Dual role of DNA intrinsic curvature and flexibility in determining nucleosome stability

A statistical mechanistic approach to evaluate the sequence-dependent thermodynamic stability of nucleosomes is proposed. The model is based on the calculation of the DNA intrinsic curvature, obtained by integrating the nucleotide step deviations from the canonical B-DNA structure, and on the evaluation of the first order elastic distortion energy to reach the nucleosomal superstructure. Literature data on the free energy of nucleosome formation as obtained by competitive nucleosome reconstitution of a significant pool of different DNA sequences were compared with the theoretical results, and a satisfactorily good correlation was found. A striking result of the comparison is the emergence of two opposite roles of the DNA intrinsic curvature and flexibility in determining nucleosome stability. Finally, the obtained results suggest that the curvature-dependent DNA hydration should play a relevant role in the sequence-dependent nucleosome stability.     

An assessment of three dinucleotide parameters to predict DNA curvature by quantitative comparison with experimental data

Curved DNA fragments are often found near functionally important sites such as promoters and origins of replication, and hence sequence-dependent DNA curvature prediction is of great utility in genomics and bioinformatics. In light of this, an assessment of three different dinucleotide step parameters (based on gel retardation as well as crystal structure data) is carried out. These parameters (BMHT, LB and CS) are evaluated quantitatively for their ability to predict correctly the experimental results of a large set of nucleic acid sequences containing A-tracts as well as GC-rich motifs. This set contained around 40 synthetic as well as natural sequences whose solution properties have been well characterized experimentally. All three models could account reasonably well for curvature in the various DNA sequences. The CS model, where dinucleotide parameters are calculated from crystal structure data, consistently shows slightly better correlation with experimental data. Our simple analysis also indicates that presently available trinucleotide parameters fail to predict curvature in some of the well-characterized sequences. The study shows that the dinucleotide parameters with some further refinement can be used to predict sequence-dependent curvature correctly in genomic sequences.     

AFM imaging and theoretical modeling studies of sequence-dependent nucleosome positioning

Telomeric chromatin has different features with respect to bulk chromatin, since nucleosomal repeat along the chain is unusually short. We studied the role of telomeric DNA sequences on nucleosomal spacing in a model system. Nucleosomal arrays, assembled on a 1500-bp-long human telomeric DNA and on a DNA fragment containing 8 copies of the 601 strong nucleosome positioning sequence, have been studied at the single molecule level, by atomic force microscopy imaging. Random nucleosome positioning was found in the case of human telomeric DNA. On the contrary, nucleosome positioning on 601 DNA is characterized by preferential positions of nucleosome dyad axis each 200 bp. The AFM-derived nucleosome organization is in satisfactory agreement with that predicted by theoretical modeling, based on sequence-dependent DNA curvature and flexibility. The reported results show that DNA sequence has a main role, not only in mononucleosome thermodynamic stability, but also in the organization of nucleosomal arrays.     

Sequence-Dependent Biophysical Modeling of DNA Amplification

A theoretical framework for prediction of the dynamic evolution of chemical species in DNA amplification reactions, for any specified sequence and operating conditions, is reported. Using the polymerase chain reaction (PCR) as an example, we developed a sequence- and temperature-dependent kinetic model for DNA amplification using first-principles biophysical modeling of DNA hybridization and polymerization. We compare this kinetic model with prior PCR models and discuss the features of our model that are essential for quantitative prediction of DNA amplification efficiency for arbitrary sequences and operating conditions. Using this model, the kinetics of PCR is analyzed. The ability of the model to distinguish between the dynamic evolution of distinct DNA sequences in DNA amplification reactions is demonstrated. The kinetic model is solved for a typical PCR temperature protocol to motivate the need for optimization of the dynamic operating conditions of DNA amplification reactions. It is shown that amplification efficiency is affected by dynamic processes that are not accurately represented in the simplified models of DNA amplification that form the basis of conventional temperature cycling protocols. Based on this analysis, a modified temperature protocol that improves PCR efficiency is suggested. Use of this sequence-dependent kinetic model in a control theoretic framework to determine the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is discussed.     

Intrinsic curvature of DNA influences LacR-mediated looping

Protein-mediated DNA looping is a common mechanism for regulating gene expression. Loops occur when a protein binds to two operators on the same DNA molecule. The probability of looping is controlled, in part, by the basepair sequence of inter-operator DNA, which influences its structural properties. One structural property is the intrinsic or stress-free curvature. In this article, we explore the influence of sequence-dependent intrinsic curvature by exercising a computational rod model for the inter-operator DNA as applied to looping of the LacR-DNA complex. Starting with known sequences for the inter-operator DNA, we first compute the intrinsic curvature of the helical axis as input to the rod model. The crystal structure of the LacR (with bound operators) then defines the requisite boundary conditions needed for the dynamic rod model that predicts the energetics and topology of the intervening DNA loop. A major contribution of this model is its ability to predict a broad range of published experimental data for highly bent (designed) sequences. The model successfully predicts the loop topologies known from fluorescence resonance energy transfer measurements, the linking number distribution known from cyclization assays with the LacR-DNA complex, the relative loop stability known from competition assays, and the relative loop size known from gel mobility assays. In addition, the computations reveal that highly curved sequences tend to lower the energetic cost of loop formation, widen the energy distribution among stable and meta-stable looped states, and substantially alter loop topology. The inclusion of sequence-dependent intrinsic curvature also leads to nonuniform twist and necessitates consideration of eight distinct binding topologies from the known crystal structure of the LacR-DNA complex.     

Sequence-Dependent Biophysical Modeling of DNA Amplification

A theoretical framework for prediction of the dynamic evolution of chemical species in DNA amplification reactions, for any specified sequence and operating conditions, is reported. Using the polymerase chain reaction (PCR) as an example, we developed a sequence- and temperature-dependent kinetic model for DNA amplification using first-principles biophysical modeling of DNA hybridization and polymerization. We compare this kinetic model with prior PCR models and discuss the features of our model that are essential for quantitative prediction of DNA amplification efficiency for arbitrary sequences and operating conditions. Using this model, the kinetics of PCR is analyzed. The ability of the model to distinguish between the dynamic evolution of distinct DNA sequences in DNA amplification reactions is demonstrated. The kinetic model is solved for a typical PCR temperature protocol to motivate the need for optimization of the dynamic operating conditions of DNA amplification reactions. It is shown that amplification efficiency is affected by dynamic processes that are not accurately represented in the simplified models of DNA amplification that form the basis of conventional temperature cycling protocols. Based on this analysis, a modified temperature protocol that improves PCR efficiency is suggested. Use of this sequence-dependent kinetic model in a control theoretic framework to determine the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is discussed.     

Sequence-dependent DNA deformability studied using molecular dynamics simulations

Proteins recognize specific DNA sequences not only through direct contact between amino acids and bases, but also indirectly based on the sequence-dependent conformation and deformability of the DNA (indirect readout). We used molecular dynamics simulations to analyze the sequence-dependent DNA conformations of all 136 possible tetrameric sequences sandwiched between CGCG sequences. The deformability of dimeric steps obtained by the simulations is consistent with that by the crystal structures. The simulation results further showed that the conformation and deformability of the tetramers can highly depend on the flanking base pairs. The conformations of xATx tetramers show the most rigidity and are not affected by the flanking base pairs and the xYRx show by contrast the greatest flexibility and change their conformations depending on the base pairs at both ends, suggesting tetramers with the same central dimer can show different deformabilities. These results suggest that analysis of dimeric steps alone may overlook some conformational features of DNA and provide insight into the mechanism of indirect readout during protein–DNA recognition. Moreover, the sequence dependence of DNA conformation and deformability may be used to estimate the contribution of indirect readout to the specificity of protein–DNA recognition as well as nucleosome positioning and large-scale behavior of nucleic acids.     

Predicting sequence-dependent melting stability of short duplex DNA oligomers

Many important applications of DNA sequence-dependent hybridization reactions have recently emerged. This has sparked a renewed interest in analytical calculations of sequence-dependent melting stability of duplex DNA. In particular, for many applications it is often desirable to accurately predict the transition temperature, or t_m, of short duplex DNA oligomers (∼ 20 base pairs or less) from their sequence and concentration. The thermodynamic analytical method underlying these predictive calculations is based on the nearest-neighbor model. At least 11 sets of nearest-neighbor sequence-dependent thermodynamic parameters for DNA have been published. These sets are compared. Use of the nearest-neighbor sets in predicting t_m from the DNA sequence is demonstrated, and the ability of the nearest-neighbor parameters to provide accurate predictions of experimental t_m's of short duplex DNA oligomers is assessed. © 1998 John Wiley & Sons, Inc. Biopoly 44: 217–239, 1997     

Studies of DNA dumbbells. I. Melting curves of 17 DNA dumbbells with different duplex stem sequences linked by T4 endloops: evaluation of the nearest-neighbor stacking interactions in DNA.

Seventeen DNA dumbbells were constructed that have duplex sequences ranging in length from 14 to 18 base pairs linked on the ends by T4 single-strand loops. Fifteen of the molecules have the core duplexes with the sequences 5'G-T-A-T-C-C-(W-X-Y-Z)-G-G-A-T-A-C3', where (W-X-Y-Z) represents a unique combination of A.T, T.A, G.C, and C.G base pairs. The remaining two molecules have the central sequence (W-X-Y-Z) = A-C and A-C-A-C-A-C. These duplex sequences were designed such that the central sequences include different combinations of the 10 possible nearest-neighbor (n-n) stacks in DNA. In this sense the set of molecules is complete and serves as a model system for evaluating sequence-dependent local stability of DNA. Optical melting curves of the samples were collected in 25, 55, 85, and 115 mM [Na+], and showed, regardless of solvent ionic strength, that the transition temperatures of the dumbbells vary by as much as 14 degrees for different molecules of the set. Results of melting experiments analyzed in terms of a n-n sequence-dependent model allowed evaluation of nine independent linear combinations of the n-n stacking interactions in DNA as a function of solvent ionic strength. Although there are in principle 10 possible different n-n interactions in DNA, these 10 are not linearly independent and therefore can not be uniquely determined. For molecules with ends, there are 9 linearly independent combinations, as opposed to circular or semiinfinite repeating copolymers where only 8 linear combinations of the 10 possible n-n interactions are linearly independent. The n-n interactions are presented as combinations of the deviations from average stacking for the 5'-3' base-pair doublets, delta Gi, and reveal several interesting features: (1) Titratable changes in the values of delta Gi with changing salt environment are observed. In all salts the most stable unique combination is delta G4 = (delta GGpC+delta GCpG)/2, and the least stable is the GpG/CpC stack, delta G2 = delta GGpG/CpC. (2) The chi 2 values of the fits of the evaluated delta Gi's to experimental data increased with decreasing [Na+], suggesting that significant interactions beyond nearest neighbors become more pronounced, particularly at 25 nM Na+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nanomechanics of Diaminopurine-Substituted DNA

2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensions. Here, we show that DAP-DNA overstretches at 52 pN. In summary, DAP substitution decreases the overall stability of the B-form double helix, biasing toward non-B-form DNA helix conformations at zero tension and facilitating the B-to-S transition at high tension.     

Comparison of sequence-dependent tiling array normalization approaches

Background  

The detection of enriched DNA or RNA fragments by tiling microarrays has become more and more popular. These microarrays contain a high number of small probes covering genomic loci. However, to achieve high coverage the probe sequences cannot be selected for their hybridization properties. The affinity of the probes towards their targets varies in a sequence-dependent manner. In order to remove this bias a number of approaches have been developed and shown to increase the detection of enriched DNA or RNA fragments. However, these approaches also employ a peak detection algorithm that is different from the one used previously. Thus, it seems possible that the enhancement of detection is due to the peak detection algorithm rather than the sequence-dependent normalization.     

Triple Helix Structures: Sequence Dependence,Flexibility and Mismatch Effects

Abstract

By means of molecular modelling, electrostatic interactions are shown to play an important role in the sequence-dependent structure of triple helices formed by a homopyrimidine oligonucleotide bound to a homopurine, homopyrimidine sequence on DNA. This is caused by the presence of positive charges due to the protonation of cytosines in the Hoogsteen-bonded strand, required in order to form C.GxC+ triplets. Energetic and conformational characteristics of triple helices with different sequences are analyzed and discussed. The effects of duplex mismatches on the triple helix stability are investigated via thermal dissociation using UV absorption.     

Wavelet Analysis of DNA Bending Profiles reveals Structural Constraints on the Evolution of Genomic Sequences

Analyses of genomic DNA sequences have shown in previous works that base pairs are correlated at large distances with scale-invariant statistical properties. We show in the present study that these correlations between nucleotides (letters) result in fact from long-range correlations (LRC) between sequence-dependent DNA structural elements (words) involved in the packaging of DNA in chromatin. Using the wavelet transform technique, we perform a comparative analysis of the DNA text and of the corresponding bending profiles generated with curvature tables based on nucleosome positioning data. This exploration through the optics of the so-called `wavelet transform microscope' reveals a characteristic scale of 100-200 bp that separates two regimes of different LRC. We focus here on the existence of LRC in the small-scale regime ( 200 bp). Analysis of genomes in the three kingdoms reveals that this regime is specifically associated to the presence of nucleosomes. Indeed, small scale LRC are observed in eukaryotic genomes and to a less extent in archaeal genomes, in contrast with their absence in eubacterial genomes. Similarly, this regime is observed in eukaryotic but not in bacterial viral DNA genomes. There is one exception for genomes of Poxviruses, the only animal DNA viruses that do not replicate in the cell nucleus and do not present small scale LRC. Furthermore, no small scale LRC are detected in the genomes of all examined RNA viruses, with one exception in the case of retroviruses. Altogether, these results strongly suggest that small-scale LRC are a signature of the nucleosomal structure. Finally, we discuss possible interpretations of these small-scale LRC in terms of the mechanisms that govern the positioning, the stability and the dynamics of the nucleosomes along the DNA chain. This paper is maily devoted to a pedagogical presentation of the theoretical concepts and physical methods which are well suited to perform a statistical analysis of genomic sequences. We review the results obtained with the so-called wavelet-based multifractal analysis when investigating the DNA sequences of various organisms in the three kingdoms. Some of these results have been announced in B. Audit et al. [1, 2].     

Biological asymmetries and the fidelity of eukaryotic DNA replication.

A diploid human genome contains approximately six billion nucleotides. This enormous amount of genetic information can be replicated with great accuracy in only a few hours. However, because DNA strands are oriented antiparallel while DNA polymerization only occurs in the 5'----3' direction, semi-conservative replication of double-stranded DNA is an asymmetric process, i.e., there is a leading and a lagging strand. This provides a considerable opportunity for non-random error rates, because the architecture of the two strands as well as the DNA polymerases that replicate them may be different. In addition, the proteins that start or finish chains may well be different from those that perform the bulk of chain elongation. Furthermore, while replication fidelity depends on the absolute and relative concentrations of the four deoxyribonucleotide precursors, these are not equal in vivo, not constant throughout the cell cycle, and not necessarily equivalent in all cell types. Finally, the fidelity of DNA synthesis is sequence-dependent and the eukaryotic nuclear genome is a heterogeneous substrate. It contains repetitive and non-repetitive sequences and can actually be considered as two subgenomes that differ in nucleotide composition and gene content and that replicate at different times. The effects that each of these asymmetries may have on error rates during replication of the eukaryotic genome are discussed.     

Comparative study of sequence-dependent hybridization kinetics in solution and on microspheres

Hybridization kinetics of DNA sequences with known secondary structures and random sequences designed with similar melting temperatures were studied in solution and when one strand was bound to 5 μm silica microspheres. The rates of hybridization followed second-order kinetics and were measured spectrophotometrically in solution and fluorometrically in the solid phase. In solution, the rate constants for the model sequences varied by almost two orders of magnitude, with a decrease in the rate constant with increasing amounts of secondary structure in the target sequence. The random sequences also showed over an order of magnitude difference in the rate constant. In contrast, the hybridization experiments in the solid phase with the same model sequences showed almost no change in the rate constant. Solid phase rate constants were approximately three orders of magnitude lower compared with the solution phase constants for sequences with little or no single-stranded structure. Sequences with a known secondary structure yielded solution phase rate constants as low as 3 × 10³ M⁻¹ s⁻¹ with solid phase rate constants for the same sequences measured at 2.5 × 10² M⁻¹ s⁻¹. The results from these experiments indicate that (i) solid phase hybridization occurs three orders of magnitude slower than solution phase, (ii) trends observed in structure-dependent kinetics of solution phase hybridization may not be applicable to solid phase hybridization and (iii) model probes with known secondary structure decrease reaction rates; however, even random sequences with no known internal single-stranded structure can yield a broad range of reaction rates.     

Triple helix structures: sequence dependence, flexibility and mismatch effects.

By means of molecular modelling, electrostatic interactions are shown to play an important role in the sequence-dependent structure of triple helices formed by a homopyrimidine oligonucleotide bound to a homopurine. homopyrimidine sequence on DNA. This is caused by the presence of positive charges due to the protonation of cytosines in the Hoogsteen-bonded strand, required in order to form C.GxC+ triplets. Energetic and conformational characteristics of triple helices with different sequences are analyzed and discussed. The effects of duplex mismatches on the triple helix stability are investigated via thermal dissociation using UV absorption.     

Comparison between chromosomal and nuclear sap RNA from Chironomus tentans salivary gland cells by RNA/DNA hybridization

Newly-synthesized, high molecular weight RNA from salivary gland polytene chromosomes and from the nuclear sap was investigated by RNA/DNA hybridization. Salivary glands were incubated for 90 min with radioactive nucleosides and afterwards fixed. Chromosomes and nuclear sap were subsequently isolated by microdissection. Labelled RNA, extracted from three different chromosomal fractions and from the nuclear sap, was subjected to different hybridization procedures under conditions which primarily allow repeated nucleotide sequences to interact.In one type of experiments RNA was hybridized by a microtechnique to filter-bound DNA at increasing RNA/DNA input ratios. Nuclear sap RNA saturated 0.25−0.30% of the DNA, while the chromosomal RNA fractions had not reached a plateau even after hybridization with 0.5−1% of the DNA. Thus chromosomal RNA appears to contain sequences which are absent from, or present in only low concentration in, the nuclear sap. Nuclear sap RNA hybrids also showed a higher thermal stability than chromosomal RNA hybrids, which may reflect a higher precision of base-pairing in hybrids formed by nuclear sap RNA.In a second type of experiments the time dependence of hybrid formation was investigated. The hybridization rate for nuclear sap RNA was about three times as high as the corresponding rate for chromosomal RNA. This result indicates a relative enrichment of rapidly hybridizing RNA sequences in the nuclear sap.The difference in hybridization properties between chromosomal and nuclear sap RNA may be due to a predominance in the nuclear sap of RNA from a special chromosomal puff, the Balbiani Ring 2 (BR2), which has been shown to contain highly repeated DNA sequences. A comparison between the hybridization properties of nuclear sap RNA and BR2 RNA indicated that 55–70% of nuclear sap RNA may be derived from BR2.The specific hybridization rate of chromosomal RNA points to an average multiplicity of about 30 for its complementary DNA sequences. On the basis of the present and previous results it is suggested that the repeated DNA is arranged in families of related sequences and that sequences belonging to a particular family are distributed in different chromosomes.     

A systematic molecular dynamics study of nearest-neighbor effects on base pair and base pair step conformations and fluctuations in B-DNA

It is well recognized that base sequence exerts a significant influence on the properties of DNA and plays a significant role in protein–DNA interactions vital for cellular processes. Understanding and predicting base sequence effects requires an extensive structural and dynamic dataset which is currently unavailable from experiment. A consortium of laboratories was consequently formed to obtain this information using molecular simulations. This article describes results providing information not only on all 10 unique base pair steps, but also on all possible nearest-neighbor effects on these steps. These results are derived from simulations of 50–100 ns on 39 different DNA oligomers in explicit solvent and using a physiological salt concentration. We demonstrate that the simulations are converged in terms of helical and backbone parameters. The results show that nearest-neighbor effects on base pair steps are very significant, implying that dinucleotide models are insufficient for predicting sequence-dependent behavior. Flanking base sequences can notably lead to base pair step parameters in dynamic equilibrium between two conformational sub-states. Although this study only provides limited data on next-nearest-neighbor effects, we suggest that such effects should be analyzed before attempting to predict the sequence-dependent behavior of DNA.