Andrew V. Colasanti

Internucleosomal Interactions Mediated by Histone Tails Allow Distant Communication in Chromatin

Background: Gene expression is regulated by DNA elements that often lie far apart along genomic sequences. Results: Novel computations and experiments provide new structural insights into long-range communication on chromatin. Conclusion: Efficient long-range association of transcriptional elements requires intact tails on the core histones. Significance: The understanding of action-at-a-distance in three dimensions helps to connect nucleosome structure/positioning to chromatin dynamics and gene regulation. Action across long distances on chromatin is a hallmark of eukaryotic transcriptional regulation. Although chromatin structure per se can support long-range interactions, the mechanisms of efficient communication between widely spaced DNA modules in chromatin remain a mystery. The molecular simulations described herein suggest that transient binary internucleosomal interactions can mediate distant communication in chromatin. Electrostatic interactions between the N-terminal tails of the core histones and DNA enhance the computed probability of juxtaposition of sites that lie far apart along the DNA sequence. Experimental analysis of the rates of communication in chromatin constructs confirms that long-distance communication occurs efficiently and independently of distance on tail-containing, but not on tailless, chromatin. Taken together, our data suggest that internucleosomal interactions involving the histone tails are essential for highly efficient, long-range communication between regulatory elements and their targets in eukaryotic genomes.

Properties of the nucleic-acid bases in free and Watson-Crick hydrogen-bonded states: computational insights into the sequence-dependent features of double-helical DNA

The nucleic-acid bases carry structural and energetic signatures that contribute to the unique features of genetic sequences. Here, we review the connection between the chemical structure of the constituent nucleotides and the polymeric properties of DNA. The sequence-dependent accumulation of charge on the major- and minor-groove edges of the Watson–Crick base pairs, obtained from ab initio calculations, presents unique motifs for direct sequence recognition. The optimization of base interactions generates a propellering of base-pair planes of the same handedness as that found in high-resolution double-helical structures. The optimized base pairs also deform along conformational pathways, i.e., normal modes, of the same type induced by the binding of proteins. Empirical energy computations that incorporate the properties of the base pairs account satisfactorily for general features of the next level of double-helical structure, but miss key sequence-dependent differences in dimeric structure and deformability. The latter discrepancies appear to reflect factors other than intrinsic base-pair structure.

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at http://w3dna.rutgers.edu, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

3DNALandscapes: a database for exploring the conformational features of DNA

3DNALandscapes, located at: http://3DNAscapes.rutgers.edu, is a new database for exploring the conformational features of DNA. In contrast to most structural databases, which archive the Cartesian coordinates and/or derived parameters and images for individual structures, 3DNALandscapes enables searches of conformational information across multiple structures. The database contains a wide variety of structural parameters and molecular images, computed with the 3DNA software package and known to be useful for characterizing and understanding the sequence-dependent spatial arrangements of the DNA sugar-phosphate backbone, sugar-base side groups, base pairs, base-pair steps, groove structure, etc. The data comprise all DNA-containing structures—both free and bound to proteins, drugs and other ligands—currently available in the Protein Data Bank. The web interface allows the user to link, report, plot and analyze this information from numerous perspectives and thereby gain insight into DNA conformation, deformability and interactions in different sequence and structural contexts. The data accumulated from known, well-resolved DNA structures can serve as useful benchmarks for the analysis and simulation of new structures. The collective data can also help to understand how DNA deforms in response to proteins and other molecules and undergoes conformational rearrangements.

Sequence-Dependent Variability of B-DNA

DNA bending is universal in biology—both the storage and the retrieval of information encoded in the base-pair sequence require significant deformations, particularly bending, of the double helix. The A-tract curvature, which modulates these processes, has thus been a subject of long-standing interest. Here we describe the ongoing evolution of models developed to account for the sequence-dependent bending and curvature of DNA, namely the AA-wedge, junction, and flexible anisotropic dimer models. We further show that recent high-resolution NMR structures of DNA A-tracts are consistent with crystallographically observed structures, and that the combined data provide a realistic basis for describing the behavior of curved DNA in solution.

Insights into Gene Expression and Packaging from Computer Simulations

Within the nucleus of each cell lies DNA—an unfathomably long, twisted, and intricately coiled molecule—segments of which make up the genes that provide the instructions that a cell needs to operate. As we near the 60th anniversary of the discovery of the DNA double helix, crucial questions remain about how the physical arrangement of the DNA in cells affects how genes work. For example, how a cell stores the genetic information inside the nucleus is complicated by the necessity of maintaining accessibility to DNA for genetic processing. In order to gain insight into the roles played by various proteins in reading and compacting the genome, we have developed new methodologies to simulate the dynamic, three-dimensional structures of long, fluctuating, protein-decorated strands of DNA. Our a priori approach to the problem allows us to determine the effects of individual proteins and their chemical modifications on overall DNA structure and function. Here, we present our recent treatment of the communication between regulatory proteins attached to precisely constructed stretches of chromatin. Our simulations account for the enhancement in communication detected experimentally on chromatin compared to protein-free DNA of the same chain length, as well as the critical roles played by the cationic ‘tails’ of the histone proteins in this signaling. The states of chromatin captured in the simulations offer new insights into the ways that the DNA, histones, and regulatory proteins contribute to long-range communication along the genome.

Weak operator binding enhances simulated lac repressor‐mediated DNA looping

The 50th anniversary of Biopolymers coincides closely with the like celebration of the discovery of the Escherichia coli (lac) lactose operon, a classic genetic system long used to illustrate the influence of biomolecular structure on function. The looping of DNA induced by the binding of the Lac repressor protein to sequentially distant operator sites on DNA continues to serve as a paradigm for understanding long‐range genomic communication. Advances in analyses of DNA structures and in incorporation of proteins in computer simulations of DNA looping allow us to address long‐standing questions about the role of protein‐mediated DNA loop formation in transcriptional control. Here we report insights gained from studies of the sequence‐dependent contributions of the natural lac operators to Lac repressor‐mediated DNA looping. Novel superposition of the ensembles of protein‐bound operator structures derived from NMR measurements reveals variations in DNA folding missed in conventional structural alignments. The changes in folding affect the predicted ease with which the repressor induces loop formation and the ways that DNA closes between the protein headpieces. The peeling of the auxiliary operators away from the repressor enhances the formation of loops with the 92‐bp wildtype spacing and hints of a structural reason behind their weak binding.

What Controls DNA Looping

The looping of DNA provides a means of communication between sequentially distant genomic sites that operate in tandem to express, copy, and repair the information encoded in the DNA base sequence. The short loops implicated in the expression of bacterial genes suggest that molecular factors other than the naturally stiff double helix are involved in bringing the interacting sites into close spatial proximity. New computational techniques that take direct account of the three-dimensional structures and fluctuations of protein and DNA allow us to examine the likely means of enhancing such communication. Here, we describe the application of these approaches to the looping of a 92 base-pair DNA segment between the headpieces of the tetrameric Escherichia coli Lac repressor protein. The distortions of the double helix induced by a second protein--the nonspecific nucleoid protein HU--increase the computed likelihood of looping by several orders of magnitude over that of DNA alone. Large-scale deformations of the repressor, sequence-dependent features in the DNA loop, and deformability of the DNA operators also enhance looping, although to lesser degrees. The correspondence between the predicted looping propensities and the ease of looping derived from gene-expression and single-molecule measurements lends credence to the derived structural picture.

Pulling and pushing the DNA double helix

Systematic, computer-simulated elongation of A- and B-DNA double helices beyond the range of normal room temperature fluctuations provides new insights into recent physical manipulations of single DNA molecules. The calculations include unusual states that are energetically disfavored under equilibrium conditions but that become favored as the DNA is highly stretched or compressed. The variation of potential energy vs. stretching provides a detailed picture of cooperative conformational change that points to the possible role played by the non-canonical states. Dramatic structural changes take place as the energies of different conformers approach one another. In particular, large-scale, concerted changes in base pair inclination, brought about by changes in backbone and glycosyl torsions, offer a model of the observed sharp increase in force required to stretch single DNA molecules more than 1.6 times their canonical extension. Small degrees of over- and under-twisting have limited effects on the over-st...

Dynamics of the Nucleosome Core Particle Revealed from a New Database of High-Resolution X-Ray Crystallographic and Simulated Structures

The nucleosome core particle is a highly conserved structure which can play diverse roles depending on the organism, cell, or part of chromatin in which it resides. The Protein Data Bank currently contains approximately 90 nucleosome core particle structures, most of which were determined in the last five years. The recent emergence of the field of epigenetics, and the increase in data available from experiments, warrants a need to develop new approaches to compare features of interest across multiple structures.The growing ensemble of structural data garnered from in vitro and in silico experiments provides a unique platform to study the mechanochemical properties of the nucleosome. We have developed a database and new computational tools to allow researchers to analyze and compare the nucleosome core particle structures deposited in the Protein Data Bank. The features of the DNA-protein assembly can be examined in novel coordinate frames placed on the structure, allowing researchers to obtain a better understanding of the organization and subtleties of the macromolecular complexes. This comparison allows one to examine the ‘motion’ of specific residues of interest, including specific sites of post-translational histone modification. The database also includes DNA-histone contact points, DNA conformational parameters, and information about protein features such as the secondary structure in the globular histone core and the ‘motion’ of the histone tails. Along with these features, we also characterize the dynamics of the global structure of the nucleosome core particle, including changes in the superhelical path of the DNA, rearrangements of the histone tetramers, and nucleosome stacking inside crystals. This information allows us to understand and model the critical role of mono-nucleosome structural propensity in processes such as carcinogenic modifications of the DNA, and nucleosome remodeling.