(Received for publication, September 5, 1995)
From the
The structural polymorphism of DNA is a vital aspect of its
biological function. However, it has become increasingly apparent in
recent years that DNA polymorphism is a complicated, multidimensional
phenomenon that includes not only static sequence-directed structures
but dynamic effects as well, including influences of counterions and
sequence context. In order to address some of these additional factors
that govern DNA conformation, we have used T4 ligase-mediated
cyclization to investigate bending in a series of DNA sequences
containing the GGGCCCGGGCCC motif in different sequence contexts
including various helical phasings with (A)
-tracts. We
present evidence for curvature in GGGCCC
GGGCCC and
(A)
-tract motifs in the presence of physiological levels of
Mg
and show that these motifs curve through similar
but oppositely directed bending angles under these ionic strength
conditions. Although these two sequence motifs appear to bend
similarly, our results suggest significant differences in stiffness and
stability of curvature between them. We also show that under the same
experimental conditions, the CTAG
CTAG sequence element possesses
unusual torsional flexibility and that this appears to be associated
with the central TA
TA dinucleotide. The results underscore the
need to include sequence context and specific ion effects as well as a
dynamic basis in more complete predictive models for functionally
related DNA polymorphism.
The structural polymorphism of DNA has attracted much attention in the past decade, primarily for its role in many regulatory processes (1, 2, 3, 4) . The early picture of DNA as a homogeneous, rod-like molecule has been replaced with one in which site-directed bending and flexibility contributes extensively to the binding affinity and specificity of bound regulatory proteins. Thus, sequence-dependent DNA curvature and flexibility are now regarded as key facets of many biological systems including chromatin (5) and specific regulatory systems(1, 3, 4, 6, 7) .
Macroscopic DNA curvature has been primarily associated with properly phased stretches of adenines, the so-called A-tracts(8) . Wedge and junction models, attempting to explain this phenomenon, proposed different basic unit lengths (dinucleotide and the whole A-tract, respectively) and different angular parameters as a source of bending (9, 10, 11, 12, 13, 14, 15) . However, a common conclusion from both models was that phased adenines are required for substantial macroscopic DNA curvature; although curvature is also associated with other sequence elements, it is invariably much smaller than that due to phased A-tracts(14, 15, 16) . A third model(17, 18, 19) , has been proposed that attributes curvature to superhelical writhe due to bent non-A-tract sequences, which is then moderated by A-tracts. The basic assumption of this model is that A-tracts, which are themselves straight, provide proper phasing for the surrounding bent sequence. Although this model was ignored for many years due to lack of experimental evidence, it has recently been revitalized by a growing body of crystallographic results(20, 21, 22) . In two nearest neighbor wedge models(14, 15) , the set of angular parameters has been expanded to include all 16 dinucleotide wedges, which suggests that sequences other than A-tracts can also be sources of bending. All of these models are static, however, and do not take the thermal fluctuations of DNA into account. To account for the dynamic character of DNA, Zhurkin and co-workers (23, 24) employed Monte Carlo simulations, and a ``flexible wedge'' model was offered as an extension of the static models.
Coincidental with the
proposals of these models, it has been shown that DNA bending
preferences and rotational orientations in covalently closed and
topologically relaxed circles mimic the same features of nucleosomal
DNA(5, 25, 26) . In this work, it was noted
that the minor grooves of AAATTT and GGC
GCC trinucleotides
were preferentially located on the inner and outer sides, respectively,
of curved nucleosomal DNA. This complemented the results of the same
authors on free, linear DNA and indicated minor and major groove
compression, respectively, for AAA
TTT and
GGC
GCC(27) . Because all DNA sequences contribute to DNA
wrapping around the nucleosome core at least to some
degree(26) , it is reasonable to expect that DNA curvature may
arise at least in part from sequence elements other than A-tracts or
exclusively so according to the bent non-A-tract model. Although gel
mobility studies, the experimental approach usually employed to detect
global DNA curvature, suggested that non-A-tract sequences might be
involved in DNA
curvature(15, 16, 28, 29) , this
conclusion retained a residual ambiguity because of uncertainties in
the interpretation of the gel mobility anomaly.
Recent studies have
shown that the GGGCCCGGGCCC sequence motif is strongly curved
toward major groove as evidenced by DNase I cutting and phasing
analysis with A-tracts(30) . Bending in the GGCC
GGCC
element has also been detected by x-ray
crystallography(20, 31) . Gel mobility experiments at
higher ionic strength conditions showed that the curvature of these
sequences is comparable in magnitude to A-tract based
curvature(32) . In this work, a surprising observation was that
the sequences containing A-tracts are much less susceptible to the
influence of elevated concentrations of certain divalent ions than are
sequences without them. This underscores the importance of specific ion
and ionic strength effects on local DNA structure and conformation and
suggests that these factors may be quite different in the cell under
physiological conditions than under the ionic conditions typically used
in standard gel mobility experiments. It also raises the interesting
possibility that under physiological conditions, the A-tract and
GGGCCC
GGGCCC motifs deflect DNA similarly but in different
directions. Whether the presence of specific ions induces fixed
curvature in GGGCCC
GGGCCC or merely stabilizes it in a curved
state by restricting flexibility is not yet clear.
In the present
work, we have employed DNA circularization assays to determine the
upper limit of curvature as 30 ° per helical repeat in DNA
containing the GGGCCCGGGCCC motif. The maximum in cyclization
efficiency, however, corresponds to a bend angle of 20-24 °,
which we propose to be its most probable value. This result is in
excellent agreement with the bending detected by x-ray crystallography
in a similar sequence(20) . In addition, certain sequences
containing the GGGCCC
GGGCCC motif are found to be virtually
independent of end alignment in cyclizing oligomers because unlike most
sequences, a change in the oligomeric repeat length did not lead to
significant differences either in occurrence or in distribution of ring
sizes. We attribute this to unusual torsional flexibility at
CTAG
CTAG elements, most probably to the TA
TA dinucleotide
within it. Torsional flexibility, defined as the facility of certain
sequence elements to vary twist angle(s), adds another dimension to DNA
polymorphism that requires additional study.
As a control, we
performed cyclization experiments using sequences containing
differentially phased GGGCCCGGGCC and A-tract elements. These
experiments demonstrated that correct helical phasing of these elements
is essential for the formation of small circles, suggesting that
A-tracts also contribute to the curvature when properly phased with
other bending elements rather than just providing the correct phasing
when placed between them. The results are discussed in the context of
current models for DNA curvature.
Figure 1:
a, sequences used for the initial
ligation experiments, with the basic repetitive unit shown in capital letters. Complementary strands were designed to
provide 3-bp overhangs at the 5` ends. The oligonucleotide lengths of
31 and 32 bp, with sequence repeats of 10.33 and 10.67, respectively,
were used to test the effect of different rotational phasings. b, sequences identical to those in a except that
thymine in the CTAGCTAG motif was replaced by guanine (underlined). c, five 42-mers with different helical
phasing relationships between poly(A)-tracts and GGGCCC
GGGCCC
motifs (both in capital letters). 4-bp overhangs were provided
at the 5` ends to ensure directed head-to-tail
ligation.
The relative amounts of circular and linear species in the various second dimension gel spots were determined either by direct scintillation counting or by particle counting from the scanned images using the public domain program NIH Image, version 1.55. The amounts of radioactivity were then normalized by the number of radioactive phosphates in each spot to get the relative number of molecules(35) . The cyclization efficiency was expressed as the ratio of normalized values for circular to linear gel spots(16) . Final results were obtained by averaging results from at least three completely independent experiments.
Our intent in the present work was to quantitate the
bending angle of the sequence containing the GGGCCCGGGCCC motif
in solution using mixed ligation
cyclization(10, 33, 36) . The second
dimension polyacrylamide gel cyclization assay for the repetitive 32-bp (
)precursor sequence 32ta in Fig. 1a is
shown in Fig. 2. It is clear that bending due to the basic
GGGCCC
GGGCCC repetitive unit must be considerable, because
microcircles as small as 128 bp (4-mers of the 32-bp precursors) were
formed in relatively large amounts. In addition, the amount of circular
products for microcircles of sizes
128 bp was comparable to the
amounts of the correspondingly sized linear fragments. The upper limit
of bending per GGGCCC
GGGCCC motif is calculated as
30 ° (i.e. 360 °/12, where 12 is the number of basic repetitive
units in the smallest circles).
Figure 2: A representative second dimension from the two-dimensional polyacrylamide gel cyclization assay for sequence 32ta (Fig. 1a). Runs of spots containing linear, closed, and nicked circles appear from bottom to top and are indicated. Numbers associated with individual spots show the number of 32-bp precursor elements (Fig. 1a) in each oligomer. Separation from left to right occurred in the 5% polyacrylamide first dimension gel and from top to bottom in the 8% polyacrylamide second dimension with added chloroquine phosphate (10, 33, 35) as described in the text.
To determine the most probable bending angle, the total radioactivity in each circular spot was normalized by the number of radioactive phosphates to obtain the relative number of circular molecules. The most probable bending angle was obtained from the maximum in the distribution of these values as a function of circle size. However, these values may be misleading in determining the relative cyclization efficiency and hence in the estimation of bending. During the initial stages of reaction, smaller linear products of self-ligation are usually present at higher concentrations, thus providing more ``substrate'' for the correspondingly sized cyclization products. This can lead to an over-representation of smaller circles, for which bending is larger, in the distribution of circle sizes and will tend to skew the distribution maximum toward smaller circle sizes. Unless differences in cyclization efficiencies as a function of circle size are very large so that such skewing of the normal thermal distribution is clearly evident, this can in turn lead to an overestimation of the bending angle. On the other hand, larger linear products predominate as the reaction goes toward infinite time, as do their corresponding circular ligation products, leading at infinite time to an underestimation of the bending angle (37) . To avoid this problem, we define the relative cyclization efficiency at a given circle size as the ratio of circular to linear molecules of that size; in other words, the number of molecules of each circular species is normalized by the number of linear substrate molecules from which it originates(16) . Because an excess of DNA is used in each experiment, a stationary state is assumed throughout the reaction with the exception of the initial time course. These methods have been reviewed and relative cyclization efficiencies, as discussed above and determined in this work, have been compared with kinetic determinations of true cyclization probabilities(36) .
Fig. 3gives
distributions of relative cyclization efficiencies (normalized as
discussed above) corresponding to circular species from 128 to 256 bp
in size for sequences 32ta and 31ta (Fig. 1). For sequence 32ta,
the maximum occurs between circle sizes of 160 and 192 bp. The
differences between these values, 1.01 and 0.99, respectively, are well
within experimental error after averaging three independent
experiments. This corresponds to a most probable bending angle in the
interval of 20-24 ° as calculated above. From the cyclization
experiments alone, it is not possible to localize this value, or any
part of it, unambiguously to any given region in the basic repetitive
sequence unit. We propose that it originates mostly in the
GGGCCCGGGCCC motif, because bending of 23 ° has already been
observed within the GGCC tetramer by x-ray
crystallography(20) .
Figure 3: Distributions of relative cyclization efficiencies for sequences 32ta and 31ta (Fig. 1a). The legend box indicates the number of base pairs in the closed circles from precursor oligonucleotide 31ta (numbers without parentheses) and 32ta (numbers with parentheses). Circular species contain the same number of repetitive precursor oligomers in each case. The experimental error in these data was ±0.04.
Sequence 31ta is identical to 32ta and
contains the same repetitive motif but is only 31 bp in length due to a
deletion of guanine 19 in the latter. This sequence has been shown to
have a smaller gel mobility anomaly than sequence 32ta(32) .
This is not surprising because although the two sequences have
virtually the same helical repeats (10.7 bp/turn), they
have different values of total twist angle (1026.1 versus 992.4 ° for 32ta and 31ta, respectively, with
=
33.7 °) as estimated from the results of Kabsch et al.(38) and from other independent
measurements(39, 40, 41) . This should impose
quite different planarity or superhelical writhe in ligated multimers.
At the same time, the two sequences had very similar gel mobilities
when 10 mM Mg
was added to the gel and
running buffers(32) . It is therefore of interest to test
whether the similar mobilities in gels containing Mg
would translate into similar cyclization profiles, because
Mg
is required for T4 ligase activity in cyclization
experiments.
Identical cyclization experiments on the two sequences
produced virtually identical gel second dimensions (sequence 32ta shown
as Fig. 2; results for 31ta not shown). Further analysis showed
that sequence 31ta has very similar distribution of cyclization
efficiencies to 32ta, with a maximum of 0.78 at 155- and 186-bp circle
sizes (Fig. 3). Thus, the two distributions in Fig. 3are
identical within experimental error except for the difference in
maximum relative cyclization efficiencies, and we conclude that both
sequences have the same most probable overall bending angles. This is
especially striking because circles that consist of 5- and 6-mer
precursor sequences differing by 1 bp differ by 5 and 6 nucleotides in
the oligomers exhibiting maximum relative cyclization efficiency, i.e. 155 versus 160 bp and 186 versus 192
bp, which is approximately half a helical turn. In other words, if the
single-stranded ends are perfectly aligned for cyclization-ligation in
one of these fragments, they are torsionally misaligned by
180 ° in the other, and the ends of 31ta and 32ta must be
rotationally displaced nearly oppositely with respect to each other.
Even if the ends are not perfectly aligned for cyclization-ligation in
32ta, they must be better positioned than in 31ta because the sequence repeat of 32ta is 10.67 bp (32 bp/3) or very nearly
the helical repeat of G/C-rich sequences (
10.7
bp/turn(39, 40, 41) ) as noted above; the
sequence repeat of 31ta is only 10.33 bp. In addition, gel mobility
experiments have demonstrated that 32ta produces more planar molecules
then 31ta (32) , in agreement with the above arguments. We
conclude, therefore, that some element within the basic repetitive unit
allows relatively unrestricted twisting, thus facilitating correct end
alignment by the ligase in both sequences. The above considerations
suggest that end alignment is achieved in sequence 31ta by
overtwisting; this hypothesis is tested in the next set of experiments.
Fig. 4compares the relative cyclization efficiency
distributions for the sequences shown in Fig. 1b.
Replacing TATA in sequence 32ta with GA
TC to produce
sequence 32ga has little effect on the circle distribution (c.f. section 32ga with section 32ta in Fig. 3). The smallest
circle shifts one oligomer toward larger circle sizes, whereas the
maximum in the cyclization distribution occurs at a 192-bp circle size
(section 32ga in Fig. 4), giving a most probable bending angle
of 20 °. This is within the range of values observed for sequences
32ta and 31ta. We conclude, therefore, that the TA
TA dinucleotide
is probably not crucial for the bending observed in sequences 32ta and
31ta. This implies that most of the bending is located in the GGGCCC
motif.
Figure 4: Distributions of relative cyclization efficiencies for sequences 32ga and 31ga (Fig. 1b). The legend box indicates the number of base pairs in the closed circles from precursor oligonucleotide 31ga (numbers without parentheses) and 32ga (numbers with parentheses). Circular species contain the same number of repetitive precursor oligomers in each case. The experimental error in these results was ±0.04.
However, the gel cyclization assay for sequence 31ga is very
much different from that for sequence 32ga (data not shown). There is
no detectable circle formation at smaller circle sizes, below 7-mers,
and the relative cyclization efficiency is very low even for the larger
oligomers (section 31ga in Fig. 4). Because sequence 32ga
exhibits both normal bending and a normal relative cyclization
probability distribution, it is likely that the weak cyclization
observed for sequence 31ga is caused by incorrect alignment of the
ends. This experiment therefore strongly supports the hypothesis that
the TATA dinucleotide in a CTAG
CTAG motif is a source of
torsional flexibility. It also validates the premise that a helical
repeat of 10.67 is closer than 10.33 for these types of sequences.
Figure 5:
Second dimension from the two-dimensional
polyacrylamide gel cyclization assay for sequence 42-1 (Fig. 1c). The absence of circles arises because the
A-tracts and GGGCCCGGGCCC motifs are separated by an integral
number of helical turns.
Relative cyclization efficiency distributions
for the set of sequences in Fig. 1c are given in Fig. 6. These results show remarkable consistency with the
published gel mobility data ( Fig. 7in (30) ), which can
be briefly summarized as follows. When GGGCCCGGGCCC motifs and
A-tracts are separated by exactly an integral number of
helical repeats, as in sequence 42-1, small microcircles are not
formed, and intermediate sized microcircles (
200 bp) are formed
only very inefficiently (Fig. 5). However, when the motifs are
separated by one and one-half helical turns, as in sequences
42-3 and 42-4, circles as small as 126 bp are formed, with
the maximum in the relative cyclization efficiency distribution at 168
bp (Fig. 6). The implications of this finding are discussed more
fully below.
Figure 6:
Distributions of relative cyclization
efficiencies for the set of 42-bp oligomers with alternative phasing of
A-tracts and GGGCCCGGGCCC motifs, sequences 42-1 through
42-5 (Fig. 1c). The legend box shows the
number of base pairs in each closed circle. The experimental error in
these results was ±0.05.
Figure 7: Computer-generated plot of a 100-bp oligomer made by self-ligation of the repeating sequence 32ta. Coordinates were calculated with the program Curvature (70) using wedge angle parameters (roll and twist or wedge angles) from Bolshoy et al.(15) (a) including the helical twist values of Kabsch et al.(38) and DeSantis et al.(14) (b). The actual plots shown were made using software developed for this purpose by one of the authors (M. D.).
In Fig. 7, we show two
computer generated plots of the 100-bp DNA sequence obtained by
successively ``ligating'' the repetitive sequence 32ta (Fig. 1a). Wedge angle (roll and tilt angle) parameters
were taken from the two most recent and extensive wedge models: Fig. 7A on the model developed by Bolshoy et al.(15) and Fig. 7B on the model of DeSantis et al.(14) . Helical twist angles in the first case
used the predicted values of Kabsch et al.(38) .
Although only the Bolshoy et al. wedge angles are based
exclusively upon experimental gel mobility data, both models predict
moderate curvature in the DNA sequence, in qualitative agreement with
experimental gel mobility assay data obtained on this sequence.
However, neither of these models can predict the results of gel
mobility assays in the presence of added Mg(32) or the results of our cyclization experiments, also
done in the presence of Mg
. It is possible that DNA
structures and conformations deduced from conventional gel mobility
assays, which are typically performed in the absence of
Mg
, are not generally equivalent to those formed in
living cells(1) , because the typical physiological level of
Mg
in most living systems is approximately equivalent
to the levels in these experiments with added Mg
. The
most recent model(54) , based upon data obtained under quasiphysiological ionic conditions(26) , is in more
general agreement with current experimental observations.
The
curvature in sequences containing A-tracts seems to be much less
dependent upon elevated divalent ion concentrations than in sequences
with GGGCCCGGGCCC motifs(32) . This observation suggests
that bending in the GGGCCC
GGGCCC motif is either induced or stabilized by physiological levels of Mg
ions. Curiously, however, these ions are not necessary for
GGGCCC
GGGCCC bending if A-tracts are also present nearby in the
DNA sequence, as deduced from the similarity between conventional gel
mobility (30) and the cyclization assays in this work for all
the 42-mer sequences listed in Fig. 1c (Fig. 6).
It appears that the results of conventional gel mobility and
cyclization experiments are in concurrence only for sequences having
stable, fixed curvature. The presence of A-tracts seems necessary in
order to observe a significant mobility anomaly in conventional gel
mobility experiments. This may explain the relative inability of the
method to detect the true degree of curvature in sequences without
A-tracts. We propose that the principal difference between A-tracts and
GGGCCCGGGCCC motifs under the conditions of the present
experiments is the stability rather than the magnitude or degree of curvature. Although it appears that A-tracts
adopt a bent conformation under various ionic strength conditions (and
possibly other environmental conditions as well) because of their
strong fixed bending and rotational
preference(5, 26, 27) , the GGGCCC
GGGCCC motif evidently requires either the nearby presence of A-tracts
in the sequence or physiological levels of certain divalent ions
including Mg
. In this respect, the utility of
conventional gel mobility assays as tools for DNA curvature studies
must be reevaluated. The loss of anomalous mobility in standard gel
assays is very likely caused by sequence-dependent flexibility in
certain DNA sequence elements(48, 55) . Thus, although
gel mobility assays have been successfully used to detect dynamic
properties of DNA in certain special
cases(48, 55, 56) , the added dimensions
imposed by specific ion and ionic strength effects can clearly no
longer be ignored.
Using
single base substitutions, we identified the TATA dinucleotide
within the CTAG
CTAG motif as a probable locus of anomalous
torsional flexibility. Analysis of the CTAG
CTAG motif in crystals (42) shows that the TA
TA dinucleotide is underwound, with
a twist angle of 21 °. The CTAG
CTAG motif can adopt an
unusual conformation with alternating high and low twist values, which
is a feature also observed in other TA
TA-containing sequence
motifs(43, 45) . An unusual tendency to overwind has
also been attributed to just the TA
TA
dinucleotide(43, 44, 45, 46, 47) ,
and such overwinding would seem even more likely in the context of the
CTAG
CTAG motif from the crystal analysis noted above. We suggest
that the driving force for this process may be the strong rotational
preference of the adjacent (G)GGC(CC)
(GG)GCC(C) motifs (see Fig. 1), which tend to position rotationally so that the minor
grooves face outward(5, 26) . This argument receives
added force because the CTAG
CTAG motif occurs in the trp operator sequence where it participates in indirect readout with
the bound Trp repressor protein (63) and with the met repressor complex (64) , where its presence is an
important component of binding specificity.
Because considerable
evidence is now available that the TATA dinucleotide is unusual
in its ability to undergo stereochemical axial kinking (48, 49, 50) and may also be a locus of high
torsional flexibility as shown here and
elsewhere(44, 65) , it is an appealing speculation
that its flexibility may represent a mechanism for facilitating
alternative modes of DNA recognition and binding by proteins (4) and for DNA positioning in nucleosomes. A similar
speculation can be made with regard to the CA
TG dinucleotide,
which is even a more ubiquitous feature than TA
TA of specific
regulatory protein operator and enhancer sites (3, 4) and for which both kinking (48, 66) and overtwisting (35, 65, 67) have been demonstrated, as well
as its bimodal distribution of twist values in solved crystal
structures(52, 68) . It is particularly tempting to
speculate that all these phenomena may somehow be related in the
sequence-dependent binding of proteins to DNA.
To understand the
contributions of the different sequence motifs to the overall curvature
in these sequences, it is necessary to examine the differential phasing
between the motifs. There are two possible ways of interpreting these
differential phasing effects: (1) A-tracts and
GGGCCCGGGCCC motifs are on the same side of the helix in sequence
42-1 and the overall curvature due to them cancels, whereas these
motifs are on opposite sides of the helix in sequences 42-3 and
42-4 and their contributions to bending are additive and the
overall curvature is increased or (2) overall curvature is
canceled when CTCTC
GAGAG motifs are located on opposite sides of
the helix from GGGCCC
GGGCCC, and the curvature is increased when
these two motifs are located on the same side of the helix. In the
first case, the assumption is that both GGGCCC
GGGCCC and A-tract
are bent but in different directions. In the second case, the A-tracts
are assumed straight, whereas both CTCTC
GAGAG and
GGGCCC
GGGCCC motifs are bent into the major groove as has been
proposed on the basis of crystallographic
results(21, 54) . These two possibilities cannot be
distinguished from this particular set of experiments. However, the
first possibility can explain the cyclization results on sequence 32ga,
whereas the second possibility cannot. In sequence 32ga,
GGGCCC
GGGCCC and GAGAG
CTCTC motifs are helically phased
oppositely to their phasing in sequences 42-3 and 42-4, and
yet the maxima in the relative cyclization efficiency distributions
occur at virtually the same circle sizes in the two cases, indicating
that the curvatures are similar. In order to satisfy both observations,
therefore, it is necessary to assume that the CTCTC
GAGAG motif
undergoes strong bending into the major groove when surrounded by
A-tracts, as in sequences 42-3 and 42-4, while
exhibiting negligible bending of this type when surrounded by
GGGCCC
GGGCCC motifs, as in sequence 32ga. We believe that this
latter explanation is unlikely based upon studies of the sequence
context-dependent behavior of the CTCTC
GAGAG motif in certain
sequence contexts (69) .(
)
Another possibility is
that A-tracts are straight, whereas the GGGCCCGGGCCC motif is
bent toward the major groove by twice as much as other non-A-tract
sequences. However, this assumption requires the GGGCCC
GGGCCC
motif to be bent more than is observed in the crystal (20) and
the other sequences to be bent much more than detected in
experiments so far reported. Additional evidence in support of the
first interpretation above is obtained from cyclization experiments on
sequences containing alternating A-tracts and GGGCCC
GGGCCC motifs
with a center-to-center separation of exactly one-half helical repeat.
This sequence is found to cyclize with extremely high efficiency,
forming microcircles as small as 105 bp whose sizes and circular
integrities have been verified by scanning tunneling
microscopy.(
)
From these results, we conclude that A-tracts are bent elements that can promote macroscopic DNA curvature when appropriately phased either with themselves or with other bent elements. The hypothesis that A-tracts are straight elements that produce macroscopic DNA curvature by providing correct helical phasing for other bent elements is not only inconsistent with the present data but would require assumptions with respect to bending in non-A-tract sequences that are not substantiated by presently available experimental results.
The observations presented here, together with
recent reports from other laboratories concerning the effects of
divalent cations on curvature in GGGCCCGGGCCC-containing
sequences, suggest that the principal differences between A-tracts and
GGGCCC
GGGCCC motifs, at least under the experimental conditions
used in conventional gel mobility assays, may be in the lability and stability of curvature, rather than in the absolute
degree of fixed curvature itself. Evidence has also been presented that
suggests that the TA
TA dinucleotide within the CTAG
CTAG
motif may be a locus of unusual torsional flexibility in
certain sequences and under certain physical and environmental
conditions. In all these cases, ionic strength and sequence context
have emerged as additional variables that must be included to define
the full dimensionality of sequence-dependent DNA structure and
conformation. All this is consistent with results from this and other
laboratories over the last few years in support of the view that a
number of sequence motifs contribute in important ways to DNA
structural polymorphism through either intrinsic or protein-induced DNA
flexibility. However, it also adds considerably to the complexity of
DNA structural polymorphism and calls for appropriately refined and
dynamically based models for the prediction of DNA trajectory and its
protein binding properties from its sequence.