Solution structure of an A-tract DNA bend

The solution structure of a DNA dodecamer d(GGCAAAAAACGG)/d(CCGTTTTTTGCC) containing an A-tract has been determined by NMR spectroscopy with residual dipolar couplings. The structure shows an overall helix axis bend of 19 degrees in a geometry consistent with solution and gel electrophoresis experiments. Fourteen degrees of the bending occurs in the GC regions flanking the A-tract. The remaining 5 degrees is spread evenly over its six AT base-pairs. The A-tract is characterized by decreasing minor groove width from the 5' to the 3' direction along the A strand. This is a result of propeller twist in the AT pairs and the increasing negative inclination of the adenine bases at the 3' side of the run of adenine bases. The four central thymine bases all have negative inclination throughout the A-tract with an average value of -6.1 degrees. Although this negative inclination makes the geometry of the A-tract different from all X-ray structures, the proton on N6 of adenine and the O4 of thymine one step down the helix are within distance to form bifurcated hydrogen bonds. The 5' bend of 4 degrees occurs at the junction between the GC flank and the A-tract through a combination of tilt and roll. The larger 3' bend, 10 degrees, occurs in two base steps: the first composed of tilt, -4.1 degrees, and the second a combination of tilt, -4.2 degrees, and roll, 6.0 degrees. This second step is a direct consequence of the change in inclination between an adjacent cytosine base, which has an inclination of -12 degrees, and the next base, a guanine, which has 3 degrees inclination. This bend is a combination of tilt and roll. The large change in inclination allows the formation of a hydrogen bond between the protons of N4 of the 3' cytosine and the O6 of the next 3' base, a guanine, stabilizing the roll component in the bend. These structural features differ from existing models for A-tract bends.For comparison, we also determined the structure of the control sequence, d(GGCAAGAAACGG)/d(CCGTTTCTTGCC), with an AT to GC transition in the center of the A-tract. This structure has no negative inclination in most of the bases within the A-tract, resulting in a bend of only 9 degrees. When ligated in phase, the control sequence has nearly normal mobility in gel electrophoresis experiments.     

Intrinsic bending and deformability at the T-A step of CCTTTAAAGG: a comparative analysis of T-A and A-T steps within A-tracts

Introduction of a T-A or pyrimidine-purine step into a straight and rigid A-tract can cause a positive roll deformation that kinks the DNA helix at that step. In CCTTTAAAGG, the central T-A step has an 8.6 degrees bend toward the major groove. We report the structural analysis of CCTTTAAAGG and a comparison with 25 other representative crystal structures from the NDB containing at least four consecutive A or T bases. On average, more local bending occurs at the disruptive T-A step (8.21 degrees ) than at an A-T step (5.71 degrees ). In addition, A-tracts containing an A-T step are more bent than are pure A-tracts, and hence A-A and A-T steps are not equivalent. All T-A steps examined exhibit positive roll, bending towards the major groove, while A-T steps display negative roll and bend slightly towards the minor groove. This illustrates how inherent negative and positive roll are, respectively, at A-T and T-A steps within A-tracts. T-A steps are more deformable, showing larger and more variable deformations of minor groove width, rise, cup, twist, and buckle. Standard deviations of twist, rise, and cup for T-A steps are 6.66 degrees, 0.55 A, and 15.90 degrees, versus 2.28 degrees, 0.21 A, and 2.99 degrees for A-T steps. Packing constraints determine which local values of these helical parameters an individual T-A step will adopt. For instance, with CCTTTAAAGG and three isomorphous structures, CGATTAATCG, CGATATATCG, and CGATCGATCG, crystal packing forces lead to a series of correlated changes: widened minor groove, large slide, low twist, and large rise. The difference in helical parameters between A-T steps lying within A-tracts, versus A-T steps within alternating AT sequences, demonstrates the importance of neighboring steps on the conformation of a given dinucleotide step.     

MPD and DNA Bending in Crystals and in Solution

Bending of 15 to 24° is observed within crystal structures ofB-DNA duplexes, is strongly sequence-dependent, and exhibits no correlation with the concentration of MPD (2-methyl-2,4-pentanediol) in the crystallizing solution. Two types of bends are observed: facultative bends or flexible hinges at junctions between regions of G·C and A·T base-pairs, and a persistent and almost obligatory bend at the center of the sequence R-G-C-Y. Only A-tracts are characteristically straight and unbent in every crystal structure examined to date. A detailed examination of normal vector plots for individual strands of a double helix provides an explanation, in terms of the stacking properties of guanine and adenine bases. The effect of high MPD concentrations, in both solution and crystal, is to decrease local bending somewhat without removing it altogether. MPD gel retardation experiments provide no basis for choosing among the three models that seek to explain macroscopic curvature of DNA by means of microscopic bending: junction bending, bent A-tracts, or bent general- sequence DNA. Crystallographic data on the straightness of A-tracts, the bendability of non-A sequences, and the identity of inclination angles in A-tract and non-A-tractB-DNA support only the general-sequence bending model. The pre-melting transition observed in A-tract DNA probably represents a relaxation of stiff adenine stacks to a flexible conformation more typical of general-sequence DNA.     

Bending and curvature calculations in B-DNA.

A simple program, BEND, has been written to calculate the magnitude of local bending and macroscopic curvature at each point along an arbitrary B-DNA sequence, using any desired bending model that specifies values of twist, roll and tilt as a function of sequence. The program has been used to evaluate six different DNA bending models in three categories. Two are bent non-A-tract models: (a) A new model based on the nucleosome positioning data of Satchwell et al 1986 (J. Mol. Biol. 191, 659-675), (b) The model of Calladine et al 1988 (J. Mol. Biol. 201, 127-137). Three are bent A-tract models: (c) The wedge model of Bolshoy et al 1991 (Proc. Natl. Acad. Sci. USA 88, 2312-2316), (d) The model of Cacchione et al 1989 (Biochem. 28, 8706-8713), (e) A reversed version of model (b). The last is a junction model: (f) The model of Koo & Crothers 1988 (Proc. Natl. Acad. Sci. USA 85, 1763-1767). Although they have widely different assumptions and values for twist, roll and tilt, all six models correctly predict experimental A-tract curvature as measured by gel retardation and cyclization kinetics, but only the new nucleosome positioning model is successful in predicting curvature in regions containing phased GGGCCC sequences. This model--showing local bending at mixed sequence DNA, strong bends at the sequence GGC, and straight, rigid A-tracts--is the only model consistent with both solution data from gel retardation and cyclization kinetics and structural data from x-ray crystallography.     

HU binding to bent DNA: a fluorescence resonance energy transfer and anisotropy study

HU, an architectural DNA-binding protein, either stabilizes DNA in a bent conformation or induces a bend upon binding to give other proteins access to the DNA. In this study, HU binding affinity for a bent DNA sequence relative to a linear sequence was investigated using fluorescence anisotropy measurements. A static bend was achieved by the introduction of two phased A4T4 tracts in a 20 bp duplex. Binding affinity for 20 bp duplexes containing two phased A-tracts in either a 5'-3' or 3'-5' orientation was found to be almost 10-fold higher than HU binding to a random sequence 20 bp duplex (6.1 vs 0.68 microM(-1)). The fluorescence technique of resonance energy transfer was used to quantitatively determine the static bend of the DNA duplexes and the HU-induced bend. DNA molecules were 5'-end labeled with fluorescein as the donor or rhodamine as the acceptor. From the efficiency of energy transfer, the end-to-end distance of the DNA duplexes was calculated. The end-to-end distance relative to DNA contour length (R/R(C)) yields a bend angle for the A-tract duplex of 45 +/- 7 degrees in the absence of HU and 70 +/- 3 degrees in the presence of HU. The bend angle calculated for the T4A4 tract duplex was 62 +/- 4 degrees after binding two HU dimers. Fluorescence anisotropy measurements reveal that HU binds in a 1:1 stoichiometry to the A4T4 tract duplex but a 2:1 stoichiometry to the T4A4 tract and random sequence duplex. These findings suggest that HU binding and recognition of DNA may be governed by a structural mechanism.     

Magnitude and direction of DNA bending induced by screw-axis orientation: influence of sequence, mismatches and abasic sites

DNA-bending flexibility is central for its many biological functions. A new bending restraining method for use in molecular mechanics calculations and molecular dynamics simulations was developed. It is based on an average screw rotation axis definition for DNA segments and allows inducing continuous and smooth bending deformations of a DNA oligonucleotide. In addition to controlling the magnitude of induced bending it is also possible to control the bending direction so that the calculation of a complete (2-dimensional) directional DNA-bending map is now possible. The method was applied to several DNA oligonucleotides including A(adenine)-tract containing sequences known to form stable bent structures and to DNA containing mismatches or an abasic site. In case of G:A and C:C mismatches a greater variety of conformations bent in various directions compared to regular B-DNA was found. For comparison, a molecular dynamics implementation of the approach was also applied to calculate the free energy change associated with bending of A-tract containing DNA, including deformations significantly beyond the optimal curvature. Good agreement with available experimental data was obtained offering an atomic level explanation for stable bending of A-tract containing DNA molecules. The DNA-bending persistence length estimated from the explicit solvent simulations is also in good agreement with experiment whereas the adiabatic mapping calculations with a GB solvent model predict a bending rigidity roughly two times larger.     

(+)-CC-1065 as a probe for intrinsic and protein-induced bending of DNA

(+)-CC -1065 is biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. This antibiotic covalently modifies DNA by alkylation of N-3 of a adenine in the minor groove. As a Structural consequence of covalent modification of DNA, the helix axis id bent into the minor groove. The drug-induced bending of DNA has similarities to intrinsic. A-tract bending and the 3′ adenine of A-tracts shows a unique reactivity to alkylation by (+) -CC-1065. Upon covalent modification of A-tracts, the magnitude of bending is increased and helix is stiffened. Using high-field NMR, hydroxyl-radical footprinting and gel electrophoresis, the molecular basis for the high reactivity of the bonding sequence 5′ - AGTTA* (an asterisk indicates the covalent modification site) to (+)-CC-1065 has been shown to involve the inherent conformational flexibility of this sequence. Furthermore, these studies also demonstrate that after alkylation the drug-induced bending is focused over the TT region. By analogy with the junction bend model for A-tracts, a ‘truncated junction bend model’ is proposed for this structure. Last, the application of (+)-CC-1065 entrapped/induced bending of DNA as a probe for the Sp1-induced bending of the 21-base-pair repeat an Mu transpose bending of the att L3 sequence is described.     

kDNA minicircles of the major sequence class of C. fasciculata contain a single region of bent helix widely separated from the two origins of replication

The major sequence class of Crithidia fasciculata minicircles is shown to have a single region of bent helical DNA widely separated from the two replication origins located 180 degrees apart on the minicircle map. The position of the bend in the DNA has been mapped both by gel electrophoretic methods and by direct electron microscopic observation of the DNA. This sequence directed bending is apparently the result of homopolymeric dA X dT tracts 4-6 base pairs long repeated in phase with the helix screw. The region of the bend contains nineteen such homopolymeric tracts in a region of about 200 base pairs with sixteen of the tracts oriented in the same direction.     

Sequence dependence of DNA conformational flexibility

By using conformational free energy calculations, we have studied the sequence dependence of flexibility and its anisotropy along various conformational variables of DNA base pairs. The results show the AT base step to be very flexible along the twist coordinate. On the other hand, homonucleotide steps, GG(CC) and AA(TT), are among the most rigid sequences. For the roll motion that would correspond to a bend, the TA step is most flexible, while the GG(CC) step is least flexible. The flexibility of roll is quite anisotropic; the ratio of fluctuations toward the major and minor grooves is the largest for the GC step and the smallest for the AA(TT) and CG steps. Propeller twisting of base pairs is quite flexible, especially of A.T base pairs; propeller twist can reach 19 degrees by thermal fluctuation. We discuss the effect of electrostatic parameters, comparison with available experimental results, and biological relevance of these results.     

Topological measurement of an A-tract bend angle: effect of magnesium

Sequences of four to six adenine residues, termed A-tracts, have been shown to produce curvature in the DNA double helix. A-tracts have been used extensively as reference standards to quantify bending induced by other sequences as well as by DNA binding proteins when they bind to their sites. However, the ability of an A-tract to serve as such a standard is hampered by the wide variation of values reported for the amount of bend conferred by an A-tract. One experimental condition that differs in these studies is the presence of divalent cation. To evaluate this effect, a new application of a topological method, termed rotational variant analysis, is used here to measure for the first time the effect of the presence of magnesium ion on the bend angle conferred by an A-tract. This method, which has the unique ability to measure a bend angle in the presence or absence of magnesium ion, demonstrates that magnesium ion markedly increases the bend angle. For example, when measured in a commonly used gel electrophoretic buffer, the bend angle conferred by a tract of six adenine residues increases from about 7 degrees in the absence of magnesium ion to 19 degrees in the presence of 3.9 mM magnesium ion. This quantitative demonstration of substantial magnesium ion dependence has several important implications. First, it explains discrepancies among bend values reported in various previous studies, particularly those employing gel electrophoretic versus other solution methods. In addition, these findings necessitate substantial revisions of the conclusions in a large number of studies that have used A-tract DNA as the bend angle reference standard in comparison measurements. Finally, any such future studies employing this comparison methodology will need to use the same sequence analyzed in the original measurements as well as replicate the original measurement conditions (e.g. ionic composition and temperature).     

A structural analysis of the bent kinetoplast DNA from Crithidia fasciculata by high resolution chemical probing.

The chemical probes potassium permanganate (KMnO4) and diethylpyrocarbonate (DEPC) have been used to study the conformation of bent kinetoplast DNA from Crithidia fasciculata at different temperatures. Chemical reactivity data shows that the numerous short A-tracts of this bent DNA adopt a similar structure at 43 degrees C. This conformation appears to be very similar to the conformation of A-tracts in DNA exhibiting normal gel mobility. The A-tract structure detected by chemical probing is characterized by a high degree of base stacking on the thymine strand, and by an abrupt conformational change at the 3' end of the adenine strand. In general, no major alteration of this A-tract specific structure was detected between 4-53 degrees C. However, probing with KMnO4 revealed two unusual features of the C. fasciculata sequence that may contribute to the highly aberrant gel mobility of this DNA: 1) the B DNA/A-tract junction 5' dC/A3-6 3'. 5' dT3-6/G 3' is disproportionately represented and is conformationally distinct from other 5' end junctions, and 2) low temperature favors a novel strand-specific conformational distortion over a 20 base pair region of the bent kinetoplast DNA. Presence of the minor groove binding drug distamycin had little detectable effect on the A-tract conformation. However, distamycin did inhibit formation of the novel KMnO4 sensitive low temperature structure and partially eliminated the anomalous gel mobility of the kinetoplast DNA. Finally, we describe a simple and reproducible procedure for the production of an adenine-specific chemical DNA sequence ladder.     

Bending and flexibility of methylated and unmethylated EcoRI DNA

We used cyclization kinetics experiments and Monte Carlo simulations to determine a structural model for a DNA decamer containing the EcoRI restriction site. Our findings agree well with recent crystal and NMR structures of the EcoRI dodecamer, where an overall bend of seven degrees is distributed symmetrically over the molecule. Monte Carlo simulations indicate that the sequence has a higher flexibility, assumed to be isotropic, compared to that of a "generic" DNA sequence. This model was used as a starting point for the investigation of the effect of cytosine methylation on DNA bending and flexibility. While methylation did not affect bend magnitude or direction, it resulted in a reduction in bending flexibility and under-winding of the methylated nucleotides. We demonstrate that our approach can augment the understanding of DNA structure and dynamics by adding information about the global structure and flexibility of the sequence. We also show that cyclization kinetics can be used to study the properties of modified nucleotides.     

Structural basis for SRY-dependent 46-X,Y sex reversal: modulation of DNA bending by a naturally occurring point mutation

The HMG-box domain of the human male sex-determining factor SRY, hSRY(HMG) (comprising residues 57-140 of the full-length sequence), binds DNA sequence-specifically in the minor groove, resulting in substantial DNA bending. The majority of point mutations resulting in 46X,Y sex reversal are located within this domain. One clinical de novo mutation, M64I in the full-length hSRY sequence, which corresponds to M9I in the present hSRY(HMG) construct, acts principally by reducing the extent of DNA bending. To elucidate the structural consequences of the M9I mutation, we have solved the 3D solution structures of wild-type and M9I hSRY(HMG) complexed to a DNA 14mer by NMR, including the use of residual dipolar couplings to derive long-range orientational information. We show that the average bend angle (derived from an ensemble of 400 simulated annealing structures for each complex) is reduced by approximately 13 degrees from 54(+/-2) degrees in the wild-type complex to 41(+/-2) degrees in the M9I complex. The difference in DNA bending can be localized directly to changes in roll and tilt angles in the ApA base-pair step involved in interactions with residue 9 and partial intercalation of Ile13. The larger bend angle in the wild-type complex arises as a direct consequence of steric repulsion of the sugar of the second adenine by the bulky S(delta) atom of Met9, whose position is fixed by a hydrogen bond with the guanidino group of Arg17. In the M9I mutant, this hydrogen bond can no longer occur, and the less bulky C(gamma)m methyl group of Ile9 braces the sugar moieties of the two adenine residues, thereby decreasing the roll and tilt angles at the ApA step by approximately 8 degrees and approximately 5 degrees, respectively, and resulting in an overall difference in bend angle of approximately 13 degrees between the two complexes. To our knowledge, this is one of the first examples where the effects of a clinical mutation involving a protein-DNA complex have been visualized at the atomic level.     

Uranyl photoprobing of nonbent A/T- and bent A-tracts. A difference of flexibility?

In this study, we have systematically compared the uranyl photocleavage of a range of bent A-tracts and nonbent TA-tracts as well as interrupted A-tracts. We demonstrate that uranyl photocleavage of A-tracts and TA-tracts is almost identical, indicating a very similar minor groove conformation. Furthermore, a 10 base pair A-tract is divided into two independent tracts by an intervening TA or GC step. Uranyl probing also clearly distinguishes the bent A4T4 and the nonbent T4A4 sequences as adopting different structures, and our interpretation of the data is consistent with a structure for the bent A4T4 sequence that resembles a continuous A-tract, whereas the nonbent T4A4 sequences are closer to two independent and opposite A-tracts that cancel each other in terms of macroscopic bending. Finally, we also note that even single TA and TAT steps are highly sensitive to uranyl photocleavage and propose that in addition to average minor groove width, uranyl also senses DNA helix flexibility/deformability. Thus, the structural difference of TA-tracts and A-tracts may to a large extent reflect a difference in flexibility, and DNA curvature may consequently require a rigid narrow minor groove conformation that creates distinct A-tract-B-DNA junctions as the predominant cause of the bending.     

Selective DNA bending by a variety of bZIP proteins.

We have investigated DNA bending by bZIP family proteins that can bind to the AP-1 site. DNA bending is widespread, although not universal, among members of this family. Different bZIP protein dimers induced distinct DNA bends. The DNA bend angles ranged from virtually 0 to greater than 40 degrees as measured by phasing analysis and were oriented toward both the major and the minor grooves at the center of the AP-1 site. The DNA bends induced by the various heterodimeric complexes suggested that each component of the complex induced an independent DNA bend as previously shown for Fos and Jun. The Fos-related proteins Fra1 and Fra2 bent DNA in the same orientation as Fos but induced smaller DNA bend angles. ATF2 also bent DNA toward the minor groove in heterodimers formed with Fos, Fra2, and Jun. CREB and ATF1, which favor binding to the CRE site, did not induce significant DNA bending. Zta, which is a divergent member of the bZIP family, bent DNA toward the major groove. A variety of DNA structures can therefore be induced at the AP-1 site through combinatorial interactions between different bZIP family proteins. This diversity of DNA structures may contribute to regulatory specificity among the plethora of proteins that can bind to the AP-1 site.