©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Bending and Torsional Flexibility of G/C-rich Sequences as Determined by Cyclization Assays (*)

(Received for publication, September 5, 1995)

Mensur Dlakic Rodney E. Harrington (§)

From the Department of Biochemistry, University of Nevada, Reno, Reno, Nevada 89557-0014

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 GGGCCCbulletGGGCCC motif in different sequence contexts including various helical phasings with (A)(5)-tracts. We present evidence for curvature in GGGCCCbulletGGGCCC and (A)(5)-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 CTAGbulletCTAG sequence element possesses unusual torsional flexibility and that this appears to be associated with the central TAbulletTA 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.


INTRODUCTION

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 AAAbulletTTT and GGCbulletGCC 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 AAAbulletTTT and GGCbulletGCC(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 GGGCCCbulletGGGCCC sequence motif is strongly curved toward major groove as evidenced by DNase I cutting and phasing analysis with A-tracts(30) . Bending in the GGCCbulletGGCC 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 GGGCCCbulletGGGCCC motifs deflect DNA similarly but in different directions. Whether the presence of specific ions induces fixed curvature in GGGCCCbulletGGGCCC 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 GGGCCCbulletGGGCCC 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 GGGCCCbulletGGGCCC 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 CTAGbulletCTAG elements, most probably to the TAbulletTA 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 GGGCCCbulletGGGCC 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.


MATERIALS AND METHODS

Purification, Labeling, and Ligation of Oligonucleotides

Oligonucleotides 31ta and 32ta and all 42-mers (Fig. 1) and their complementary strands were synthesized using an Applied Biosystems 381A oligonucleotide synthesizer, whereas oligomers 31ga and 32ga were obtained from the Midland Reagent Co. All were purified on 20% urea denaturing polyacrylamide gels, followed by desalting on Sephadex columns and ethanol precipitation. In a total volume of 50 µl, which contained kinase buffer supplied by New England Biolabs, 20-30 µg of each strand was 5` end-labeled with [-P]ATP and 10 units of T4 polynucleotide kinase for 30 min. Cold ATP was added to 2 mM, and incubation continued with a fresh batch of the kinase for another 15 min. The reaction was ended by phenol extraction, and the DNA was precipitated with ethanol in the presence of carrier tRNA. Complementary strands were mixed in Tris-EDTA buffer, and the mixture was heated to 90 °C and cooled slowly to room temperature. After the annealing, samples were run on a 5% polyacrylamide gel to separate duplex from labeled single-stranded DNA and from any alternative structures that might be formed due to the high G/C content of the DNA. After elution and desalting, DNA duplexes were self-ligated in the ligase buffer using 400 units of T4 DNA ligase (New England Biolabs). The reaction was allowed to proceed on ice for 12-16 h and was stopped by raising the temperature to 75 °C for 10 min, followed by phenol extraction and ethanol precipitation. Conditions for steady-state ligation reaction were tested prior to the analytical gel runs. We found that a change of reaction time over a range from 10 to 20 h does not affect the ratio of corresponding circular and linear fragments, indicating that stationary state conditions have been attained. We also found that initial DNA quantities of 5 µg or more are required to ensure that the extent of circularization is not concentration-dependent.


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 CTAGbulletCTAG motif was replaced by guanine (underlined). c, five 42-mers with different helical phasing relationships between poly(A)-tracts and GGGCCCbulletGGGCCC motifs (both in capital letters). 4-bp overhangs were provided at the 5` ends to ensure directed head-to-tail ligation.



Two-dimensional Gel Electrophoresis

Products of ligation were analyzed by two-dimensional gel electrophoresis using methods described elsewhere(10, 33) . Separation in the first dimension was on a 5% polyacrylamide gel (19:1 ratio of mono-acrylamide:bis-acrylamide) containing Tris-borate buffer (89 mM Tris/89 mM boric acid/2.5 mM EDTA, pH 8.3). Glycerol was added to the gel mixture to a final concentration of 25%(34) . Electrophoresis was for 12 h at 7 V/cm. The lane containing the ligation products was excised and placed horizontally between the slab gel plates to be used for the second dimension separation. Chloroquine phosphate was added both to the running buffer and the 8% polyacrylamide used for the second dimension gel to a total concentration of 50 µg/ml. Results were compared with 10% polyacrylamide second dimension gels, but in our hands, the best resolution of DNA microcircles was obtained with 8% gels. After 12-15 h of electrophoresis at 7 V/cm, linear and circular fragments were located by autoradiography of the wet gel on x-ray film at room temperature.

Size Determination and Quantitative Analysis

Various representative spots were excised from the second dimension polyacrylamide gel to determine both sizes and relative intensities. For size determination, both linear and circular DNA fragments were eluted overnight from the macerated gel slices, denatured by heating, and compared with the original ligation products on 8% denaturing polyacrylamide gels. BamHI linker ligation products were used as markers for an additional level of control.

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.


RESULTS

Determination of the Bending Angle

The sequences in Fig. 1a were originally designed to investigate the curvature of the GGGCCCbulletGGGCCC basic repetitive sequence element (shown in uppercase letters) in the absence of A-tracts including AA bullet TT dinucleotides. Indeed, they were shown to possess moderate curvature as determined by polyacrylamide gel mobility assays(29) . However, the observed curvature was much smaller than in corresponding sequences containing A-tracts. Subsequent experiments revealed that the GGGCCCbulletGGGCCC sequence motif can completely abolish the curvature caused by A-tracts when properly phased with them (30) and that divalent cations increase the gel retardation of these sequences much more than that of sequences containing A-tracts(32) . Almost simultaneously, a very similar sequence was found to be locally bent by x-ray crystallography (20) .

Our intent in the present work was to quantitate the bending angle of the sequence containing the GGGCCCbulletGGGCCC motif in solution using mixed ligation cyclization(10, 33, 36) . The second dimension polyacrylamide gel cyclization assay for the repetitive 32-bp (^1)precursor sequence 32ta in Fig. 1a is shown in Fig. 2. It is clear that bending due to the basic GGGCCCbullet 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 geq128 bp was comparable to the amounts of the correspondingly sized linear fragments. The upper limit of bending per GGGCCCbulletGGGCCC 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 GGGCCCbulletGGGCCC 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 Delta = 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.

Identification of Sequence Elements Responsible for the Observed Torsional Flexibility

In order to identify the locus of torsional flexibility, experiments were conducted in which sequence elements in the two precursor sequences, 32ta and 31ta (Fig. 1), were changed systematically, and the effect of these changes on the cyclization properties of both sequences was determined. The TAbulletTA dinucleotide in the spacer between the repeating GGGCCCbulletGGGCCC motifs is a likely possibility as a torsional flexibility site. The TAbulletTA dinucleotide within a CTAGbulletCTAG motif has been found to be undertwisted in a crystal(42) , and much additional evidence suggests that it may be a site of anisotropic axial as well as torsional flexibility(43, 44, 45, 46, 47) . In addition, the TAbulletTA dinucleotide is known to have relatively high roll values in crystals(21, 45) , and it has been shown to kink under certain conditions(48, 49, 50, 51) . Thus, it is also necessary to determine whether TAbulletTA contributes in any way to the bending observed in the sequences discussed above. Two additional precursor sequences were synthesized that are identical to 31ta and 32ta except for the substitution of guanine for thymine in the CTAGbulletCTAG motif, replacing TAbulletTA with a GAbulletTC dinucleotide at that site (Fig. 1b). The assumption was that change from TAbulletTA to GAbulletTC would not introduce any major difference in helical repeat because twist values for these two dinucleotides are very similar(14, 38, 52) .

Fig. 4compares the relative cyclization efficiency distributions for the sequences shown in Fig. 1b. Replacing TAbulletTA in sequence 32ta with GAbulletTC 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 TAbulletTA 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 TAbulletTA dinucleotide in a CTAGbulletCTAG 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.

Cyclization of Sequences Containing Differentially Phased GGGCCCbulletGGGCCC Motifs with A-Tracts

The sequences shown in Fig. 1c have already been used to test the phasing between GGGCCCbulletGGGCCC motifs and A-tracts (30) (for a detailed description of the logic behind their design also see Fig. 5from the same paper). The conclusion of the earlier work was that the bending in a GGGCCCbulletGGGCCC motif is of similar magnitude to the bending in A-tracts but opposite in directional sense. The latter point is also supported by our own results on the bending in GGGCCCbulletGGGCCC if we compare these with the most recent estimates for the bending angle in A-tracts(13) . Our use of cyclization methods and the results presented above also suggested that additional experiments on the earlier sequences could address the following two important questions. (i) Are cyclization results using these sequences in agreement with the published gel mobility assays, because we show above that this is not the case for sequences having only a GGGCCCbulletGGGCCC motif(30) ? (ii) Can these experiments contribute to the current dichotomy on whether A-tracts are curved or straight under solution conditions?


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 GGGCCCbulletGGGCCC 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 GGGCCCbulletGGGCCC 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 (geq200 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 GGGCCCbulletGGGCCC 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.).




DISCUSSION

Stability of DNA Curvature in Conventional Gel Mobility Assays

Unlike x-ray crystallography, which generally provides structural information at atomic levels of resolution for single static structures, gel mobility experiments measure time-averaged conformational properties of DNA molecules. In spite of the fact that the gel mobility anomaly is not well characterized by physical theory, the methods have made critical contributions to our present understanding of DNA conformational polymorphism. Thus, almost all predictive models for DNA sequence-dependent trajectories are based on gel mobility experiments(12, 14, 15) . The angular parameters proposed in each of these models vary considerably, showing the explicit dependence upon model, but, as a rule, all of them show good agreement with experimental data on sequences containing A-tracts. However, they all fail, in one or another respect, to account fully for the conformational behavior of certain sequences without A-tracts(53, 54) .

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 GGGCCCbulletGGGCCC motifs(32) . This observation suggests that bending in the GGGCCCbulletGGGCCC motif is either induced or stabilized by physiological levels of Mg ions. Curiously, however, these ions are not necessary for GGGCCCbulletGGGCCC 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 GGGCCCbulletGGGCCC 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 GGGCCCbullet 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.

The Implications of Bending in the GGGCCCbulletGGGCCC Motif

The possibility of sequence context-dependent behavior of the GGCbulletGCC trinucleotide was proposed almost a decade ago (11) in an attempt to rationalize DNase I cutting preferences in nucleosomal DNA(26) . A variability in roll values for the GCbulletGC dinucleotide has also been suggested based upon theoretical arguments(57) . This same theme has surfaced also in recent experimental studies concerned with the directionality of DNA bending(20, 30) . In this work, we have shown that the bending angle in sequences containing the GGGCCCbulletGGGCCC motif is in the range of 20-24 °, which is comparable to estimates for A-tract bending(13) . From the present results, we cannot rule out small contributions of flexibility or of phasing to the value calculated above, nor can we assign this entire bending angle unambiguously to only the GGGCCCbulletGGGCCC sequence motif. However, its association with at least a GGCCbulletGGCC or a GGCbulletGCC sequence element is strongly suggested by recent crystallographic results (20, 31) and by nucleosomal packaging data(26) . These findings collectively suggest that the GGGCCCbulletGGGCCC motif, or some part of it, may be a general site of structural polymorphism in DNA. Sequence elements based upon this motif are found in a number of specific binding sites for regulatory proteins(4, 7) including GC boxes of Sp1 (58) and high affinity binding sites for the p53 protein(59, 60) .

Torsional Flexibility in DNA

Cyclization assays are very powerful methods for studying torsional flexibility in DNA because of their sensitivity to the relative modulus of torsion (55) (also reviewed and discussed in Crothers et al.(61) ). Studies have shown that most DNA sequence elements have almost identical values of torsional rigidity(62) . This is implicit also in results that suggest that nearly perfect alignment of DNA ends is a very important general determinant of cyclization efficiency(37) . However, we show for the first time in the present work that two DNA oligonucleotides having nearly identical sequences but differing by a half helical turn over their 160-bp lengths nevertheless cyclize with very similar cyclization efficiencies (Fig. 3). Because such small microcircles cannot exhibit large superhelical writhes, this result can evidently only be explained if the sequences contain elements having anomalously high torsional flexibility. This adds yet another dimension to the role of flexibility in DNA structural polymorphism.

Using single base substitutions, we identified the TAbulletTA dinucleotide within the CTAGbulletCTAG motif as a probable locus of anomalous torsional flexibility. Analysis of the CTAGbulletCTAG motif in crystals (42) shows that the TAbulletTA dinucleotide is underwound, with a twist angle of 21 °. The CTAGbulletCTAG motif can adopt an unusual conformation with alternating high and low twist values, which is a feature also observed in other TAbulletTA-containing sequence motifs(43, 45) . An unusual tendency to overwind has also been attributed to just the TAbulletTA dinucleotide(43, 44, 45, 46, 47) , and such overwinding would seem even more likely in the context of the CTAGbulletCTAG 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)bullet(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 CTAGbulletCTAG 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 TAbulletTA 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 CAbulletTG dinucleotide, which is even a more ubiquitous feature than TAbulletTA 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.

Phasing Experiments between A-Tracts and GGGCCCbullet GGGCCC Motifs in a View of Current Models for DNA Curvature

A close inspection of Fig. 3and Fig. 6shows that the cyclization efficiency distributions are very similar between sequences 32ta and 31ta (Fig. 3) and 42-3 and 42-4 (Fig. 6). These pairs of sequences differ in the size of the central repetitive unit and share a common GGGCCCTAGAbulletTCTAGGGCCC motif (shown in capital letters in Fig. 1a). In sequences 32ta and 31ta, this is the only repeating motif, whereas the 42-mers have this motif as well as a twice repeated AAAAACTCTCbullet GAGAGTTTTT motif. The maximum in the cyclization efficiency distributions for both sequence pairs falls within the same range: 160-168 bp. Because these circles corresponding to the distribution maxima are formed from 15 repeating sequences with a GGGCCCbulletGGGCCC motif for sequence 32ta and with 8 GGGCCCbulletGGGCCC motifs as well as 8 A-tracts in case of the 42-mers, approximately half the curvature in the 42-mers must originate in the parts of the sequence containing the A-tracts. In addition, the relative cyclization efficiencies are somewhat higher for the 42-mers, which suggests that the parts of the sequence containing A-tracts must possess a greater overall stability of curvature.

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 GGGCCCbulletGGGCCC 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 CTCTCbulletGAGAG motifs are located on opposite sides of the helix from GGGCCCbulletGGGCCC, 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 GGGCCCbullet GGGCCC and A-tract are bent but in different directions. In the second case, the A-tracts are assumed straight, whereas both CTCTCbulletGAGAG and GGGCCCbulletGGGCCC 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, GGGCCCbulletGGGCCC and GAGAGbulletCTCTC 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 CTCTCbulletGAGAG 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 GGGCCCbulletGGGCCC motifs, as in sequence 32ga. We believe that this latter explanation is unlikely based upon studies of the sequence context-dependent behavior of the CTCTCbulletGAGAG motif in certain sequence contexts (69) .(^2)

Another possibility is that A-tracts are straight, whereas the GGGCCCbulletGGGCCC motif is bent toward the major groove by twice as much as other non-A-tract sequences. However, this assumption requires the GGGCCCbulletGGGCCC 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 GGGCCCbulletGGGCCC 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.(^3)

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 GGGCCCbulletGGGCCC-containing sequences, suggest that the principal differences between A-tracts and GGGCCCbulletGGGCCC 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 TAbulletTA dinucleotide within the CTAGbulletCTAG 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.


FOOTNOTES

*
Supported by research Grant MCB 9117488 from the National Science Foundation and Grant 1 R55 HG00656-01 from the National Institutes of Health and by the U. S. Department of Agriculture Hatch Project NEV032D through the Nevada Agricultural Experiment Station (to R. E. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviation used is: bp, base pair(s).

(^2)
M. Dlakic and R. E. Harrington, submitted for publication.

(^3)
M. Dlakic, T.-W. Jing, S. M. Lindsay, and R. E. Harrington, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Ivan Brukner for generous provision of certain sequences used in this work and for many comments and suggestions and Dr. Ilga Winicov for many helpful discussions.


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