lyophilized, redissolved in D2O and lyophilized again. Finally, 0.4 ml of
99.99% D2O was added and the solution transferred into a 5 mm NMR tube. For NMR
studies in H2O the sample was lyophilized and taken up in 0.4 ml of 90% H2O/10% D2O.
The samples were >95% pure as judged by their 'H NMR spectra at 500 MHz.
NMR Spectroscopy. NMR spectra in D2O were acquired on a home-built spectrometer
operating at a field of 11.75T corresponding to a proton resonance frequency of 500 MHz.
Details of this spectrometer are described in Gladden and Drobny (14). All NMR spectra
in D2O solution were recorded at 30°C. One dimensional spectra of the exchangeable
protons in H2O solution were acquired on a Broker WM-500. These spectra were obtained
at several temperatures to investigate the exchange-broadening of the peaks due to fraying
of the helix ends (vide infra).
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Nucleic Acids Research
All 2D NMR spectra were collected with 512 complex points in t2 and 300-400 points
in t j . The dwell time was 200 //sec and the relaxation delay was 2 sec over and above the
acquisition time. Typically. 64 scans were obtained for each tj point and the 90° pulse
length was 12 /xsec. COSY and RELAY spectra were acquired in absolute magnitude
mode. Phase-sensitive COSY and NOESY spectra were recorded using the hypercomplex
methods of States et al. (15). The mixing times for the RELAY and NOESY experiments
were 40 and 300 msec, respectively. Spectra of exchangeable protons in H2O solution were
obtained with a Redfield 21412 soft pulse (16) to suppress the water peak, with the carrier
frequency set at 12.6 ppm from TMS. NOE difference spectra were acquired as described
previously (17).
The absolute magnitude COSY and RELAY data were apodized with a 512 point
sinebell, zero-filled to 1024 points and Fourier transformed in the t2 dimension. The ti
data were apodized with a sinebell of as many points as there were experiments, zero-filled
to 1024 points and Fourier transformed. Phase-sensitive data were processed similarly
except that 60° phase-shifted sinebell apodization and the appropriate phase corrections
were applied in both the tj and t2 dimensions. For purposes of comparing intensities,
the NOESY data were reprocessed with right-shifted 90° phase-shifted sinebell-squared
apodization functions having a value of one out to 384 points in the t2 dimension or a
value of one out to 225 points in the tj dimension. All two-dimensional data processing
was performed on a DEC MicroVax II using software developed by Dennis Hare.
RESULTS
The >H NMR spectrum of d(CGAAAAATCGG) + d(CCGATTTTTCG) in D2O
buffer at 30°C is shown in Figure 1. We expect a total of 193 resonance lines due to
non-exchangeable protons consisting of 154 sugar protons, 33 base protons and 6 methyl
groups. The peaks fall into six reasonably discrete regions according to chemical type, as
is typical for DNA, and these are labelled in the Figure. Also shown in the inset is the
numbering scheme for this 11-mer duplex. Close inspection of Figure 1 reveals a number of
minor impurity peaks. The majority of these are from by-products of the DNA synthesis
that co-purify with the DNA using our procedure. They are seen in all our samples and are
easily distinguished from DNA resonances. Some of the impurity peaks are due to short
failure sequences or oligonucleotides that do not form duplex. These are at a very low
level (<5%) as shown by gel electrophoresis, and are of no consequence in the 2D NMR
spectra.
The principles of the two-dimensional NMR assignment method for right-handed helical DNA have been described in detail elsewhere (18-20) and will be outlined only briefly
here. In Figure 2, an expanded portion of the 2D NOESY plot is shown, corresponding
to the cross-peaks between aromatic C6 or C8 base protons and Cl'/C5 protons. The five
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Nucleic Acids Research
S'-C-G-A-A-A-A-A-T-C-G-G
G-C-T-T-T-T-T-A-C-C-C-s'
•n Jl 20 19 18 17 16 IS H 13 1!
CMj
v
JH.2X
8R8H.2H
9.8
8.0
7.2
6.4
5.6
4.8
PPM
4.0
3.2
2.4
1 .6
0.8
0.0
Figure 1. 500 MHz 'H NMR spectrum of d(CGAAAAATCGG) ->- d(CCGATTTTTCG)
in D2O/20 mM sodium phosphate (pH 7|. 100 m.M sodium chloride and 0.1 mM EDTA,
at 30° C.
V
1
•
very intense cross-peaks correspond to the C6 and C5 proton resonances of thefivecytidine
residues in the duplex. These protons are separated by a fixed distance of 2.45A and are
thus expected to give rise to some of the most intense N'OE cross-peaks. The cytidine H5
and H6 resonances are J-coupled and these five peaks also appear in the corresponding
region of the COSY spectrum (Figure 3a).
In right-handed helical DNA. the purine C8H or pyrimidine C6H is not only close to
its intranucleotide Cl'H but also to the Cl'H of the preceding (5') nucleotide. Therefore,
each aromatic proton gives an NOE cross-peak to its own Cl'H as well as to the Cl'H
of the nucleotide on its 5' side. This forms the basis of one of the sequential assignment
connectivities in DNA since one can start at either end of the polynucleotide and assign
the C8H/C6H and l'H resonances to specific atoms in the macromolecule.
The complete C8H/C6H to l'H walk for both strands is outlined in Figure 2a,b. There
are a few chemical shift redundancies in this region, e.g. the A5 and A7 H8 resonances
are overlapped, as are the T18 and T19 C6H resonances; but the correct assignments are
easily verified in other regions of the spectrum. The assignment of the Cl' protons of
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Nucleic Acids Research
a
1 « : I • !•
i i i *
ICC
i C C A A A A 1
i
c
IT: I A S S
0
$
i
•
I*1
J
5
1
4
t9
a A
D
$r
0
••
8.0
•
I)
*w i
B.2
1
»
a 0
•
7.8
Figure 2. Expanded region of the 'H NMR NOESY spectrum of the DNA duplex in D2O
buffer at 30°C showing the cross-peaks between C6/C8 'C2 protons and Cl'/C5 protons,
(a) Trace of the aromatic/Cl' proton walk for strand 1 (Cl-Gll). The numbers label
the intranucleotide cross-peaks, (b) Same trace for strand 2 (C12-G22). (c) Cross-peaks
between the adenine C2H and the Cl' proton of the nucleoside on its 3' side and between
the adenine C2H and the Cl'H on the 3' side of its cross-strand partner are connected by
vertical dashed lines. A3 C2H has a single cross-peak to A4 Cl'H which is indicated by a
diamond ( • ). The cross-peaks between the cytidine C5H and its own C6H and the Cl'H
of its 5' neighbor are joined by horizontal bars.
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Nucleic Acids Research
3-2
8.0
7.8
7.6
7.4
7.2
T\0
1.2
Figurec 3. Expansion of the 'H NMR COSY spectrum of the DNA duplex in D2O buffer
at 30 C. (a) Region showing the cytidine C5H-C6H cross-peaks, (b) Region showing the
thymidine C6H-rnethyl cross-peaks.
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Nucleic Acids Research
2.8
1 .2
Figure 4. Expansion of the NOESY spectrum showing the cross-peaks between C6/C8
protons and 2"/2'/methyl protons. On the left is a trace of the aromatic/2"H walk for
strand 2 (C12-G22). On the right, the cross-peaks between a thymidine methyl group and
its own and 5' neighbor's C6H/C8H are joined by vertical bars. The diamond ( • ) denotes
the cross-peaks between the T19 methyl and the C6 protons of T18 and T19, which have
the same chemical shift.
A5 and A6 is potentially ambiguous but this ambiguity was resolved by comparison with
the NOESY spectrum of d(GCCAAAAATGCC) and its complement strand under similar
conditions (data not shown). In the latter sequence the l'H assignments are unambiguous
and the Cl'H resonance of the third A residue in the tract (corresponding to A5 in our
target sequence) is clearly upfield of the Cl'H resonance of the A residue on its 3' side.
The assignments in Figure 2 can be made with a high degree of confidence since
the spectrum contains additional information that provides self-consistency checks. For
example, the cytidine C5 protons give rise to an NOE cross-peak with the C8H/C6H
protons of the nucleotide on its 5' side. These are shown by horizontal bars in Figure 2c.
Notice that only three such contacts are seen since two of the cytidine residues of this
Nucleic Acids Research
•>
V
2.8
1.2
Figure 5. Expansion of the NOESY spectrum showing the cross-peaks between Cl'H and
2"/2'/methyl protons, (a) The cross-peaks between l'H and 2"H/2'H are connected by
horizontal bars. Note the weaker intensity of the 2'H cross-peaks, (b) The horizontal
dashed lines indicate the NOE connectivity of a cytidine C5H proton with the 2"H and
2'H of its 5' flanking residue.
r
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Nucleic Acids Research
duplex are at the 5' ends; the two cytidine resonances are thus independently identified as
either Cl or C12.
The thymidine C6H resonances can likewise be identified by their cross-peaks to
methyl groups in the upfield region of the spectrum (Figure 4). In right-handed helical
*
DNA, thymidine methyl groups are close in space to their own C6 proton and to the base
proton of the residue on the 5' side, and are thus expected to have two NOE cross-peaks
^
to aromatic resonances. The intranucleotide C6H and methyl resonances are identified
t
by their cross-peaks (through an easily detectable 4-bond scalar coupling) in the COSY
spectrum (Figure 3b). In the NOESY spectrum, four of the thymidine methyls have pairs
<
of cross-peaks in which both of the aromatic components are thymidine H6 resonances and
-«*
these must be T17, T18, T19 or T20. Of the other two methyl resonances at 1.07 ppm and
1.23 ppm, the former is easily identified as T8 by its cross-peak to the C8H of A7 and the
latter is T16 from its cross-peak to A15 C8H. Furthermore, it is also possible to carry out
a sequential 2"H to C8H/C6H walk in the region shown in Figure 4 in the same fashion as
*
the Cl'H to base proton walk shown in Figure 2, thereby confirming the aromatic proton
^„
'
assignments and simultaneously assigning the 2" protons.
An additional, and often more reliable, way to assign the 2" protons, as well as the
2' protons, is by the COSY and NOESY cross-peaks to their own Cl' protons, which
have already been assigned. This region of the NOESY spectrum is shown expanded in
Figure 5, with horizontal bars connecting the l'H-2'H and l'H-2"H cross-peaks of each
sugar. In addition to being a valuable assignment tool, the intensity (proximity) of the
*
1
-
l'-2' cross-peak compared to the l'-2"cross-peak is diagnostic of the sugar conformation
^"
in that these two distances change in the various allowed conformations of D-deoxyribose;
in most conformations the 2"H is closer to the l'H producing a more intense cross-peak.
These assignments can be made just as easily in the corresponding region of the COSY
spectrum (data not shown). For the most common C2'-endo sugar conformation in B-
«•
DNA, the 2"H is very weakly scalar coupled to 3'H allowing it to be distinguished from
the 2'H. In general, the 2'H of a given nucleotide is upfield of the 2"H. For this particular
oligonucleotide, the only exceptions occur in residue G2 where the 2'H and 2"H chemical
shifts are virtually identical, and in the 3' terminal residues (Gil and G22) where the 2'
<
A
*"
protons are downfield of the 2" protons.
The C3' and C4' protons were assigned by their NOE cross-peaks to Cl' protons. This
region is shown in Figure 6a. The C3'H/Cl'H cross-peak is generally less intense than the
corresponding C4'H/C1'H cross-peak, and the C3'H resonances are usually downfield of
the C4'H resonances. The l'H to (n +l)5'H/5"H cross-peaks are either extremely weak
or absent. The .assignments of the C3'H resonances from the Cl'H resonances can be
independently corroborated by C3'H cross peaks to the aromatic region, where we can
carry out a complete C3'H/base proton sequential walk (data not shown). Finally, the
i
y
'H
,
••
*
0
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Nucleic Acids Research
1
f
f
6
a
(
0
5
1
9
o
f
0
T
9
I
i
i
S
1)
P
*
<
i
g
13
*i
>
9
S3
is
f
©
Oc
6.2
6.0
5.8
@
5.6
PPM
0
0
5.4
5.2
5.0
Figure 6. (a) Expansion of the NOESY spectrum showing the cross-peaks between Cl'H
and the intranucleotide C3'H and C4'H, indicated by vertical bars, (b) Corresponding
region of the RELAY spectrum showing the intranucleotide Cl'H/C3'H cross-peaks. The
diamonds ( • ) denote impurity peaks or artifacts which are not present in either the COSY
or NOESY spectra.
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Nucleic Acids Research
I ! ]( S C ! M
1011
a
5' CGAAAAATCGG
GCTTTTTAGCC 5'
HDCBAEFIC
4
<
J.
14
—r~
12
10
Figure 7. (a) Downfield region of the 'H NMR spectrum of the duplex DNA in H2O buffer
at 32°C. Peaks A to I correspond to imino protons hydrogen-bonded between base pairs.
The peaks on the right correspond to amino protons (broad peaks) and carbon-bonded
aromatic protons of the bases. The sequence of base pairs numbered 1 to 11 is shown
in the inset together with the assignment of each imino proton, (b) One-dimensional
NOE difference spectrum showing the cross-relaxation between peak E (which has been
saturated) and imino peaks F. A and C2H peaks f and e. (c) Same as in (b) except
resonance F has been saturated.
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Nucleic Acids Research
ri"
C3'H assignments are made completely ironclad by observing coherence RELAY cross••«•
peaks from the C l ' proton as shown in Figure 6b. Unfortunately, coherence RELAY from
*
2'H or 2"H to the C4' proton is extremely weak and provides only partial verification of
the C4' assignments (data not shown). RELAY cross-peaks from 3'H to 5' and 5" protons
were not observed even after trying several mixing times, possibly indicating rather weak
'A
coupling between 3'H and 4'H. The C4'H assignments can still be confirmed by their crosspeaks to the C 3 ' protons in the COSY or RELAY spectrum near the diagonal (data not
shown).
%
r*+
•
The assignment of the adenine C2 protons was made from their NOE contacts to
imino protons in H2O solution. The one-dimensional ' H NMR spectrum of the DNA
sample in H2O at 32°C, showing the imino, amino and aromatic regions, is displayed
»..
each end of the helix have been exchange-broadened. Terminal fraying of the duplex was
confirmed by recording spectra at various temperatures down to 5°C (data not shown).
The imino protons were assigned using one-dimensional NOE difference techniques (21,22).
in Figure 7a. Only nine resolved imino peaks were observed: the imino resonances from
Representative difference spectra are shown in Figure 7b,c. For example, upon saturating
»
resonance E in the imino proton region, cross-saturation of imino peaks F and A and of
C2H resonances f and e was observed (Figure 7b). These protons must be separated by
•*
'*
one. Similarly, when resonance F was saturated, cross-saturation of peak E in the imino
region and of peaks f and e in the C2H region was observed (Figure 7c). Repeating the
experiment for peaks A to I establishes the linear relationship of the imino resonances in
less than 5A and therefore must belong to either the same base pair or to an adjacent
..
the DNA duplex as HDCBAEFIG. Resonances G, H, and I correspond to imino protons
in CG base pairs since they exhibit NOEs only to broad amino resonances in the 8-9 ppm
region, and not to narrow C2H lines.
^
The polarity of the assignments is determined by several means. One line of evidence is
^
that resonance G is the third residue to broaden as the temperature is raised. This coupled
to the fact that the guanine resonances H and I are adjacent to A-T base pairs establishes
^
the assignment of resonance G to the imino proton of residue GlO. Moreover, the imino
proton in an A-T base pair is expected to have NOE contact with the C2H of its own
t
adenosine and with the C2 of the A residue on the 3' side in B-DNA. Observation of these
types of contacts in the tract of A residues allows us to confirm the imino assignments.
These are shown in the inset of Figure 7. Furthermore, the C2H resonances of specific
*
adenosine residues can now be assigned since it is the stronger of the NOE contacts (crossstrand) upon irradiation of the complementary thymidine imino resonance. Going from
downfield to upfield, the C2H resonances are assigned as fedcab at 32°C where the small
••
letters have the same correspondence to nucleotides as the capital letters in the inset of
^
Figure 7. Assuming this order is not changed at 30°C, we can transfer these assignments
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Nucleic Acids Research
Table 1. Assignment of the Bent DNA Undecamer
1
11
C-G-A-A-A-A-A-T-C-G-G
G-C-T-T-T-T-T-A-G-C- C
22
12
Residue
C8H
Cl
G2
A3
A4
A5
A6
A7
C12
C13
G14
A15
T16
T17
T18
7.45
5.75
C2H
CH 3
7.53
6.94
6.84
6.90
7.49
6.92
7.25
5.42
1.07
7.60
7.38
5.79
5.54
7.70
7.66
7.79
8.10
7.68
7.04
7.33
7.36
7.36
7.29
7.39
T19
T20
C21
G22
C5H
7.77
7.99
7.91
7.85
7.81
7.85
T8
C9
GlO
Gil
C6H
7.81
1.23
1.42
1.50
1.53
1.55
5.61
Cl'H
C2"H
C2'H
C3'H
C4'H
5.56
5.08
5.68
5.63
5.71
5.80
5.97
5.77
5.54
5.55
6.00
2.13
2.48
2.67
2.66
2.74
2.79
2.77
2.31
2.19
2.56
2.22
1.57
2.51
2.51
2.43
2.40
2.41
2.32
1.81
1.78
2.49
2.40
4.50
4.80
4.91
4.89
4.88
4.90
4.85
4.71
4.70
4.84
4.52
3.90
4.11
4.25
4.28
4.29
4.32
4.34
4.07
3.98
4.19
4.07
5.84
5.35
5.48
6.17
5.83
6.07
6.07
6.02
5.95
5.60
6.01
2.37
2.23
2.69
2.85
2.48
2.54
2.57
2.56
2.38
2.24
2.25
1.90
1.90
2.59
2.55
1.92
2.12
2.12
2.06
1.99
1.90
2.50
4.53
4.70
4.89
4.92
4.73
4.80
4.82
4.81
4.78
4.72
4.56
3.98
3.97
4.23
4.38
4.09
4.11
4.13
4.12
4.06
4.02
4.04
The DNA duplex was in D2O solution at 30°C. Solution conditions are described in
the text.
to the 2D NOESY spectrum where we observe C2H to C2H cross-peaks near the diagonal
in the base proton region (data not shown) and C2H to Cl'H cross-peaks (Figure 2). The
chemical shifts of all non-exchangeable protons, except 5'H and 5"H, at 30°C are listed in
Table I.
DISCUSSION
In general terms, the NOESY spectrum of this double-helical DNA undecamer is
consistent with a solution structure within the broad family of B-DNA in that qualitatively
the intranucleotide and internucleotide connectivities characteristic of B-type DNA are all
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observed. There are, however, some unusual and subtle features in the spectra that may
be correlated to structural anomalies in this putatively bent DNA duplex.
First, the G2 Cl' proton was found to have an exceptionally upfield-shifted resonance
position of 5.08 ppm, almost overlapping the 3' protons. To our knowledge, this is the
highest upfield Cl'H yet observed in unmodified DNA. Patel et al. (23) have observed
Cl'H resonances even further upfield but only in DNA duplexes containing a chemically
modified guanosine nucleotide. The most likely cause of the anomalous position of G2
Cl'H is a 0.3-0.4 ppm upfield ring-current shift from a heterocyclic base. Such upfield
shifts derive from stacking geometry, which eliminates the G2 purine as the cause of the
shift. The cytosine in position one has a much weaker ring current, is quite far away
from G2 l'H in right-handed DNA, and does not shift the G2 l'H resonance in several
other duplexes we have studied that begin with CG in the first and second positions.
This leaves the strong ring-current of the adenine moiety of A3 as the only remaining
candidate causing the upfield shift on G2 Cl'H. This effect is not seen on the analogous
G4 Cl'H in (CGCGAATTCGCG)2 which resonates at 5.42 ppm (20) in a sequence that
does not exhibit bending. However in a different bent sequence, GCCAAAAATGCC, we
have observed an analogous dramatic upfield shift of C3 Cl'H, suggesting that the effect is
peculiar to the residue proceeding the oligo-A tract in bent DNA sequences. In the present
CGAAAAATCGG sequence the most likely way for the A3 base to have this upfield effect
on G2 Cl'H is for it to markedly propeller-twist or to side-slip, or both.
The sugar of the G2 residue also appears to be in an unusual conformation. Evidence
that this sugar is structurally perturbed ib given by the unusual chemical shift positions of
the 2' and 2" protons of G2, in which 2'H resonates very slightly downfield of 2"H. This is
in contrast to all the other nucleoside residues in this macromolecule (except the terminal
Gil and G22) and in other DNA duplexes reported in the literature; in all these cases
the 2'H almost always resonates upfield of the 2"H. The only exceptions are at helix ends
(24,25) or at sites of chemical modification (23).
Another unusual and interesting feature of the NMR data is the presence of relatively
strong NOE cross-peaks between adenosine C2 protons and Cl' protons of the 3' neighbor on the same strand and across the strand to the 3' neighbor of the complementary
residue. Such cross-peaks have been observed in previous 2D NMR studies of B-form DNA
(23,26,27), and they are usually very weak. Interestingly, these cross-peaks are seen in the
NOESY spectrum of poly(dA)-poly(dT) which one might expect to have a similar structure to our oligo-A tract (27). In the NOESY spectrum of the present duplex (acquired
with a mixing time of 300 msec), we observe at least 10 C2H/Cl'H cross-peaks of which
two are quite strong (comparable in intensity to some C8H/C6H to l'H cross-peaks), six
are intermediate, and two are weak (Figure 2c). At least four of these cross-peaks are
still visible in a NOESY spectrum acquired with 150 msec mixing time (data not shown)
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Nucleic Acids Research
indicating reasonably short distances between these C2H and Cl'H atoms.
From the coordinates of classical B-form DNA determined by Arnott and co-workers
(28), one would not expect to see these cross-peaks since there are no C l ' protons within
5A of any adenine C2 protons. The work of Dickerson et al. (29) on a single crystal
of the self-complementary DNA d(CGCGAATTCGCG) however, reveals extensive local
deviation of the structure from idealized B-form DNA. For example, there is considerable
variation of the helix pitch and propeller twist of the base pairs at each position in the
sequence. Using the coordinates from Dickerson and co-workers' study, we find that an
adenosine C2H may be within ~4 A of the Cl'H of the 3'flanking residue and the Cl'H of
»*
..
4
*
the nucleotide to the 3'side of the cross-strand partner may also approach this close, thus
giving rise to NOE cross-peaks. Our observation of these cross-peaks suggests that there
is substantial deviation from classical B-DNA structure in the oligo-A tract of our DNA
duplex. Morever, we note that the intensities of the different C2H/C1'H cross-peaks vary
and are strongest for A6 C2H to T18 Cl'H and for A7 C2H to T17 Cl'H. This suggests
"*• -
that the deformation in the helix that allows these cross-peaks to appear is greatest near
the fourth and fifth adenosine residues from the 5'end of the tract. In the 2D NMR study of
poly(dA)-poly(dT) by Behling and Reams (27), the interstrand A 2H to T l'H cross-peak
^,
was also found to be more intense than the corresponding intrastrand cross-peak. This
•*•*
observation is consistent with the compression of the minor groove in B-form DNA.
Further unusual aspects of the NMR spectra are observed in the intensities of the NOE
cross-peaks between base protons and Cl'protons. In idealized B-DNA. the intensities of
all of these cross-peaks are expected to be quite similar. In the NOESY spectrum of the
undecamer, however, seven of these cross-peaks are exceptionally weak. These are G2
C8H/C1 Cl'H, A6 C8H/A6 Cl'H. C9 C6H/T8 Cl'H, C9 C6H/C9 Cl'H, G14 C8H/C13
Cl'H, C13 C6H/C13 Cl'H, Cl3 C6H/C12 Cl'H. We note that these anomalous NOE
intensities occur primarily for residues at the 3' end of the A tract, although anomalies
also occur at the 5' end and within the oligo-A tract at the fourth adenosine residue. It
is tempting, but somewhat dangerous, to draw conclusions about the molecular structure
from NOE intensities measured at only one mixing time, since numerous factors can affect
these values. For example, the observed weak NOE intensities may indicate that the
relevant protons are quite distant, or that there may be complex cross-relaxation pathways
between them (spin-diffusion), or that there is large-amplitude motion of one of these
moieties, or even some combination of these factors. Nevertheless, these preliminary results
suggest that there is a substantial perturbation of the helix structure at both ends of the
stretch of A residues with perhaps smaller perturbations at the fourth A nucleotide of the
oligo-A tract.
In summary, we have assigned all of the non-exchangeable protons (except 5'H and
5"H) in the 'H NMR spectrum of d(CGAAAAATCGG) + d(CCGATTTTTCG) in so5860
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r
'
,,,
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Nucleic Acids Research
lution. Qualitative analysis of the NMR parameters indicate that although the overall
structure of this duplex is B-form there are several significant local structural deviations.
In particular, there is a structural perturbation at the 5' end of the oligo-A tract and a
more extensive one at the 3' end. There is also a deviation of the helix parameters in the
A tract itself which is maximum for A6 and A7. These observations are consistent with
models of DNA bending but lack the molecular level detail to significantly increase our
understanding of the phenomenon. Therefore we are currently collecting a time-dependent
set of 2D NOESY experiments which will ultimately yield relatively precise proton-proton
distances. From this data set, we hope to obtain a detailed three-dimensional structure of
this macromolecule using distance geometry methods, and a clearer picture of the molecular basis of DNA bending.
ACKNOWLEDGEMENTS
We thank Susan Ribeiro, Jerilyn Beltman, Ponni Rajagopal, Leh-Jame Lin, and Kelly
Horng for their help in preparing the DNA sample, and Mary Coventry for her help in
preparing the manuscript.
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