A-Tract bending: insights into experimental structures by computational models

While solution structures of adenine tract (A-tract) oligomers have indicated a unique bend direction equivalent to negative global roll (commonly termed "minor-groove bending"), crystallographic data have not unambiguously characterized the bend direction; nevertheless, many features are shared by all A-tract crystal and solution structures (e.g. propeller twisting, narrow minor grooves, and localized water spines). To examine the origin of bending and to relate findings to the crystallographic and solution data, we analyze molecular dynamics trajectories of two solvated A-tract dodecamers: 1D89, d(CGCGA(6)CG), and 1D98, d(CGCA(6)GCG), using a new general global bending framework for analyzing bent DNA and DNA/protein complexes. It is significant that the crystallographically-based initial structures are converted from dissimilar to similar bend directions equivalent to negative global roll, with the average helical-axis bend ranging from 10.5 degrees to 14.1 degrees. The largest bend occurs as positive roll of 12 degrees on the 5' side of the A-tracts (supporting a junction model) and is reinforced by gradual curvature at each A-tract base-pair (bp) step (supporting a wedge model). The precise magnitude of the bend is subtly sequence dependent (consistent with a curved general sequence model). The conversion to negative global roll only requires small local changes at each bp, accumulated over flexible moieties both outside and inside the A-tract. In contrast, the control sequence 1BNA, d(CGCGA(2)TTCGCG), bends marginally (only 6.9 degrees ) with no preferred direction. The molecular features that stabilize the bend direction in the A-tract dodecamers include propeller twisting of AT base-pairs, puckering differences between A and T deoxyriboses, a narrow minor groove, and a stable water spine (that extends slightly beyond the A-tract, with lifetimes approaching 0.2 ns). The sugar conformations, in particular, are proposed as important factors that support bent DNA. It is significant that all these curvature-stabilizing features are also observed in the crystallographic structures, but yield overall different bending paths, largely due to the effects of sequences outside the A-tract. These results merge structural details reported for A-tract structures by experiment and theory and lead to structural and dynamic insights into sequence-dependent DNA flexibility, as highlighted by the effect of an A-tract variant of a TATA-box element on bending and flexibility required for TBP binding.     

(+)-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.     

Molecular dynamics studies of sequence-directed curvature in bending locus of trypanosome kinetoplast DNA.

The macroscopic curvature induced in the double helical B-DNA by regularly repeated adenine tracts (A-tracts) plays an exceptional role in structural studies of DNA because this effect presents the most well documented example of sequence specific conformational modulations. Recently, a new hypothesis of its physical origin has been put forward. According to it, the intrinsic bends in B-DNA may represent one of the consequences of the compressed frustrated state of its backbone. The compressed backbone hypothesis agrees with many data and explains some controversial experimental observations. The original arguments of this theory came out from MD simulations of a DNA fragment with a strong bending propensity. Its sequence, however, was not experimental. It was constructed empirically so as to maximize the magnitude of bending in calculations. To make sure that our computations reproduce the experimental effect we carried out similar simulations with an A-tract repeat of a natural base pair sequence found in a bent locus of a minicircle DNA. We demonstrate spontaneous development of static curvature in the course of MD simulations excluding any initial bias except the base pair sequence. Its direction and magnitude agree with experimental estimates. The results confirm earlier qualitative conclusions and agree with the hypothesis of a compressed backbone as the origin of static bending in B-DNA.     

Molecular Dynamics Studies of Sequence-directed Curvature in Bending Locus of Trypanosome Kinetoplast DNA

Abstract

The macroscopic curvature induced in the double helical B-DNA by regularly repeated adenine tracts (A-tracts) plays an exceptional role in structural studies of DNA because this effect presents the most well documented example of sequence specific conformational modulations. Recently, a new hypothesis of its physical origin has been put forward. According to it, the intrinsic bends in B-DNA may represent one of the consequences of the compressed frustrated state of its backbone. The compressed backbone hypothesis agrees with many data and explains some controversial experimental observations. The original arguments of this theory came out from MD simulations of a DNA fragment with a strong bending propensity. Its sequence, however, was not experimental. It was constructed empirically so as to maximize the magnitude of bending in calculations. To make sure that our computations reproduce the experimental effect we carried out similar simulations with an A-tract repeat of a natural base pair sequence found in a bent locus of a minicircle DNA. We demonstrate spontaneous development of static curvature in the course of MD simulations excluding any initial bias except the base pair sequence. Its direction and magnitude agree with experimental estimates. The results confirm earlier qualitative conclusions and agree with the hypothesis of a compressed backbone as the origin of static bending in B-DNA.     

Monovalent cation binding by curved DNA molecules containing variable numbers of a-tracts

Monovalent cation binding by DNA A-tracts, runs of four or more contiguous adenine or thymine residues, has been determined for two curved ∼200 basepair (bp) restriction fragments, one taken from the M13 origin of replication and the other from the VP1 gene of SV40. These two fragments have previously been shown to contain stable, centrally located bends of 44° and 46°, respectively, located within ∼60 bp “curvature modules” containing four or five irregularly spaced A-tracts. Transient electric birefringence measurements of these two fragments, sequence variants containing reduced numbers of A-tracts in the SV40 curvature module or changes in the residues flanking the A-tracts in the M13 curvature module, have been combined with the free solution electrophoretic mobilities of the same fragments using known equations to estimate the effective charge of each fragment. The effective charge is reduced, on average, by one-third charge for each A-tract in the curvature module, suggesting that each A-tract binds a monovalent cation approximately one-third of the time. Monovalent cation binding to two or more A-tracts is required to observe significant curvature of the DNA helix axis.     

A-tract clusters may facilitate DNA packaging in bacterial nucleoid

Molecular mechanisms of bacterial chromosome packaging are still unclear, as bacteria lack nucleosomes or other apparent basic elements of DNA compaction. Among the factors facilitating DNA condensation may be a propensity of the DNA molecule for folding due to its intrinsic curvature. As suggested previously, the sequence correlations in genome reflect such a propensity [Trifonov and Sussman (1980) Proc. Natl Acad. Sci. USA, 77, 3816–3820]. To further elaborate this concept, we analyzed positioning of A-tracts (the sequence motifs introducing the most pronounced DNA curvature) in the Escherichia coli genome. First, we observed that the A-tracts are over-represented and distributed ‘quasi-regularly’ throughout the genome, including both the coding and intergenic sequences. Second, there is a 10–12 bp periodicity in the A-tract positioning indicating that the A-tracts are phased with respect to the DNA helical repeat. Third, the phased A-tracts are organized in ~100 bp long clusters. The latter feature was revealed with the help of a novel approach based on the Fourier series expansion of the A-tract distance autocorrelation function. Since the A-tracts introduce local bends of the DNA duplex and these bends accumulate when properly phased, the observed clusters would facilitate DNA looping. Also, such clusters may serve as binding sites for the nucleoid-associated proteins that have affinities for curved DNA (such as HU, H-NS, Hfq and CbpA). Therefore, we suggest that the ~100 bp long clusters of the phased A-tracts constitute the ‘structural code’ for DNA compaction by providing the long-range intrinsic curvature and increasing stability of the DNA complexes with architectural proteins.     

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.     

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.     

Mechanical properties of symmetric and asymmetric DNA A-tracts: implications for looping and nucleosome positioning

A-tracts are functionally important DNA sequences which induce helix bending and have peculiar structural properties. While A-tract structure has been qualitatively well characterized, their mechanical properties remain controversial. A-tracts appear structurally rigid and resist nucleosome formation, but seem flexible in DNA looping. In this work, we investigate mechanical properties of symmetric A_nT_n and asymmetric A_2n tracts for n = 3, 4, 5 using two types of coarse-grained models. The first model represents DNA as an ensemble of interacting rigid bases with non-local quadratic deformation energy, the second one treats DNA as an anisotropically bendable and twistable elastic rod. Parameters for both models are inferred from microsecond long, atomic-resolution molecular dynamics simulations. We find that asymmetric A-tracts are more rigid than the control G/C-rich sequence in localized distortions relevant for nucleosome formation, but are more flexible in global bending and twisting relevant for looping. The symmetric tracts, in contrast, are more rigid than asymmetric tracts and the control, both locally and globally. Our results can reconcile the contradictory stiffness data on A-tracts and suggest symmetric A-tracts to be more efficient in nucleosome exclusion than the asymmetric ones. This would open a new possibility of gene expression manipulation using A-tracts.     

Increased temperature and 2-methyl-2,4-pentanediol change the DNA structure of both curved and uncurved adenine/thymine-rich sequences

DNA curvature is affected by elevated temperature and dehydrating agents such as 2-methyl-2,4-pentanediol (MPD) (used in crystallization). This effect of MPD has been ascribed to a specific distortion of the structure of adenine tracts (A-tracts), probably through a deformation of the characteristic narrow minor groove. Uranyl photoprobing indicates that a narrowed minor groove is present in all A/T regions containing four or more A/T base pairs. Consequently, this technique may be employed to study conformational changes in other A/T-rich sequences than pure A-tracts. In this study we use uranyl photoprobing to demonstrate that the effect of elevated temperature and MPD is analogous on both "normal" and curve-inducing A/T-rich sequences. The results therefore indicate that under these conditions the minor groove is widened in all A/T sequences and not only in pure A-tracts as previously suggested. Thus, the rather subtle structural difference of AT regions and A-tracts in nonbent DNA versus A-tracts in bent DNA may be quantitative rather than qualitative; i.e., the structure is more persistent and/or rigid in bent DNA.     

Chemical reactivity of potassium permanganate and diethyl pyrocarbonate with B DNA: specific reactivity with short A-tracts

We have examined the reactivity of B DNA with two chemical probes of DNA structure, potassium permanganate (KMnO4; thymine specific) and diethyl pyrocarbonate (DEPC; purine specific, A greater than G). The DNA probed is from the beta-lactamase promoter region of the vector pBR322, and from the 3' noncoding region of a chicken embryonic myosin heavy chain gene. The chemical probes display variable reactivity with the susceptible bases in these fragments, suggesting that modification of these bases by KMnO4 and DEPC is quite sequence dependent. In contrast, these probes react with the short A-tracts present in these DNA fragments in a reproducible fashion, generating two related patterns of reactivity. In the majority of the A-tracts, all but the 3'-terminal thymine are protected from KMnO4 attack, while DEPC reacts significantly with all but the 3'-terminal adenine of the A-tracts. Some A-tracts also display a very high DEPC reactivity at the adenine adjacent to the 3'-terminal unreactive adenine. Little qualitative difference in the KMnO4 reactivity of the A-tracts was found between 12 and 43 degrees C. However, at lower temperatures the elevated KMnO4 reactivity at the 3'-terminal A-tract thymine is sometimes lost. Raising the temperature of the KMnO4 reaction can cause relatively large increases in the reactivity of some single thymines, suggesting that significant local changes in stacking occur at these thymines at elevated temperatures. The data presented suggest that many short A-tracts embedded in long fragments of DNA can assume a number of related structures in solution, each of which possess distinct junctions with the flanking DNA. This result is consistent with high-resolution structural studies on oligonucleotides containing short A-tracts. The relevance of these results to current models of A-tract structure and DNA bending is discussed. Our data also indicate that KMnO4 and DEPC are potentially useful reagents for the study of sequence-dependent variations in B DNA structure.     

Detection of an unusual distortion in A-tract DNA using KMnO4: effect of temperature and distamycin on the altered conformation.

The chemical probes potassium permanganate (KMnO4) and diethylpyrocarbonate (DEPC) can be used to study the conformational flexibility of short tracts of adenine (A-tracts) present in DNA. With these probes, we demonstrate that a novel distortion is induced in a 5 base pair A-tract at low temperature. Formation of this distorted A-tract structure, which occurs in a DNA fragment from the promoter region of the plasmid pBR322, is distinguished by a dramatic increase in the KMnO4 reactivity of the central thymines in this tract at 12 degrees C. This alteration occurs in the absence of any detectable rearrangement in the conformation of the adenines in the complementary strand. Induction of this low temperature A-tract structure is blocked by the minor groove binding drug distamycin. Hydroxyl radical footprinting of distamycin binding to the fragment containing the d(A)5 tract at 12 degrees C suggests that this drug has two different modes of binding to DNA in agreement with recent NMR data. These experiments show that short A-tracts are capable of forming more than one structural variant of B DNA in solution. The possible relationship between the intrinsic bending of DNA containing short phased A-tracts and the low temperature A-tract conformation is discussed.     

Analysis of local helix bending in crystal structures of DNA oligonucleotides and DNA-protein complexes.

Sequence-dependent bending of the helical axes in 112 oligonucleotide duplex crystal structures resident in the Nucleic Acid Database have been analyzed and compared with the use of bending dials, a computer graphics tool. Our analysis includes structures of both A and B forms of DNA and considers both uncomplexed forms of the double helix as well as those bound to drugs and proteins. The patterns in bending preferences in the crystal structures are analyzed by base pair steps, and emerging trends are noted. Analysis of the 66 B-form structures in the Nucleic Acid Database indicates that uniform trends within all pyrimidine-purine and purine-pyrimidine steps are not necessarily observed but are found particularly at CG and GC steps of dodecamers. The results support the idea that AA steps are relatively straight and that larger roll bends occur at or near the junctions of these A-tracts with their flanking sequences. The data on 16 available crystal structures of protein-DNA complexes indicate that the majority of the DNA bends induced via protein binding are sharp localized kinks. The analysis of the 30 available A-form DNA structures indicates that these structures are also bent and show a definitive preference for bending into the deep major groove over the shallow minor groove.     

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.     

Molecular dynamics simulations of DNA curvature and flexibility: helix phasing and premelting

Recent studies of DNA axis curvature and flexibility based on molecular dynamics (MD) simulations on DNA are reviewed. The MD simulations are on DNA sequences up to 25 base pairs in length, including explicit consideration of counterions and waters in the computational model. MD studies are described for ApA steps, A-tracts, for sequences of A-tracts with helix phasing. In MD modeling, ApA steps and A-tracts in aqueous solution are essentially straight, relatively rigid, and exhibit the characteristic features associated with the B'-form of DNA. The results of MD modeling of A-tract oligonucleotides are validated by close accord with corresponding crystal structure results and nuclear magnetic resonance (NMR) nuclear Overhauser effect (NOE) and residual dipolar coupling (RDC) structures of d(CGCGAATTCGCG) and d(GGCAAAAAACGG). MD simulation successfully accounts for enhanced axis curvature in a set of three sequences with phased A-tracts studied to date. The primary origin of the axis curvature in the MD model is found at those pyrimidine/purine YpR "flexible hinge points" in a high roll, open hinge conformational substate. In the MD model of axis curvature in a DNA sequence with both phased A-tracts and YpR steps, the A-tracts appear to act as positioning elements that make the helix phasing more precise, and key YpR steps in the open hinge state serve as curvature elements. Our simulations on a phased A-tract sequence as a function of temperature show that the MD simulations exhibit a premelting transition in close accord with experiment, and predict that the mechanism involves a B'-to-B transition within A-tracts coupled with the prediction of a transition in key YpR steps from the high roll, open hinge, to a low roll, closed hinge substate. Diverse experimental observations on DNA curvature phenomena are examined in light of the MD model with no serious discrepancies. The collected MD results provide independent support for the "non-A-tract model" of DNA curvature. The "junction model" is indicated to be a special case of the non-A-tract model when there is a Y base at the 5' end of an A-tract. In accord with crystallography, the "ApA wedge model" is not supported by MD.     

Preferential counterion binding to A-tract DNA oligomers

The free solution mobility of four 20 bp DNA oligomers, with and without A-tracts, has been measured by capillary electrophoresis in Tris-acetate buffer, to test the hypothesis that site-specific binding of monovalent counterions can occur in the narrow minor groove of A-tract DNAs. Preferential counterion binding has been proposed to cause A-tract bending because of asymmetric charge neutralization and collapse of the helix backbone toward the minor groove. Preferential counterion binding in A-tract DNAs should be manifested by a decrease in the electrophoretic mobility observed in free solution, compared to that of non-A-tract DNAs of the same size. Of the four sequences studied here, the slowest absolute mobility, indicative of the greatest counterion binding, was observed for a 20 bp oligomer containing two runs of A3T3 in phase with the helix repeat. A 20-mer containing phased CACA sequences migrated with the fastest mobility; 20-mers containing phased A5 tracts or phased runs of T3A3 migrated with intermediate mobilities. Very similar mobility differences were observed when 1-20 mM NaCl was added to the buffer. The results suggest that preferential counterion binding occurs in A-tract DNAs, especially those containing the AnTn sequence motif.     

A Refined Prediction Method for Gel Retardation of DNA Oligonucleotides from Dinucleotide Step Parameters: Reconciliation of DNA Bending Models with Crystal Structure Data

Abstract

The development and assessment of a prediction method for gel retardation and sequence dependent curvature of DNA based on dinucleotide step parameters are described. The method is formulated using the Babcock-Olson equations for base pair step geometry (1) and employs Monte Carlo simulated annealing for parameter optimization against experimental data. The refined base pair step parameters define a structural construct which, when the width of observed parameter distributions is taken into account, is consistent with the results of DNA oligonucleotide crystal structures. The predictive power of the method is demonstrated and tested via comparisons with DNA bending data on sets of sequences not included in the training set, including A-tracts with and without periodic helix phasing, phased A₄T₄ and T₄A₄ motifs, a sequence with a phased GGGCCC motif, some “unconventional” helix phasing sequences, and three short fragments of kinetoplast DNA from Crithidia fasiculata that exhibit significantly different behavior on non-denaturing polyacrylamide gels. The nature of the structural construct produced by the methodology is discussed with respect to static and dynamic models of structure and representations of bending and bendability. An independent theoretical account of sequence dependent chemical footprinting results is provided. Detailed analysis of sequences with A-tract induced axis bending forms the basis for a critical discussion of the applicability of wedge models, junction models and non A-tract, general sequence models for understanding the origin of DNA curvature at the molecular level.