Anita Scipioni

A Theoretical Model for the Prediction of Sequence-Dependent Nucleosome Thermodynamic Stability

A theoretical model for predicting nucleosome thermodynamic stability in terms of DNA sequence is advanced. The model is based on a statistical mechanical approach, which allows the calculation of the canonical ensemble free energy involved in the competitive nucleosome reconstitution. It is based on the hypothesis that nucleosome stability mainly depends on the bending and twisting elastic energy to transform the DNA intrinsic superstructure into the nucleosomal structure. The ensemble average free energy is calculated starting from the intrinsic curvature, obtained by integrating the dinucleotide step deviations from the canonical B-DNA and expressed in terms of a Fourier series, in the framework of first-order elasticity. The sequence-dependent DNA flexibility is evaluated from the differential double helix thermodynamic stability. A large number of free-energy experimental data, obtained in different laboratories by competitive nucleosome reconstitution assays, are successfully compared to the theoretical results. They support the hypothesis that the stacking energies are the major factor in DNA rigidity and could be a measure of DNA stiffness. A dual role of DNA intrinsic curvature and flexibility emerges in the determination of nucleosome stability. The difference between the experimental and theoretical (elastic) nucleosome-reconstitution free energy for the whole pool of investigated DNAs suggests a significant role for the curvature-dependent DNA hydration and counterion interactions, which appear to destabilize nucleosomes in highly curved DNAs. This model represents an attempt to clarify the main features of the nucleosome thermodynamic stability in terms of physical-chemical parameters and suggests that in molecular systems with a large degree of complexity, the average molecular properties dominate over the local features, as in a statistical ensemble.

A theoretical model of DNA curvature.

Distortions from the uniform idealized B-DNA structure are investigated in terms of differential interactions between adjacent nucleotide pairs on the basis of conformational energy calculations. A theoretical model of DNA curvature is proposed based on the evaluation of the curvature vector defined in the complex plane and the corresponding variance. The model appears to contain the basic physical features for translating the deterministic fluctuations of DNA sequences in superstructure elements. It allows the quantitative reproduction of all the available gel electrophoresis experiments on both periodical polynucleotides and tracts of DNAs as well as the theoretical prediction of the sequence dependent DNA writhing in good agreement with the experimental data. The general pattern of agreement between the theoretical and experimental data and the biological significance of the results obtained allow an extensive application of the model for the screening of DNA regions which are possible candidates for protein recognition.

Mapping the intrinsic curvature and flexibility along the DNA chain

The energy of DNA deformation plays a crucial and active role in its packaging and its function in the cell. Considerable effort has gone into developing methodologies capable of evaluating the local sequence-directed curvature and flexibility of a DNA chain. These studies thus far have focused on DNA constructs expressly tailored either with anomalous flexibility or curvature tracts. Here we demonstrate that these two structural properties can be mapped also along the chain of a “natural” DNA with any sequence on the basis of its scanning force microscope (SFM) images. To know the orientation of the sequence of the investigated DNA molecules in their SFM images, we prepared a palindromic dimer of the long DNA molecule under study. The palindromic symmetry also acted as an internal gauge of the statistical significance of the analysis carried out on the SFM images of the dimer molecules. It was found that although the curvature modulus is not efficient in separating static and dynamic contributions to the curvature of the population of molecules, the curvature taken with its direction (its sign in two dimensions) permits the direct separation of the intrinsic curvature from the flexibility contributions. The sequence-dependent flexibility seems to vary monotonically with the chains intrinsic curvature; the chain rigidity was found to modulate as its local thermodynamic stability and does not correlate with the dinucleotide chain rigidities evaluation made from x-ray data by other authors.

Sequence-Dependent DNA Curvature and Flexibility from Scanning Force Microscopy Images

This paper reports a study of the sequence-dependent DNA curvature and flexibility based on scanning force microscopy (SFM) images. We used a palindromic dimer of a 1878-bp pBR322 fragment and collected a large pool of SFM images. The curvature of each imaged chain was measured in modulus and direction. It was found that the ensemble curvature modulus does not allow the separation of static and dynamic contributions to the curvature, whereas the curvature, when its direction in the two dimensions is taken into account, permits the direct separation of the intrinsic curvature contributions static and dynamic contributions. The palindromic symmetry also acted as an internal gauge of the validity of the SFM images statistical analysis. DNA static curvature resulted in good agreement with the predicted sequence-dependent intrinsic curvature. Furthermore, DNA sequence-dependent flexibility was found to correlate with the occurrence of A.T-rich dinucleotide steps along the chain and, in general, with the normalized basepair stacking energy distribution.

From the sequence to the superstructural properties of DNAs.

A theoretical model for predicting intrinsic and induced DNA superstructures as well as their thermodynamic properties is presented. Intrinsic sequence-dependent superstructures are evaluated by integrating local deviations from the canonical B-DNA of the different dinucleotide steps. Induced superstructures are obtained by adopting the principle of minimum deformation free energy, evaluated in the Fourier space, in the framework of first-order elasticity. Finally dinucleotide stacking energies and melting temperatures are considered to account for local flexibility. In fact the two scales are strongly correlated. The model works very satisfactorily in predicting the sequence-dependent effects on the DNA experimental behavior, such as the gel electrophoresis retardation, the writhe transitions in topologically constrained domains, the thermodynamic constants of circularization reactions as well as the nucleosome thermodynamic stability constants.

Generation of RNA Molecules by a Base-Catalysed Click-Like Reaction

The problem of the abiotic origin of RNA from prebiotically plausible compounds remains unsolved. As a potential partial solution, we report the spontaneous polymerization of 3′,5′‐cyclic GMP in water, in formamide, in dimethylformamide, and (in water) in the presence of a Brønsted base such as 1,8‐diazabicycloundec‐7‐ene. The reaction is untemplated, does not require enzymatic activities, is thermodynamically favoured and selectively yields 3′,5′‐bonded ribopolymers containing as many as 25 nucleotides. We propose a reaction pathway on the basis of 1) the measured stacking of the 3′,5′‐cyclic monomers, 2) the activation by Brønsted bases, 3) the determination (by MALDI‐TOF mass spectrometry, by 31P NMR, and by specific ribonucleases) of the molecular species produced. The reaction pathway has several of the attributes of a click‐like reaction.

Theoretical prediction of the gel electrophoretic retardation changes due to point mutations in a tract of SV40 DNA.

The changes of gel electrophoretic retardation due to single base substitutions in a 173 bp fragment of Sv40 DNA were predicted by using a theoretical model based on conformational energy calculations. As described in previous papers, this model allows successful prediction of the gel electrophoretic retardation of synthetic as well as natural DNAs reported in literature. The experimental retardations related to 195 point-mutated DNAs were reproduced with a standard deviation of 0.05 comparable with the experimental one of 0.04. This result, which represents a very critical test for the proposed model, indicates that DNA superstructures can be satisfactorily predicted on the simple physical basis of the integration of the nearest-neighbour perturbations in the dinucleotide steps. Thus, cooperative effects appear, in the majority of cases investigated, to play a second order role.

Prediction of nucleosome positioning in genomes: limits and perspectives of physical and bioinformatic approaches.

Abstract Nucleosomes, the fundamental repeating subunits of all eukaryotic chromatin, are responsible for packaging DNA into chromosomes inside the cell nucleus and controlling gene expression. While it has been well established that nucleosomes exhibit higher affinity for select DNA sequences, until recently it was unclear whether such preferences exerted a significant, genome-wide effect on nucleosome positioning in vivo. For this reason, an increasing interest is arising on a wide-ranging series of experimental and computational analyses capable of predicting the nucleosome positioning along genomes. Toward this goal, we propose a theoretical model for predicting nucleosome thermodynamic stability in terms of DNA sequence. Based on a statistical mechanical approach, the model allows the calculation of the sequence-dependent canonical ensemble free energy involved in nucleosome formation. The theoretical free energies were evaluated for 90 single nucleosome DNA tracts and successfully compared with those obtained with nucleosome competitive reconstitution. These results, obtained for single nucleosomes, could in principle allow the calculation of the intrinsic affinity of nucleosomes along DNA sequences virtually opening the possibility of predicting the nucleosome positioning along genomes on physical basis. The theoretical nucleosome distribution was compared and validated with that of yeast and human genome experimentally determined. The results interpret on a physical basis the experimental nucleosome positioning and are comparable with those obtained adopting models based on the identification of some recurrent sequence features obtained from the statistical analysis of a very large pool of nucleosomal DNA sequences provided by the positioning maps of genomes.

Recognition of the DNA sequence by an inorganic crystal surface

The sequence-dependent curvature is generally recognized as an important and biologically relevant property of DNA because it is involved in the formation and stability of association complexes with proteins. When a DNA tract, intrinsically curved for the periodical recurrence on the same strand of A-tracts phased with the B-DNA periodicity, is deposited on a flat surface, it exposes to that surface either a T- or an A-rich face. The surface of a freshly cleaved mica crystal recognizes those two faces and preferentially interacts with the former one. Statistical analysis of scanning force microscopy (SFM) images provides evidence of this recognition between an inorganic crystal surface and nanoscale structures of double-stranded DNA. This finding could open the way toward the use of the sequence-dependent adhesion to specific crystal faces for nanotechnological purposes.

Dual role of sequence-dependent DNA curvature in nucleosome stability: the critical test of highly bent Crithidia fasciculata DNA tract

In spite of the knowledge of the nucleosome molecular structure, the role of DNA intrinsic curvature in determining nucleosome stabilization is still an open question. In this paper, we describe a general model that allows the prediction of the nucleosome stability, tested on 83 different DNA sequences, in surprising good agreement with the experimental data, carried out in ours as well as in many other laboratories. The model is based on the dual role of DNA curvature in nucleosome thermodynamic stabilization. A critical test is the evaluation of the nucleosome free energy relative to a Crithidia fasciculata kinetoplast DNA fragment, which represents the most curved DNA found so far in biological systems and, therefore, is generally believed to form a highly stable nucleosome.