Analysis and Development of the Computer Methods of Nucleosome Localization on DNA Fragments with Different AT-content

Abstract

Trinucleotide parameter sets published previously were used for the development of the predictive method for the determining the nucleosome positions along the DNA. The choice of the type of parameter sets used depends upon AT-content of the fragment. Some limitations are imposed on these predictions due to the presence of A_n, T_n tracts (in our case n>5 or =5) within the 145 bp fragment leading to the displacement or even the prohibition for the corresponding site to be occupied by nucleosomes. The predicted nucleosome positioning site with the large potential may influence on the choice of the proximal nucleosome positions with the weaker bending potentials as is revealed by the comparison with the micrococcal nuclease digestion map. Trinucleotide methods may be considered as advantageous in the comparison with the dinucleotide ones.     

A measure of bending in nucleic acids structures applied to A-tract DNA

A method is proposed to measure global bending in DNA and RNA structures. It relies on a properly defined averaging of base-fixed coordinate frames, computes mean frames of suitably chosen groups of bases and uses these mean frames to evaluate bending. The method is applied to DNA A-tracts, known to induce considerable bend to the double helix. We performed atomistic molecular dynamics simulations of sequences containing the A₄T₄ and T₄A₄ tracts, in a single copy and in two copies phased with the helical repeat. Various temperature and salt conditions were investigated. Our simulations indicate bending by roughly 10° per A₄T₄ tract into the minor groove, and an essentially straight structure containing T₄A₄, in agreement with electrophoretic mobility data. In contrast, we show that the published NMR structures of analogous sequences containing A₄T₄ and T₄A₄ tracts are significantly bent into the minor groove for both sequences, although bending is less pronounced for the T₄A₄ containing sequence. The bending magnitudes obtained by frame averaging are confirmed by the analysis of superhelices composed of repeated tract monomers.     

Structural Basis of Stable Bending in DNA Containing An Tracts. Different Types of Bending

Abstract

Structural determinants of DNA bending of different types have been studied by theoretical conformational analysis of duplexes. Their terminal parts were fixed either in an ordinary low-energy B-like conformation or in “anomalous” conformations with a narrowed minor groove typical of A_n tracts. The anomalous conformations had different negative tilt angles (up to about zero), different propeller twists and minor groove widths. Calculations have been performed for DNA fragments A_nT_m, T_nA_m, A_nGCT_m, A_nCGT_m, T_mGCA_n, T_mCGA_n which are the models of the junction of two anomalous structures on A_n and T_m tracts. On the AT step of the A_nT_m fragment the minor groove can be easily narrowed so that a whole unbent fragment of anomalous structure is formed on A_n T_m. According to our energy estimates, there should not be any reliable bending on A_nT_m. In contrast, in all other cases there was a pronounced roll-like bending into the major groove in the chemical symmetry region. Calculations of the junction between the anomalous and ordinary B-like structure for G_nT_m and C_nA_m have shown that there is an equilibrium bending with a tilt component towards the chain having the anomalous structure at the 5′-end. From our calculations it is impossible to determine precisely the direction of bending, though it can be suggested that the roll component of bending might be directed towards the major groove. The anomalous structure is the main reason of bending; alternations of pyrimidines and purines can modulate the value and the direction of equilibrium bending (only the value in the case of self-complemantary fragments).

The results are consistent with the experimental data and promote a better understanding of the problem of DNA bending.     

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.     

Unmethylated and methylated CpG dinucleotides distinctively regulate the physical properties of DNA

In eukaryotic cells, DNA has to bend significantly to pack inside the nucleus. Physical properties of DNA such as bending flexibility and curvature are expected to affect DNA packaging and partially determine the nucleosome positioning patterns inside a cell. DNA CpG methylation, the most common epigenetic modification found in DNA, is known to affect the physical properties of DNA. However, its detailed role in nucleosome formation is less well‐established. In this study, we evaluated the effect of defined CpG patterns (unmethylated and methylated) on DNA structure and their respective nucleosome‐forming ability. Our results suggest that the addition of CpG dinucleotides, either as a (CG)_n stretch or (CGX₈)_n repeats at 10 bp intervals, lead to reduced hydrodynamic radius and decreased nucleosome‐forming ability of DNA. This effect is more predominant for a DNA stretch ((CG)₅) located in the middle of a DNA fragment. Methylation of CpG sites, surprisingly, seems to reduce the difference in DNA structure and nucleosome‐forming ability among DNA constructs with different CpG patterns. Our results suggest that unmethylated and methylated CpG patterns can play very different roles in regulating the physical properties of DNA. CpG methylation seems to reduce the DNA conformational variations affiliated with defined CpG patterns. Our results can have significant bearings in understanding the nucleosome positioning pattern in living organisms modulated by DNA sequences and epigenetic features. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 517–524, 2014.     

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.     

Poly-dA:dT Tracts Form an In Vivo Nucleosomal Turnstile

Nucleosomes regulate many DNA-dependent processes by controlling the accessibility of DNA, and DNA sequences such as the poly-dA:dT element are known to affect nucleosome binding. We demonstrate that poly-dA:dT tracts form an asymmetric barrier to nucleosome movement in vivo, mediated by ATP-dependent chromatin remodelers. We theorize that nucleosome transit over poly-A elements is more energetically favourable in one direction, leading to an asymmetric arrangement of nucleosomes around these sequences. We demonstrate that different arrangements of poly-A and poly-T tracts result in very different outcomes for nucleosome occupancy in yeast, mouse, and human, and show that yeast takes advantage of this phenomenon in its promoter architecture.     

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.     

A-tract polarity dominate the curvature in flanking sequences

It is well-known, but little understood, that the nucleotide sequences between phased A(4-6)-tracts (at 10-11 bp intervals) have only a slight effect on overall curvature. To explore this phenomenon, we have examined the gel-migration properties of sequences containing both A-tracts as well as G-tracts (i.e., sequences of the form G(n)C(m) or C(n)G(m), n + m > 4) in various relative positioning. We show that the composite bend of these sequences depends on their relative arrangement. When G-tracts are placed between two A-tracts, such that both tracts are repeated in phase to themselves (e.g., G(5)A(6)G(5)A(5)), or adjacent to the 3'-side of A-tracts (e.g., A(6)G(5)N(10)), they have minimal influence on the extent of bending of the composite sequence. When G-tracts are placed one helical repeat away from A-tracts (e.g., G(5)N(5)A(6)N(6)), or are adjacent only to the 5'-side of A-tracts (e.g., G(5)A(6)N(10)) their influence on the composite bend is larger. The differential behavior of AG- versus GA-tracts means that A-tracts influence their flanking sequences in a polar manner. Whereas they suppress, or make constant, the intrinsic bending characteristics of any sequence placed immediately 3' to them (and hence by definition any sequence placed between two phased A-tracts), sequences adjoining them on their 5'-side are free to modulate the overall curvature. We interpret these results as evidence for the dominant nature of the unique and nonuniform structure adopted by tracts of four adenines or more. The effects of A-tracts extend at least five base pairs into the adjoining 3' region. This is further evidence for the complexity of DNA structure and the inadequacy of simple nearest-neighbor models to explain all its manifestations.     

Function and Assembly of DNA Looping,Clustering, and Microtubule Attachment Complexes within a Eukaryotic Kinetochore

The kinetochore is a complex protein–DNA assembly that provides the mechanical linkage between microtubules and the centromere DNA of each chromosome. Centromere DNA in all eukaryotes is wrapped around a unique nucleosome that contains the histone H3 variant CENP-A (Cse4p in Saccharomyces cerevisiae). Here, we report that the inner kinetochore complex (CBF3) is required for pericentric DNA looping at the Cse4p-containing nucleosome. DNA within the pericentric loop occupies a spatially confined area that is radially displaced from the interpolar central spindle. Microtubule-binding kinetochore complexes are not involved in pericentric DNA looping but are required for the geometric organization of DNA loops around the spindle microtubules in metaphase. Thus, the mitotic segregation apparatus is a composite structure composed of kinetochore and interpolar microtubules, the kinetochore, and organized pericentric DNA loops. The linkage of microtubule-binding to centromere DNA-looping complexes positions the pericentric chromatin loops and stabilizes the dynamic properties of individual kinetochore complexes in mitosis.     

A determining influence for CpG dinucleotides on nucleosome positioning in vitro

DNA sequence information that directs the translational positioning of nucleosomes can be attenuated by cytosine methylation when a short run of CpG dinucleotides is located close to the dyad axis of the nucleosome. Here, we show that point mutations introduced to re-pattern methylation at the (CpG)₃ element in the chicken β^A-globin promoter sequence themselves strongly influenced nucleosome formation in reconstituted chromatin. The disruptive effect of cytosine methylation on nucleosome formation was found to be determined by the sequence context of CpG dinucleotides, not just their location in the positioning sequence. Additional mutations indicated that methylation can also promote the occupation of certain nucleosome positions. DNase I analysis demonstrated that these genetic and epigenetic modifications altered the structural characteristics of the (CpG)₃ element. Our findings support a proposal that the intrinsic structural properties of the DNA at the −1.5 site, as occupied by (CpG)₃ in the nucleosome studied, can be decisive for nucleosome formation and stability, and that changes in anisotropic DNA bending or flexibility at this site explain why nucleosome positioning can be exquisitely sensitive to genetic and epigenetic modification of the DNA sequence.     

Design and calibration of a semi-synthetic DNA phasing assay

Electrophoretic assays of intrinsic DNA shape and shape changes induced by ligand binding are extremely useful because of their convenience and simplicity. The development of calibrations and empirical quantitative relationships permits highly accurate measurement of DNA shape using electrophoresis. Many conventional analyses employ the unidirectional ligation of short DNA duplexes. However, many oligonucleotides (typically more than 20) must often be synthesized for a single experiment. Additionally, the length of the DNA duplex can become limiting, preventing the analysis of certain DNA sequences. We now describe a semi-synthetic electrophoretic phasing method that offers several advantages, including a reduced number of required synthetic oligonucleotides, the ability to analyze longer DNA duplexes and a simplified approach for data analysis. We characterize semi-synthetic DNA probes in electrophoretic phasing assays by ligation of synthetic duplexes containing A₅ tracts between two longer restriction fragments. Upon electrophoresis, the gel mobility is strongly correlated with the predicted DNA curvature provided by the reference A₅ tracts. Having obtained this calibration, we show that the semi-synthetic phasing assay can be readily and economically applied to analyze DNA curvature induced by DNA charge modifications and DNA bending due to peptide binding.     

DNA sequence and methylation prescribe the inside-out conformational dynamics and bending energetics of DNA minicircles

Eukaryotic genome and methylome encode DNA fragments’ propensity to form nucleosome particles. Although the mechanical properties of DNA possibly orchestrate such encoding, the definite link between ‘omics’ and DNA energetics has remained elusive. Here, we bridge the divide by examining the sequence-dependent energetics of highly bent DNA. Molecular dynamics simulations of 42 intact DNA minicircles reveal that each DNA minicircle undergoes inside-out conformational transitions with the most likely configuration uniquely prescribed by the nucleotide sequence and methylation of DNA. The minicircles’ local geometry consists of straight segments connected by sharp bends compressing the DNA’s inward-facing major groove. Such an uneven distribution of the bending stress favors minimum free energy configurations that avoid stiff base pair sequences at inward-facing major grooves. Analysis of the minicircles’ inside-out free energy landscapes yields a discrete worm-like chain model of bent DNA energetics that accurately account for its nucleotide sequence and methylation. Experimentally measuring the dependence of the DNA looping time on the DNA sequence validates the model. When applied to a nucleosome-like DNA configuration, the model quantitatively reproduces yeast and human genomes’ nucleosome occupancy. Further analyses of the genome-wide chromatin structure data suggest that DNA bending energetics is a fundamental determinant of genome architecture.     

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.     

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.     

7-Deaza-2′-deoxyadenosine and 3-deaza-2′-deoxyadenosine replacing dA within d(A6)-tracts: differential bending at 3′- and 5′-junctions of d(A6)·d(T6) and B-DNA

7-Deaza-2′-deoxyadenosine (1, c⁷A_d) and 3-deaza-2′-deoxyadenosine (2, c³A_d) have been incorporated into d(AAAAAA) tracts replacing dA at various positions within oligonucleotides. For this purpose suitably protected phosphonates have been prepared and oligonucleotides were synthsized on solid-phase. The oligomers were hybridized with their cognate strands. The duplexes were phosphorylated at OH-5′ by polynucleotide kinase and self-ligated to multimers employing T4 DNA ligase. Oligomerized DNA-fragments were analyzed by polyacrylamide gel electrophoresis and the bending was determined from anomalies of electrophoretic mobility. Replacement of dA by c³A_d decreased the bending more than replacement by c⁷A_d. Reduction of bending was much stronger when the modified nucleosides replaced one or several dA residues at the 3′-site of an d(AAAAAA)-tract whereas replacement at the 5′-site showed no significant influence [1, 2].     

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.     

The sequence-directed bent DNA detected in the replication origin of Chlamydomonas reinhardtii chloroplast DNA is important for the replication function

Summary We demonstrated that the 1055 by restriction fragment containing OriA, a chloroplast DNA replication origin of Chlamydomonas reinhardtii, has electrophoretic anomalies characteristic of bent DNA. A tandem dimer of the region was constructed. Quantitative measurement of the relative gel mobility of a set of permuted fragments was used to extrapolate the approximate position of the bent DNA segment. By analyzing the gel mobility of short, sequenced fragments of the bent DNA region, the putative bending locus was identified. Two A₄ tracts and two A5 tracts were located in the bending locus. Oligonucleotide-directed mutagenesis was then used to disrupt the A tract or the spacing between A tracts and the effect of site-specific mutation on electrophoretic mobility was analyzed. To assess the functional role of the bent DNA region, subclones containing the bending locus, mutated bending locus, and regions flanking the bending locus were constructed. Each subclone was used as template in an in vitro DNA replication system which preferentially initiated DNA replication at OriA. A 224 by subclone with the bending locus positioned in the middle displayed the highest replication function and was sufficient to initiate DNA replication in vitro. Site-specific mutations or alterations of the A tracts resulted in decreased DNA bending and decreased DNA replication activity.