Conformation and rigidity of DNA microcircles containing waf1 response element for p53 regulatory protein.

The tumor-suppressor activity of p53 is closely related to its DNA-binding properties. It binds a number of DNA response-elements and it is likely that these share a common structural feature. Here, we present a new, general method to determine the absolute twist of flexible DNA promoter sequences based on direct imaging of the topology of microcircles containing the sequences. We have used magnetically driven dynamic force microscopy ("MacMode" AFM) to observe, in solution, the conformation of 168 base-pair DNA microcircles, each containing four equally spaced copies of the waf1/cip1/p21 p53 response-element. Analysis of the images showed that the microcircles are markedly puckered with a small excess of negatively writhed molecules. The average measured values of writhe are 0.109+/-0.013 (for 60 positively writhed molecules) and -0.098+/-0.011 (for 65 negatively writhed molecules). These values lead directly to a difference in linking number for the positively and negatively writhed molecules prior to ligation, from which we derive a twist mismatch of 178 degrees (overtwist). This is 44.5 degrees for each 42-mer precursor containing a single waf1/cip1/p21 p53 response-element, in good agreement with the range of values deduced by indirect biochemical techniques. The two values of writhe may also be used to determine the ratio of the bending (B) to twisting (C) rigidity, yielding B/C=0.23. This is about one-third of the value for long, random-sequence DNA, suggesting that the waf1/cip1/p21 p53 response-element is extremely flexible, a result that is also consistent with indirect biochemical experiments. These results support the idea, proposed by us earlier, that torsional stress may play a role in the regulation of p53 binding through modulation of twist at the binding site.

Conformational transition in DNA on a cold surface.

The contour length of DNA fragments, deposited and imaged on mica under buffer, was measured as a function of deposition temperature. Extended DNA molecules (on Ni- and silane-treated surfaces) contract rapidly with falling temperature, approaching the contour length of A-DNA at 2 degrees C. The contraction is not unique to a specific sequence and does not occur in solution at 2 degrees C or on a surface at 25 degrees C, indicating that it arises from a combination of low temperature and surface contact. It is probably a consequence of reduced water activity at a cold surface.

p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting.

DNA binding activity of p53 is crucial for its tumor suppressor function. Our recent studies have shown that four molecules of the DNA binding domain of human p53 (p53DBD) bind the response elements with high cooperativity and bend the DNA. By using A-tract phasing experiments, we find significant differences between the bending and twisting of DNA by p53DBD and by full-length human wild-type (wt) p53. Our data show that four subunits of p53DBD bend the DNA by 32-36 degrees, whereas wt p53 bends it by 51-57 degrees. The directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element. More sophisticated phasing analyses also demonstrate that p53DBD and wt p53 overtwist the DNA response element by approximately 35 degrees and approximately 70 degrees, respectively. These results are in accord with molecular modeling studies of the tetrameric complex. Within the constraints imposed by the protein subunits, the DNA can assume a range of conformations resulting from correlated changes in bend and twist angles such that the p53-DNA tetrameric complex is stabilized by DNA overtwisting and bending toward the major groove at the CATG tetramers. This bending is consistent with the inherent sequence-dependent anisotropy of the duplex. Overall, the four p53 moieties are placed laterally in a staggered array on the external side of the DNA loop and have numerous interprotein interactions that increase the stability and cooperativity of binding. The novel architecture of the p53 tetrameric complex has important functional implications including possible p53 interactions with chromatin.

Unconventional helical phasing of repetitive DNA motifs reveals their relative bending contributions.

A novel, multiple DNA phasing analysis is described in which three sequence motifs associated with bent DNA are clustered together in oligomers of identical base composition, but with different phasing relationships of these motifs to each other. Synthetic oligonucleotides containing different combinations of AAAAA(A), GGGCCC and GAGAG sequence motifs were ligated and analyzed by gel mobility and cyclization experiments to determine their global curvature. These assays were used to obtain relative bending contributions of the analyzed sequence motifs. The experimental results also provide a rigorous test of predictive models for DNA bending. We report, using molecular modeling, that none of the most widely used dinucleotide (nearest neighbor) models can accurately describe the conformational properties of these DNA sequences and that more complex models, at least at the trinucleotide level, are required.

DIAMOD: display and modeling of DNA bending.

MOTIVATION: DIAMOD (Displayandmodeling ofDNA) was created as a user-friendly software for exploring and better understanding DNA structural variations, particularly DNA bending. It was intended to be as open as possible so that any of the existing or future predictive models can be used with it. RESULTS: DIAMOD features graphic display and interactive manipulation of DNA molecules on the screen. Since it works with di-, tri- or tetranucleotide models supplied as external files of angular parameters, it was recently used to evaluate critically all available predictive models for DNA bending. The program has a unique option to insert bends at defined positions in DNA sequence independently of the currently used model, which enables the simulation of both intrinsic and protein-induced kinking. Finally, many output file formats facilitate the sharing of data with other programs and the creation of visually pleasing images. AVAILABILITY: The program is available on request to academic users free of charge. It will be distributed via the WWW (http://www-personal.umich.edu/ mensur/software.html). Users with no network access can get a copy directly from the author. CONTACT: mensur@umich.edu

Strained DNA is kinked by low concentrations of Zn2+.

A novel atomic force microscope with a magnetically oscillated tip has provided unprecedented resolution of small DNA fragments spontaneously adsorbed to mica and imaged in situ in the presence of divalent ions. Kinks (localized bends of average angle 78 degrees) were observed in axially strained minicircles consisting of tandemly repeated d(A)5 and d(GGGCC[C]) sequences. The frequency of kinks in identical minicircles increased 4-fold in the presence of 1 mM Zn2+ compared with 1 mM Mg2+. Kinking persisted in mixed Mg2+/Zn2+ electrolytes until the Zn2+ concentration dropped below 100 microM, indicating that this type of kinking may occur under physiological conditions. Kinking appears to replace intrinsic bending, and statistical analysis shows that kinks are not localized within any single sequence element. A surprisingly small free energy is associated with kink formation.

Architectural accommodation in the complex of four p53 DNA binding domain peptides with the p21/waf1/cip1 DNA response element.

High resolution chemical footprinting and cross-linking experiments have provided a basis for elucidating the overall architecture of the complex between the core DNA binding domain of p53 (p53DBD, amino acids 98-309) and the p21/waf1/cip1 DNA response element implicated in the G1/S phase cell cycle checkpoint. These studies complement both a crystal structure and earlier biophysical studies and provide the first direct experimental evidence that four subunits of p53DBD bind to the response element in a regular staggered array having pseudodyad symmetry. The invariant guanosines in the highly conserved C(A/T)|(T/A)G parts of the consensus half-sites are critical to the p53DBD-DNA binding. Molecular modeling of the complex using the observed peptide-DNA contacts shows that when four subunits of p53DBD bind the response element, the DNA has to bend approximately 50 degrees to relieve steric clashes among different subunits, consistent with recent DNA cyclization studies. The overall lateral arrangement of the four p53 subunits with respect to the DNA loop comprises a novel nucleoprotein assembly that has not been reported previously in other complexes. We suggest that this kind of nucleoprotein superstructure may be important for p53 binding to response elements packed in chromatin and for subsequent transactivation of p53-mediated genes.

DNA bending is essential for the site-specific recognition of DNA response elements by the DNA binding domain of the tumor suppressor protein p53.

We have used circular permutation assays to determine the extent and location of the DNA bend induced by the DNA binding domain of human wild type p53 (p53DBD) upon binding to several naturally occurring DNA response elements. We have found that p53DBD binding induces axial bending in all of the response elements investigated. In particular, response elements having a d(CATG) sequence at the junction of two consensus pentamers in each half-site favor highly bent complexes (bending angle is approximately 50 degrees ), whereas response elements having d(CTTG) bases at this position are less bent (bending angles from approximately 37 to approximately 25 degrees ). Quantitative electrophoretic mobility shift assays of different complexes show a direct correlation between the DNA bending angle and the binding affinity of the p53DBD with the response elements, i.e. the greater the stability of the complex, the more the DNA is bent by p53DBD binding. The study provides evidence that the energetics of DNA bending, as determined by the presence or absence of flexible sites in the response elements, may contribute significantly to the overall binding affinity of the p53DBD for different sequences. The results therefore suggest that both the structure and the stability of the p53-DNA complex may vary with different response elements. This variability may be correlated with variability in p53 function.

The organic crystallizing agent 2-methyl-2,4-pentanediol reduces DNA curvature by means of structural changes in A-tracts.

Contemporary predictive models for sequence-dependent DNA structure provide a good estimation of overall DNA curvature in most cases. However, the two current models differ fundamentally in their view of the origin of DNA curvature. An earlier model that associates DNA bending primarily, although not exclusively, with stretches of adenines (A-tracts) is based on results of comparative gel retardation, cyclization kinetics, hydroxyl radical cutting, and other solution measurements. It represents an intersection of wedge and junction models. More recently, a non-A-tract bending model has been proposed, built on structural results from x-ray crystallography and molecular modeling. In this view, A-tracts are proposed to be straight and rigid, whereas mixed sequence DNA is bent. Because a key premise of the non-A-tract bending model is the crystallographic observation that A-tracts are straight, we have examined the effect in solution of 2-methyl-2,4-pentanediol (MPD), an organic solvent used in crystal preparation for crystallographic DNA structure determinations. Using cyclization analysis, DNase I cutting, chemical probing, and electron microscopy on DNA oligomers with and without A-tracts, we show that the presence of MPD in solution dramatically affects A-tracts and that the effect is specific to these sequence elements. Combined with the previous observation that MPD affects gel mobility of curved sequences with A-tracts, our findings support the bent A-tract model and call for caution in the interpretation of crystallographic results on DNA structure as these are presently obtained.

The effects of sequence context on DNA curvature.

Recent experiments have exposed significant discrepancies between experimental data and predictive models for DNA structure. These results strongly suggest that DNA structural parameters incorporated in the models are not always sufficient to account for the influence of sequence context and of specific ion effects. In an attempt to evaluate these two effects, we have investigated repetitive DNA sequences with the sequence motif GAGAG.CTCTC located in different helical phasing arrangements with respect to poly(A) tracts and GGGCCC.GGGCCC sequence motifs. Methods used are ligase-mediated cyclization and gel mobility experiments along with DNase I cutting and chemical probe studies. The results provide new evidence for curvature in poly(A) tracts. They also show that the sequence context in which bending and flexible sequence elements are found is an important aspect of sequence-dependent DNA conformation. Although dinucleotide models generally have good predictive power, this work demonstrates that in some instances sequence elements larger than the dinucleotide must be taken into account, and hence it provides a starting point for the appropriate modification and refinement of existing structural models for DNA.

Bending and torsional flexibility of G/C-rich sequences as determined by cyclization assays.

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 GGGCCC.GGGCCC motif in different sequence contexts including various helical phasings with (A)5-tracts. We present evidence for curvature in GGGCCC.GGGCCC and (A)5-tract motifs in the presence of physiological levels of Mg2+ 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.

Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA.

Recent structural studies of the minimal core DNA-binding domain of p53 (p53DBD) complexed to a single consensus pentamer sequence and of the isolated p53 tetramerization domain have provided valuable insights into their functions, but many questions about their interacting roles and synergism remain unanswered. To better understand these relationships, we have examined the binding of the p53DBD to two biologically important full-response elements (the WAF1 and ribosomal gene cluster sites) by using DNA circularization and analytical ultracentrifugation. We show that the p53DBD binds DNA strongly and cooperatively with p53DBD to DNA binding stoichiometries of 4:1. For the WAF1 element, the mean apparent Kd is (8.3 +/- 1.4) x 10(-8) M, and no intermediate species of lower stoichiometries can be detected. We show further that complex formation induces an axial bend of at least 60 degrees in both response elements. These results, taken collectively, demonstrate that p53DBD possesses the ability to direct the formation of a tight nucleoprotein complex having the same 4:1 DNA-binding stoichiometry as wild-type p53 which is accompanied by a substantial conformational change in the response-element DNA. This suggests that the p53DBD may play a role in the tetramerization function of p53. A possible role in this regard is proposed.

Structure of three-way DNA junctions. 2. Effects of extra bases and mismatches.

The structure of three-way DNA junctions, containing two linear double helices (arms) and a hairpin as a third arm, was studied by means of a cyclization technique. In addition to branched molecules containing perfect base-pairing in helical parts, three-way junctions with mismatches and extra non-complementary nucleotides (bulges) at junction points were studied. Molecules thus designed were ligated at identical conditions and their geometry was compared through the analysis of the efficiency of circle formation. The analysis showed that irregularities in base pairing listed above dramatically change the static and dynamic structural characteristics of the three-way junctions. All mismatches facilitate the kink between linear arms, but quantitatively, the effect depends on the position of the mismatch. The effect is maximal for GG-mismatch placed at the hairpin junction point. The results for bulges are of different kind, and they lead us to conclude that the three-way DNA junction with unpaired nucleotides adopts a T-like geometry with an angle around 90 degrees between arms containing the bulge and two other arms coaxially stacked. Broad distribution of circles indicates that this T-form geometry of bulge-containing junction is more flexible than initial pyramidal structure predominantly due to high mobility of the third arm.

Structure of three-way DNA junctions. 1. Non-planar DNA geometry.

Three-way junctions were obtained by annealing two synthetic DNA-oligomers. One of the strands contains a short palindrome sequence, leading to the formation of a hairpin with four base pairs in the stem and four bases in the loop. Another strand is complementary to the linear arms of the first hairpin-containing strand. Both strands were annealed to form a three-way branched structure with sticky ends on the linear arms. The branched molecules were ligated, and the ligation mixture was analysed on a two-dimensional gel in conditions which separated linear and circular molecules. Analysis of 2D-electrophoresis data shows that circular molecules with high mobility are formed. Formation of circular molecules is indicative of bends between linear arms. We estimate the magnitude of the angle between linear arms from the predominant size of the circular molecules formed. When the junction-to-junction distance is 20-21 bp, trimers and tetramers are formed predominately, giving an angle between linear arms as small as 60-90 degrees. Rotation of the hairpin position in the three-way junction allowed us to measure angles between other arms, yielding similar values. These results led us to conclude that the three-way DNA junction possesses a non-planar pyramidal geometry with 60-90 degrees between the arms. Computer modeling of the three-way junction with 60 degrees pyramidal geometry showed a predominantly B-form structure with local distortions at the junction points that diminish towards the ends of the helices. The size distributions of circular molecules are rather broad indicating a dynamic flexibility of three-way DNA junctions.

Structure of hydrated oligonucleotides studied by in situ scanning tunneling microscopy.

We have used the scanning tunneling microscope (STM) to image several synthetic oligonucleotides adsorbed onto a positively charged Au(111) electrode. The molecules were deposited and imaged in aqueous electrolyte under potential control, a procedure that eliminated the problem of the substrate artifacts that had limited some previous STM studies. Experiments were carried out with two types of single-stranded molecules (11 and 20 bases long) and three types of double-stranded molecules (20 and 61 base pairs and 31 bases with 25 bases paired and 6-base "sticky" ends). The molecules lie along symmetry directions on the reconstructed (23 x square root of 3) gold surface, and length measurements indicate that they adopt simple base-stacked structures. The base stacking distances are, within experimental uncertainty, equal to the 0.33 nm measured for polymeric aggregates of stacked purines by direct imaging in identical conditions. The images show features consistent with helical structures. Double helices have a major-groove periodicity that is consistent with a 36 degrees twist. The single helices appear to be more tightly twisted. A simple tunneling model of STM contrast generates good agreement between measured and calculated images.

Studies of DNA bending and flexibility using gel electrophoresis.

Gel electrophoretic methods have become established as primary tools in the study and elucidation of sequence-directed curvature both in free DNA and in the operator DNA of several site-specific nucleoprotein complexes. Results using them have been generally consistent with physical methods sensitive to DNA structure and conformation in those instances where direct comparisons can be made, and in a number of cases, gel methods have provided unique information not presently available from other techniques. Two basic strategies have been used: one based upon anomalous gel mobility effects; and a second based upon cyclization properties of curved DNA. Within each of these categories, various approaches have been used, some of which can lead, in favorable cases, to quantitative estimation of bending angles. In this review, the various gel-based methods that have been used to date are critically discussed and the qualitative and quantitative information that can be obtained from them is evaluated. A number of possible structural models for DNA curvature are described and a distinction is drawn between static or fixed bending and bending due to anisotropic flexibility at specific sequence loci. The importance and roles of gel electrophoretic methods in providing experimental approaches to this question are discussed. It is suggested that both static curvature and anisotropic flexibility in operator DNA may provide much of the basis for indirect readout of sequence information by specific site-binding regulatory proteins.

CA runs increase DNA flexibility in the complex of lambda Cro protein with the OR3 site.

The alternating pyrimidine-purine elements CA, CAC, and CACA are anisotropically flexible, as deduced from gel circularization assays on point mutations and single-base mismatches in the OR3 site of lambda phage alone and in the specific complex with the Cro protein. These sequences evidently promote DNA bending in the specific binding region of the complex and may also facilitate overwinding in the central nonbinding region. Effects for CACA are exceptionally large and suggest that an alternative DNA structure may occur in this element.