From the Institut für Molekularbiologie,
Friedrich-Schiller-Universität, Winzerlaer Strasse 10, D-07745 Jena, Germany and the § Department of
Biophysics, Arrhenius Laboratory, Stockholm University,
S-106 91 Stockholm, Sweden
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ABSTRACT |
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Sequence-dependent structural
features of the DNA double helix have a strong influence on the base
pair opening dynamics. Here we report a detailed study of the kinetics
of base pair breathing in tracts of GC base pairs in DNA duplexes
derived from 1H NMR measurements of the imino proton
exchange rates upon titration with the exchange catalyst ammonia. In
the limit of infinite exchange catalyst concentration, the exchange
times of the guanine imino protons of the GC tracts extrapolate to much
shorter base pair lifetimes than commonly observed for isolated GC base
pairs. The base pair lifetimes in the GC tracts are below 5 ms for
almost all of the base pairs. The unusually rapid base pair opening
dynamics of GC tracts are in striking contrast to the behavior of AT
tracts, where very long base pair lifetimes are observed. The
implication of these findings for the structural principles governing
spontaneous helix opening as well as the DNA-binding specificity of the
cytosine-5-methyltransferases, where flipping of the cytosine base has
been observed, are discussed.
Many DNA-binding proteins are highly selective in their
recognition of particular DNA sequences. Besides sequence-specific hydrogen bonding and van der Waals interactions,
sequence-dependent structure and dynamics of DNA are likely
to play an important role in DNA-protein interaction. In addition, the
adaptability of a DNA sequence element to structural changes necessary
for sequence-specific interaction is important in recognition (1).
Base pair opening is required in many fundamental processes
in the cell, for example, transcription and recombination. Recently, base pair opening was found to participate in a novel mode of protein-DNA interaction. The crystal structures of the
M.HhaI1 and
M.HaeIII cytosine-5-methyltransferases in complex with their DNA recognition sequences showed the target base completely flipped out
from the helix (2, 3). Cytosine-5-methyltransferases usually recognize
a sequence of four GC base pairs (4). M.HhaI and
M.HaeIII recognize 5'-GCGC-3' and 5'-GGCC-3', respectively. A pertinent question is whether these enzymes actively expel the target
base from the helix stack or capture a transient spontaneous opening.
It has been shown that M.HhaI binds more tightly when a
mismatch is created in the recognition sequence by replacing the target
cytosine by any other base or an abasic site but not 5mC (5, 6). The
enhanced binding was attributed to the lower energy required for
opening a mismatched base pair upon formation of the binary complex.
Hence, the base pair dynamics at the cytosine target site seems to
contribute to the specificity of the cytosine-5-methyltransferases. These findings prompted us to investigate the base pair dynamics of
tracts of GC base pairs.
Measurements of base pair dynamics yield information about stability
and structure of the double helix. Furthermore, studies of base pair
opening in DNA interacting with drugs (7, 8) and hybridized with
uncharged PNA (peptide nucleic acids) (9) have provided new clues to
the mechanisms of spontaneous helix breathing. Another important
finding is that tracts of AT base pairs exhibit anomalously long base
pair lifetimes. Although the origin of this effect is uncertain, it
seems likely that AT tracts form a particularly stable structure
cooperatively, a so-called B'-DNA helix (10). Hence, increased base
pair lifetimes are indicative of this type of structure.
In general AT base pair lifetimes have been found to be in the range
1-5 ms at 15 °C, except for AT tracts where lifetimes longer than
100 ms have been observed (11). For GC base pairs, lifetimes about 10 times longer than for AT base pairs usually have been observed (12), as
one might expect from the presence of an additional hydrogen bond in
the GC base pair. However, most studies have concerned isolated GC base
pairs (9, 11, 13-16). Representative values for isolated GC base pair
lifetimes are compiled in Table I. No study has been undertaken of the
base pair opening dynamics in tracts of GC base pairs.
Since the binding affinity of M.HhaI increase with the
lability of the target base pair (5, 6), one would not expect base pair
dynamics to contribute to the specificity of the
cytosine-5-methyltransferases in view of the hitherto observed higher
stability of GC base pairs. However, in the present study it is shown
that tracts of GC base pair have unusually rapid base pair dynamics
contrary to isolated GC base pairs and in striking contrast to AT tracts.
Sample Preparations and Base-catalyst Titrations--
All
oligonucleotides were either synthesized by using automated
phosphoramidite chemistry on a DNA synthesizer (Applied Biosystems model 394) or purchased from Cybergene Inc. (Sweden). The
oligonucleotides were purified by reverse-phase high performance liquid
chromatography and desalted by Sephadex G-25 column chromatography. The
NMR samples were prepared by dissolving the oligonucleotides in a 3 mM borate buffer at pH 8.8 containing 100 mM
NaCl (90% H2O and 10% D2O). The duplex
concentrations were in the range 1.1-1.8 mM. Ammonia was
added in appropriate amounts from a 6.6 M stock solution at pH 8.8.
Two separate titrations were carried out for each duplex and the
exchange-time ( Spectroscopy--
The imino protons were assigned from
sequential imino-imino connectivities in two-dimensional NOESY
experiments in H2O solution (18) using either the WATERGATE
(19) or the Jump-Return (20) observe pulse for water suppression. The
experiments were performed with a mixing time of 200-250 ms at
5-15 °C.
Inversion recovery experiments were performed using a 0.633-ms Gaussian
(21) or a 1-ms iBURP (22) pulse for inversion followed by a variable
delay and a 1-ms Gaussian observe pulse. Right shift and linear
prediction of the free induction decay were employed to correct for
magnetization evolution during the observe pulse. The spectral width
varied between 1500 and 2000 Hz, depending on the NMR spectrometer (500 or 600 MHz) used. The carrier frequency was centered in the imino
proton region. The exchange time at a particular base concentration
[B],
Saturation transfer experiments utilized a rapid removal of the water
magnetization by a 1-1.2-ms Gaussian 90°-pulse followed by a pulsed
field gradient. This was repeated in a loop 1-10 times. In each loop
element the gradient strength and length was randomly varied with 10%
around 10 Gauss/cm and 1 ms, respectively, yielding an effective
solvent suppression without refocusing effects. Each loop contained a
1-ms gradient recovery delay. The initial loop was followed by a
presaturation period of variable length and finally a jump-return
observe pulse.2 The decay of
the imino proton resonances was fitted to a single exponential function
to yield the exchange time and the magnetic spin-lattice relaxation
time according to published procedures (23).
The infrared spectra in H2O and D2O were
measured with 6 mM oligomer duplex concentration at pH 8.8 and 20 °C as described previously (24, 25).
Imino Proton NMR Spectra and IR Spectroscopy--
In the
following, GC tracts is synonymous for sequences of GC base pairs with
no particular order of guanine and cytosine bases and with a length of
at least four base pairs. G tracts are sequences of the type
5'-GnCn-3'. Isolated GC
base pairs will mean at most two consecutive GC base pairs. The studied
DNA sequences are shown in Scheme 1. Imino proton spectra of the five
DNA duplexes at 15 °C are displayed in Fig. 1, without addition of exchange
catalyzing base (left) and in presence of 0.1 M
ammonia (right). The assignment of the imino proton
resonances is based on two-dimensional 1H NOESY experiments
(data not shown). All thymine and guanine imino proton resonances can
be observed except those of the terminal base pairs and, notably,
I:T2 and II:T2. The second AT base pairs from
the ends of the decamers, III:T2, IV:T2, and
V:T2 are clearly visible. As is seen from the
right panels of Fig. 1, the three outermost base
pairs of all duplexes have vanished at 0.1 M ammonia, displaying the typical higher mobility of terminal base pairs, the
so-called end-fraying.
The larger broadening observed for III, IV:T2 and III,
IV:T3 as compared with V:T2 and V:T3 indicates that the
5'-T3G4-3' step exerts a destabilizing effect
on the ends. This is consistent with the reduced stacking and higher
flexibility suggested for this step from the NMR solution structure of
sequence III (26). The effect becomes even larger in the dodecamer
sequences I and II, where the T2 imino proton resonances
have completely disappeared by exchange broadening. This may indicate a
cooperative formation of a homogenous G tract type of structure
promoted by a flexible 5'-TG-3' step at the G tract ends.
The infrared spectra in D2O and H2O of duplex
III in the absence of added catalyst and at the ammonia concentration
reached at the end point of the titration are shown in Fig.
2. The difference between the two spectra
is very small, indicating that no structural alterations occur in
course of the titration even at the high duplex concentration used in
the IR experiments. The influence of the strong buffer conditions
during the titrations was further investigated by performing NOESY
experiments on sequence V at an ammonia base concentration of 20 mM and 0.9 M, which corresponds to the
titration range. Only minor differences are observed in the chemical
shifts and the relative intensity of the cross-peaks in the two spectra
(data not shown).
Imino Proton Exchange--
The exchange times of the imino protons
were obtained from inversion recovery times of the NMR resonances as
described previously (9). Addition of an exchange catalyst yields in
the limit of infinite catalyst concentration the kinetic parameters for
the base pair opening (Equation 1). In Fig.
3, the exchange times of the guanine
imino protons of the five GC tracts are displayed as a function of the
inverse ammonia concentration at 15 °C. The exchange times display
the linear dependence on the inverse base-concentration expected from
Eq. 1. The base pair lifetime
Only the averaged lifetimes of the G5 and G6 imino protons of duplex I
and the G4 and G5 imino protons of duplex II could be determined due to
spectral overlap at high ammonia base concentration (i.e.
the last three titration steps). However, at low and intermediate catalyst concentration, it is clear that the exchange properties are
very similar for these base pairs (data not shown).
Several sequence-dependent characteristics of the base pair
dynamics of the tracts are apparent. In all sequences the outermost GC
base pairs display similar behavior with base pair lifetimes around 4 ms and a relatively high base pair dissociation constant in the range
5-14×10
In principle, the increased recovery rates of the imino proton
resonances upon addition of the catalyst could be due to an increase of
the magnetic relaxation rates as well as increased exchange rates. The
former could be the result of aggregation induced by the high ionic
strength present at the high buffer concentrations necessary to reach
near opening-limited exchange conditions in the course of the
titration. In most studies of base pair dynamics, it has been
implicitly assumed that any changes in the magnetic relaxation during a
catalyst titration remain so small that they can be neglected. Indeed,
this holds true in most cases (15). However, the unexpected rapid
dynamics inferred from the observed exchange behavior of the guanine
imino-protons in the G tracts prompted us to investigate this
possibility by carrying out saturation transfer experiments on sequence
V. In this type of experiment, the exchange and the magnetic relaxation contributions are separated and the exchange is directly measured (23).
In Fig. 4, the exchange times derived
from the inversion recovery experiments and those derived from the
saturation transfer experiments display a close similarity, strongly
indicating that changes in magnetic relaxation do not significantly
influence the results of the inversion recovery experiments.
Sequence-dependent structural features of the DNA
double helix have a strong influence on the base pair opening dynamics. For instance, the base pair lifetime of the central GC base pairs of
the self-complementary duplex d(CCTTTCGAAAGG)2 is 40 ms,
while that of the reverse sequence is only 7 ms (Table I). This
difference was interpreted as caused by a kink in the center of the
helix in the latter case (14). In the DNA dodecamer duplex
d(CGCACATGTGCG)2, the base pair opening rates in the
CACA/GTGT motif were 3-8 times higher than in a same dodecamer with
the central CG base pair reversed to a GC base pair (16). Despite these
differences, GC base pair lifetimes below 5 ms at 15 °C have to our
knowledge never been observed in the interior of DNA oligomers where
end-fraying effects are negligible. In the present study, base pair
lifetimes below 5 ms are observed in almost all of the GC tracts.
Structural properties specific for G/GC tracts have been proposed to
originate from stacking effects (26, 27), the unmethylated state of the
major groove (28, 29), or the pattern of hydrogen-bond donor and
acceptor groups in the major groove (30). By comparative studies of
different DNA oligomer crystal structures, Dickerson and co-workers
(27) concluded that guanine bases prefer to stack flat atop one
another, without the helix-following roll observed in A tracts. To
maintain hydrogen-bonding with the complementary strand, it becomes
necessary to break the stack after a certain distance. Hence, a
competing situation occurs where the hydrogen-bonding may be
compromised for the benefit of optimized stacking. The reason for the
difference in stacking properties was suggested to be the presence of a
"projecting" N2 amine in guanine (27). The crystal structure of the
GC base pair decamer d(CCGGCGCCGG)2 display an unusually
deep and wide minor groove and a shallow and accessible major groove
(31). It was implied that shallowness may be a characteristic feature
of a major groove devoid of methyl groups. A further manifestation of
the accessibility of the major groove in G tract is the unusual
groove-backbone interactions observed in the crystal packing of G tract
containing oligomers (31, 32). In fact, A tracts and G tracts exhibit a
striking reciprocity with respect to groove dimensions, hydration and
base pair dynamics (10, 28, 31). These features have been summarized in
Table III.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
ex) data were combined and linearly fit
versus the inverse base-concentration (1/[B]) via Equation 1 (13).
(Eq. 1)
op is the base pair lifetime,
is an
accessibility parameter, Kd is the dissociation
constant for the base pair, and ki is the proton
transfer rate from the mononucleoside, taken to be 2 × 108 s
1 M
1 at
15 °C (17). The base pairs of the duplexes are numbered according to
Scheme 1, where the dodecamer numbering
is shown on top and the decamer numbering on the
bottom. Symmetry-related base pairs are denoted by
underlined numbers. The duplexes are referred to
by their Roman numerals.
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Scheme 1.
ex[B], was obtained from the
recovery time Trec[B] as described
(9, 13).
RESULTS
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Fig. 1.
1H NMR imino proton spectra with
assignments at 15 °C. No addition of catalyst (left)
and with ammonia buffer added to yield a base concentration of 0.1 M (right). The Roman
numerals refer to the numbering in Scheme 1.
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Fig. 2.
Infrared spectra of duplex III in
D2O and H2O (inset). No
addition of catalyst (------) and in the presence of 4 M
ammonia buffer at pH 8.8 (
). The duplex concentration was 6 mM, and the spectra were measured at 20 °C.
op, as well as the apparent dissociation constant
Kd, obtained from
the linear fits, are given in Table II. Unexpectedly, the base pair lifetimes are below 15 ms for all GC base pairs in the GC tracts. The
general pattern is that the lifetimes are much smaller than observed
for isolated GC base pair (cf. Table
I), and the lifetimes decrease even
further when the tract are of the type
GnCn. On the average,
base pair lifetimes in GC tracts are about 10 times shorter than for
isolated GC base pairs (Tables I and
II).
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Fig. 3.
Exchange times
ex of the guanine imino protons in the
five DNA duplexes at 15 °C. A, I:G4 (
, - - -),
II:G4/G5 (
, ------); B, I:G5/G6 (
, - - -),
II:G6 (
, ------); C, III:G4 (
, - - -),
IV:G4 (
, ------), V:G4 (×,
); D,
III:G5 (
, - - -), IV:G5 (
, ------), and V:G5 (×,
). The lines were obtained by fitting to Equation 1, with the
exchange times weighted according to their errors.
Base-pair lifetimes (op) of isolated GC base pairs at
15 °C
Base pair lifetimes (op) and apparent dissociation constants
kd of GC tract GC base pair at 15 °C
7 (Fig. 3, left panel).
Notably, from Fig. 3 (A and C), it is seen that
the dissociation constant increase with the length of the tract (Table
II). In duplex II the central GC step of duplex I has been reversed to
a CG step. This leads to an increase of the base pair lifetime and a
decrease of the dissociation constant for this base pair as compared
with sequence I with roughly a factor of 2 (Fig. 3, A and
B; Table II). This is consistent with GnCn-type tracts having
unique properties leading to higher base pair dissociation constants
(see below). Although the dissociation constants and base pair
lifetimes of the outermost GC base pairs in the decamer duplexes are
similar (Fig. 3C), the innermost GC base pairs display
different kinetics in the three decamers. The central base pairs of the
alternating tract 5'-GCGC-3' of duplex IV and 5'-CGCG-3' of duplex V
are more stable than any other base pairs, with dissociation constants
3.3 × 10
7 and 2.3 × 10
7,
respectively. These values are close to what is commonly observed for
isolated GC base pairs (12). The central base pair of sequence III
retains the typical properties observed for the longer G tracts with a
high dissociation constant (Fig. 3, C and D;
Table II).
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Fig. 4.
Comparison of the exchange times derived from
saturation transfer from the solvent to the imino protons and from the
inversion recovery rates of the imino protons of duplex V. Exchange times derived from saturation transfer experiments, V:G4
( ), V:G5 (
) and from inversion recovery experiments, V:G4 (
),
and V:G5 (
).The lines were obtained by fitting to Equation 1, with
the exchange times weighted according to their errors.
DISCUSSION
Properties of A and G tract DNA
It seems probable that one or several of the structural properties listed in Table III are responsible for the very different base pair dynamics observed in A tracts and G tracts. For example, an accessible major groove where the stacking properties of the guanine bases leads to a tendency to transiently break the hydrogen bonding with the complementary strand can give rise to a more rapid base pair dynamics observed in G tracts.
The base pair dissociation constant is higher for G tracts than GC tracts and increases with the length of the G tract. In addition, upon increasing the length of the G tract, the ends of the helix become more labile. A possible explanation is a cooperative formation of a structure in the G tract part with bifurcated hydrogen bonds between the cytosine amino group and the guanine carbonyl group but with poor stacking with the ends. This may lead to a base-pairing shift in the major groove, as has been observed in crystal structures of DNA oligomers with similar sequences (30), which may stabilize the open state of the base pair leading to a higher base pair dissociation constant.
On the basis of the similarity of the NMR relaxation of the 19F resonances of 5-fluorocytosine at the target site of the cytosine-5-methyltransferase M.HhaI and at a reference position unaffected by protein binding, it was suggested that the protein does not accelerate base pair opening (33). However, fluorination at the 5-position of uridine leads to an increase of the imino proton exchange by almost a factor of 60 (34), indicating a substantial increase in the base pair opening rate. A similar effect by fluorination of cytosine at the same position could potentially be the dominating contribution to the dynamic behavior in both the absence and presence of the protein. In the present study, we have shown that tracts of four or more GC base pairs exhibit a unique rapid opening dynamics that may contribute to the specificity of the cytosine-5-methyltransferases. It should be noted that it is the guanine base that carries the solvent exchangeable imino proton that is used in deriving the base pair dynamics. In case the fluctuations of the two bases in the GC base pair are independent, i.e. the opening is asymmetric, no information is provided on the fluctuations of the cytosine base. However, no evidence exists that base pair opening is asymmetric in the DNA double helix, although this appears to be the case in PNA-DNA hybrid (9). Furthermore, the cytosine base is not recognized itself by the methyltransferases. For example, M.HhaI binds with higher affinity to an abasic site than to the cognate sequence (5).
Interestingly, M.HhaI and M.HaeIII cause
only quite modest deformation of the DNA helix, whereas
adenine-N6-methyltransferase
(M.EcoRI) with the recognition sequence
5'-GAATTC-3' causes a severe 52° bend of the DNA helix
(35). It is possible that this reflects the intrinsic tendency for the
G tract base pair to open. On the other hand, the stable, A tract type
of recognition sequence of M.EcoRI may require larger
deformations of the helix to facilitate the base pair opening.
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FOOTNOTES |
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* This work was supported by the Swedish Natural Science Research Council, the Magnus Bergvall Foundation, the Lars Hierta Memorial Foundation, the Swedish Institute, the Deutscher Akademischer Austauschdienst, the Deutsche Forschungsgemeinschaft and the Fonds of the Chemical Industry.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 46-8-16-24-47; Fax: 46-8-15-55-97; E-mail: leijon{at}biophys.su.se.
2 M. Leijon, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: M.HhaI, M.HaeIII, and M.EcoRI, methyltranferases; 5mC, 5-methylcytosine; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; PNA, peptide nucleic acid.
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REFERENCES |
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