Structural Mechanics of DNA Wrapping in the Nucleosome

Experimental X-ray crystal structures and a database of calculated structural parameters of DNA octamers were used in combination to analyse the mechanics of DNA bending in the nucleosome core complex. The 1kx5 X-ray crystal structure of the nucleosome core complex was used to determine the relationship between local structure at the base-step level and the global superhelical conformation observed for nucleosome-bound DNA. The superhelix is characterised by a large curvature (597°) in one plane and very little curvature (10°) in the orthogonal plane. Analysis of the curvature at the level of 10-step segments shows that there is a uniform curvature of 30° per helical turn throughout most of the structure but that there are two sharper kinks of 50° at ± 2 helical turns from the central dyad base pair. The curvature is due almost entirely to the base-step parameter roll. There are large periodic variations in roll, which are in phase with the helical twist and account for 500° of the total curvature. Although variations in the other base-step parameters perturb the local path of the DNA, they make minimal contributions to the total curvature. This implies that DNA bending in the nucleosome is achieved using the roll-slide-twist degree of freedom previously identified as the major degree of freedom in naked DNA oligomers. The energetics of bending into a nucleosome-bound conformation were therefore analysed using a database of structural parameters that we have previously developed for naked DNA oligomers. The minimum energy roll, the roll flexibility force constant and the maximum and minimum accessible roll values were obtained for each base step in the relevant octanucleotide context to account for the effects of conformational coupling that vary with sequence context. The distribution of base-step roll values and corresponding strain energy required to bend DNA into the nucleosome-bound conformation defined by the 1kx5 structure were obtained by applying a constant bending moment. When a single bending moment was applied to the entire sequence, the local details of the calculated structure did not match the experiment. However, when local 10-step bending moments were applied separately, the calculated structure showed excellent agreement with experiment. This implies that the protein applies variable bending forces along the DNA to maintain the superhelical path required for nucleosome wrapping. In particular, the 50° kinks are constraints imposed by the protein rather than a feature of the 1kx5 DNA sequence. The kinks coincide with a relatively flexible region of the sequence, and this is probably a prerequisite for high-affinity nucleosome binding, but the bending strain energy is significantly higher at these points than for the rest of the sequence. In the most rigid regions of the sequence, a higher strain energy is also required to achieve the standard 30° curvature per helical turn. We conclude that matching of the DNA sequence to the local roll periodicity required to achieve bending, together with the increased flexibility required at the kinks, determines the sequence selectivity of DNA wrapping in the nucleosome.     

Strong bending of the DNA double helix

During the past decade, the issue of strong bending of the double helix has attracted a lot of attention. Here, we overview the major experimental and theoretical developments in the field sorting out reliably established facts from speculations and unsubstantiated claims. Theoretical analysis shows that sharp bends or kinks have to facilitate strong bending of the double helix. It remains to be determined what is the critical curvature of DNA that prompts the appearance of the kinks. Different experimental and computational approaches to the problem are analyzed. We conclude that there is no reliable evidence that any anomalous behavior of the double helix happens when DNA fragments in the range of 100 bp are circularized without torsional stress. The anomaly starts at the fragment length of about 70 bp when sharp bends or kinks emerge in essentially every molecule. Experimental data and theoretical analysis suggest that kinks may represent openings of isolated base pairs, which had been experimentally detected in linear DNA molecules. The calculation suggests that although the probability of these openings in unstressed DNA is close to 10⁻⁵, it increases sharply in small DNA circles reaching 1 open bp per circle of 70 bp.     

Sequence-dependent DNA structure: a database of octamer structural parameters

We have constructed the potential energy surfaces for all unique tetramers, hexamers and octamers in double helical DNA, as a function of the two principal degrees of freedom, slide and shift at the central step. From these potential energy maps, we have calculated a database of structural and flexibility properties for each of these sequences. These properties include: the values of each of the six step parameters (twist roll, tilt, rise, slide and shift), for each step of the sequence; flexibility measures for both decrease and increase in each property value from the minimum energy conformation for the central step; and the deviation from the path of a hypothetical straight octamer. In an analysis of structural change as a function of sequence length, we observe that almost all DNA tends to B-DNA and becomes less flexible. A more detailed analysis of octamer properties has allowed us to determine the structural preferences of particular sequence elements. GGC and GCC sequences tend to confer bistability, low stability and a predisposition to A-form DNA, whereas AA steps strongly prefer B-DNA and inhibit A-structures. There is no correlation between flexibility and intrinsic curvature, but bent DNA is less stable than straight. The most difficult deformation is undertwisting. The TA step stands out as the most flexible sequence element with respect to decreasing twist and increasing roll. However, as with the structural properties, this behavior is highly context-dependent and some TA steps are very straight.     

Inhibition of cancer cell growth by cyclin dependent kinase 4 inhibitors synthesized based on the structure of fascaplysin

Tryptamine derivatives, a new structural class of cyclin dependent kinase 4 inhibitors, have been identified during extensive biological screening of synthetic molecules. The molecules were synthesized based on the structure of fascaplysin, which is not only a specific inhibitor of the Cdk4-cyclin D1 enzyme but also a relatively toxic molecule, probably because it binds and intercalates DNA. Interestingly, the new structural analogues of fascaplysin do not interact or intercalate with double-stranded DNA, although they inhibit Cdk4-cyclin D1 specifically. We found that compound CA199 was the most potent molecule, showing at least 25-fold specificity towards Cdk4-cyclin D1 (IC50 for Cdk4-cyclin D1 = 20 microM, Cdk2 > 500 microM). CA199 inhibits the growth of different cancer cell lines at concentrations ranging from 10-40 microM. It blocks growth of asynchronous cells at G0/G1 in a retinoblastoma protein (pRb) dependent manner. Moreover, CA199 blocks growth only at early G1 in synchronised cells released from a mimosine-induced G1/S block. These observations are reminiscent of a true Cdk4 inhibitor.     

Imaging of kinked configurations of DNA molecules undergoing orthogonal field alternating gel electrophoresis by fluorescence microscopy

The dynamics of individual DNA molecules undergoing orthogonal field alternating gel electrophoresis (OFAGE) have been studied by use of T2 DNA molecules labeled with a dye and visualized with a fluorescence microscope. The mechanism of reorientation used by a molecule to align itself in the direction of the new orthogonal field depends on the degree of extension of the chain immediately before the application of this field. The formation of kinks is promoted when time is allowed between the application of the two orthogonal fields so that the molecule attains a partially relaxed configuration. In this case, the chain appears bunched up in domains moving along the contour of the molecule. These regions are found to be the locations where the kinks are formed upon application of the second field perpendicular to the chain. The formation of kinks provides a significant retardation of the reorientation of the molecules, relative to molecules that do not form kinks, and appears to play an important role in the fractionation attained with OFAGE. A classification of various reorientation mechanisms observed in molecules that form kinks is presented.     

All 16 centromere DNAs from Saccharomyces cerevisiae show DNA curvature

All 16 centromere DNA regions of Saccharomyces cerevisiae including 90 bp framing sequences on either side were cloned. These 300 bp long centromere regions were analysed by native polyacrylamide gel electrophoresis and found to display a reduced mobility indicative of DNA curvature. The degree of curvature is centromere dependent. The experimental data were confirmed by computer analysis of the 3-dimensional structure of the CEN DNAs. Altogether these data provide further evidence for a model for budding yeast centromeres in which CEN DNA structure could be important for the assembly, activity and/or regulation of the centromere protein-DNA complex.     

Evidence for opposite groove-directed curvature of GGGCCC and AAAAA sequence elements.

The repetitive sequence (AGGGCCCTAGAGGGGCCC-TAG)n was previously shown to be curved by gel mobility assays. Here we show, using hydroxy radical/DNase I digestion and differential helical phasing experiments that the curvature is directed towards the major groove and is located in the GGGCCC, but not the CTAGAG segments. The effect of the GC step in the context of the GGGCCC motif is apparently about as large as that of AA/TT, i.e. enough to cancel the macroscopic curvature of helically phased A-tracts. These data are in agreement with positive roll-like curvature of the GCC/GGC motif, predicted from nucleosome packing data and the 3D structure of the GGGGCCCC octamer, but they are not in agreement with the dinucleotide-based roll angle values predicted for AG/CT, TA, GG/CC and GC steps. Our results thus indicate the importance of interactions beyond the dinucleotide steps in predictive models of DNA curvature.     

Characterization of inherent curvature in DNA lacking polyadenine runs.

Sequence-directed DNA curvature is most commonly associated with AA dinucleotides in the form of polyadenine runs. We demonstrate inherent curvature in DNA which lacks AA/TT dinucleotides using the criteria of polyacrylamide gel mobility and efficiency of DNA cyclization. These studies are based upon two 21-base pair synthetic DNA fragments designed to exhibit fixed curvature according to deflections made to the helical axis by non-AA dinucleotide stacks. Repeats of these sequences display anomalously slow migration in polyacrylamide gels. Moreover, both sequences describe helical conformations that are closed into circles by DNA ligase at much smaller sizes than is typical of nondeformed DNA. Chemical cleavage of these DNA molecules with hydroxyl radical is also consistent with local variation in helical conformation at specific dinucleotide steps.     

Model and computer simulations of the motion of DNA molecules during pulse field gel electrophoresis

A model is presented for the motion of individual molecules of DNA undergoing pulse field gel electrophoresis (PFGE). The molecule is represented by a chain of charged beads connected by entropic springs, and the gel is represented by a segmented tube surrounding the beads. This model differs from earlier reptation/tube models in that the tube is allowed to leak in certain places and the chain can double over and flow out of the side of the tube in kinks. It is found that these kinks often lead to the formation of U shapes, which are a major source of retardation in PFGE. The results of computer simulations using this model are compared with real DNA experimental results for the following cases: steady field motion as seen in fluorescence microscopy, mobility in steady fields, mobility in transverse field alternation gel electrophoresis (TFAGE), mobility in field inversion gel electrophoresis (FIGE), and linear dichroism (LD) of DNA in agarose gels during PFGE. Good agreement between the simulations and the experimental results is obtained.     

Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites.

The integration protein (IN) of Moloney murine leukemia virus (MuLV), purified after being produced in yeast cells, has been analyzed for its ability to bind its putative viral substrates, the att sites. An electrophoretic mobility shift assay revealed that the Moloney MuLV IN protein binds synthetic oligonucleotides containing att sequences, with specificity towards its cognate (MuLV) sequences. The terminal 13 base pairs, which are identical at both ends of viral DNA, are sufficient for binding if present at the ends of oligonucleotide duplexes in the same orientation as in linear viral DNA. However, only weak binding was observed when the same sequences were positioned within a substrate in a manner simulating att junctions in circular viral DNA with two long terminal repeats. Binding to att sites in oligonucleotides simulating linear viral DNA was dependent on the presence of the highly conserved CA residues preceding the site for 3' processing (an IN-dependent reaction that removes two nucleotides from the 3' ends of linear viral DNA); mutation of CA to TG abolished binding, and a CA to TA change reduced affinity by at least 20-fold. Removal of either the terminal two base pairs from both ends of the oligonucleotide duplex or the terminal two nucleotides from the 3' ends of each strand did not affect binding. The removal of three 3' terminal nucleotides, however, abolished binding, suggesting an essential role for the A residue immediately upstream of the 3' processing site in the binding reaction. These results help define the sequence requirements for att site recognition by IN, explain the conservation of the subterminal CA dinucleotide, and provide a simple assay for sequence-specific IN activity.     

Structural rearrangements and insertions of dispersed elements in pericentromeric alpha satellites occur preferably at kinkable DNA sites

Centromeric region of human chromosome 21 comprises two long alphoid DNA arrays: the well homogenized and CENP-B box-rich alpha21-I and the alpha21-II, containing a set of less homogenized and CENP-B box-poor subfamilies located closer to the short arm of the chromosome. Continuous alphoid fragment of 100 monomers bordering the non-satellite sequences in human chromosome 21 was mapped to the pericentromeric short arm region by fluorescence in situ hybridization (alpha21-II locus). The alphoid sequence contained several rearrangements including five large deletions within monomers and insertions of three truncated L1 elements. No binding sites for centromeric protein CENP-B were found. We analyzed sequences with alphoid/non-alphoid junctions selectively screened from current databases and revealed various rearrangements disrupting the regular tandem alphoid structure, namely, deletions, duplications, inversions, expansions of short oligonucleotide motifs and insertions of different dispersed elements. The detailed analysis of more than 1100 alphoid monomers from junction regions showed that the vast majority of structural alterations and joinings with non-alphoid DNAs occur in alpha satellite families lacking CENP-B boxes. Most analyzed events were found in sequences located toward the edges of the centromeric alphoid arrays. Different dispersed elements were inserted into alphoid DNA at kinkable dinucleotides (TG, CA or TA) situated between pyrimidine/purine tracks. DNA rearrangements resulting from different processes such as recombination and replication occur at kinkable DNA sites alike insertions but irrespectively of the occurrence of pyrimidine/purine tracks. It seems that kinkable dinucleotides TG, CA and TA are part of recognition signals for many proteins involved in recombination, replication, and insertional events. Alphoid DNA is a good model for studying these processes.     

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.     

A refined prediction method for gel retardation of DNA oligonucleotides from dinucleotide step parameters: reconciliation of DNA bending models with crystal structure data

The development and assessment of a prediction method for gel retardation and sequence dependent curvature of DNA based on dinulcleotide 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 stuctural 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.     

Long-range organization and sequence-directed curvature of Xenopus laevis satellite 1 DNA.

We have investigated the long-range organization and the intrinsic curvature of satellite 1 DNA, an unusual tandemly-repeated DNA family of Xenopus laevis presenting sequence homologies to SINEs. PFGE was used in combination with frequent-cutter restriction enzymes not likely to cut within satellite 1 DNA and revealed that almost all the repeating units are tandemly organized to form large arrays (200 kb to 2 Mb) that are marked by restriction length polymorphism and contain intra-array domains of sequence variation. Besides that, we have analysed the secondary structure of satellite 1 DNA by computer modelling. Theoretical maps of curvature obtained from three independent models of DNA bending (the dinucleotide wedge model of Trifonov, the junction model of Crothers and the model of de Santis) showed that satellite 1 DNA is intrinsically curved and these results were confirmed experimentally by polyacrylamide gel electrophoresis. Moreover, we observed that this bending element is highly conserved among all the members of the satellite 1 DNA family that are accessible to analysis. A potential genetic role for satellite 1 DNA based on this unusual structural feature is discussed.     

Atomistic simulations reveal bubbles, kinks and wrinkles in supercoiled DNA

Although DNA is frequently bent and supercoiled in the cell, much of the available information on DNA structure at the atomistic level is restricted to short linear sequences. We report atomistic molecular dynamics (MD) simulations of a series of DNA minicircles containing between 65 and 110 bp which we compare with a recent biochemical study of structural distortions in these tight DNA loops. We have observed a wealth of non-canonical DNA structures such as kinks, denaturation bubbles and wrinkled conformations that form in response to bending and torsional stress. The simulations show that bending alone is sufficient to induce the formation of kinks in circles containing only 65 bp, but we did not observe any defects in simulations of larger torsionally relaxed circles containing 110 bp over the same MD timescales. We also observed that under-winding in minicircles ranging in size from 65 to 110 bp leads to the formation of single stranded bubbles and wrinkles. These calculations are used to assess the ability of atomistic MD simulations to determine the structure of bent and supercoiled DNA.     

Detection of satellite DNA in Palorus ratzeburgii: analysis of curvature profiles and comparison with Tenebrio molitor satellite DNA.

Very abundant and homogenous satellite DNA has been found in the flour beetle Palorus ratzeburgii, representing 40% of its genome. Sequencing of 14 randomly cloned satellite monomers revealed a conserved monomer length of 142 bp and an average A+T content of 68%. Sequence variation analysis showed that base substitutions, appearing with a frequency of 2.3%, are predominant differences among satellite monomers. The satellite sequence is unique without significant direct repeats and with only two potentially stable inverted repeats. After electrophoresis of satellite monomers on native polyacrylamide gel retarded mobilities characteristic for curved DNA molecules are observed. The curvature profiles and DNA helix axis trajectory are calculated on the basis of three different algorithms. These calculations predict that P ratzeburgii satellite DNA forms a left-handed solenoid superstructure. Comparison of described features with other satellite DNAs reveals some striking similarities with satellite DNA from related species Tenebrio molitor, which belongs to the same family of Tenebrionidae. Both satellites are very abundant and homogenous with the same, highly conserved monomer length, although there is no homology at the nucleotide level. Their monomers, as well as multimers, exhibit very similar retarded electrophoretic mobilities. The calculated curvature profiles predict two bend centers in monomers of each satellite, resulting in a model of left-handed solenoid superstructures of similar appearance.     

Effects of diaminopurine and inosine substitutions on A-tract induced DNA curvature. Importance of the 3'-A-tract junction.

Gel migration and uranyl photoprobing have been used to study the effects of inosine and 2,6-diaminopurine (2,6-DAP) substitution on adenine-tract (A-tract) induced DNA curvature. Using a 10mer repeated sequence including five inosines we show by uranyl photoprobing that a narrow minor groove varying in phase with the helix repeat is not the cause of DNA curvature. Further, we have systematically studied by gel migration the effects on A-tract induced curvature of either single or full substitution with inosine and/or 2,6-DAP in a 5'-AAAAAGCCGC-3'sequence. DNA curvature is shown to increase when inosines are substituted for the guanosines in the sequence between the A-tracts. By comparing the effects of each monosubstitution it can be seen that when the G closest to the 3'-end of the A-tract is substituted the effect on DNA curvature is much stronger than when substitution is made at any other position. By contrast, curvature is abolished when 2,6-DAP residues are substituted for all adenines, and monosubstitution reveals that the effect of substituting a single adenine is strongest at the 3'-end of the A-tract. These results favor a model in which the curvature induced by an A-tract in DNA molecules is primarily located at the junction with the 3'-end of the A-tract, and this peculiar junction is created because the A-tract has a preference to form a non-B-DNA structure which builds up from the 5'-end.     

DNA bending: the prevalence of kinkiness and the virtues of normality.

DNA bending in 86 complexes with sequence-specific proteins has been examined using normal vector plots, matrices of normal vector angles between all base pairs in the helix, and one-digit roll/slide/twist tables. FREEHELIX, a new program especially designed to analyze severely bent and kinked duplexes, generates the foregoing quantities plus local roll, tilt, twist, slide, shift and rise parameters that are completely free of any assumptions about an overall helix axis. In nearly every case, bending results from positive roll at pyrimidine-purine base pair steps: C-A (= T-G), T-A, or less frequently C-G, in a direction that compresses the major groove. Normal vector plots reveal three well-defined types of bending among the 86 examples: (i) localized kinks produced by positive roll at one or two discrete base pairs steps, (ii) three-dimensional writhe resulting from positive roll at a series of adjacent base pairs steps, or (iii) continuous curvature produced by alternations of positive and negative roll every 5 bp, with side-to-side zig-zag roll at intermediate position. In no case is tilt a significant component of the bending process. In sequences with two localized kinks, such as CAP and IHF, the dihedral angle formed by the three helix segments is a linear function of the number of base pair steps between kinks: dihedral angle = 36 degrees x kink separation. Twenty-eight of the 86 examples can be described as major bends, and significant elements in the recognition of a given base sequence by protein. But even the minor bends play a role in fine-tuning protein/DNA interactions. Sequence-dependent helix deformability is an important component of protein/DNA recognition, alongside the more generally recognized patterns of hydrogen bonding. The combination of FREEHELIX, normal vector plots, full vector angle matrices, and one-digit roll/slide/twist tables affords a rapid and convenient method for assessing bending in DNA.     

Detection of satellite DNA in Palorus ratzeburgii: Analysis of curvature profiles and comparison with Tenebrio molitor satellite DNA

Very abundant and homogenous satellite DNA has been found in the flour beetle Palorus ratzeburgii, representing 40% of its genome. Sequencing of 14 randomly cloned satelite monomers revealed a conserved monomer length of 142 bp and an average A+T content of 68%. Sequence variation analysis showed that base substitutions, appearing with a frequency of 2.3%, are predominant differences among satellite monomers. The satellite sequence is unique without significant direct repeats and with only two potentially stable inverted repeats. After electrophoresis of satellite monomers on native polyacrylamide gel retarded mobilities characteristic for curved DNA molecules are observed. The curvature profiles and DNA helix axis trajectory are calculated on the basis of three different algorithms. These calculations predict that P ratzeburgii satellite DNA forms a left-handed solenoid superstructure. Comparison of described features with other satellite DNAs reveals some striking similarities with satellite DNA from related species Tenebrio molitor, which belongs to the same family of Tenebrionidae. Both satellites are very abundant and homogenous with the same, highly conserved monomer length, although there is no homology at the nucleotide level. Their monomers, as well as multimers, exhibit very similar retarded electrophoretic mobilities. The calculated curvature profiles predict two bend centers in monomers of each satellite, resulting in a model of left-handed solenoid superstructures of similar appearance.