DNA topology confers sequence specificity to nonspecific architectural proteins.

Topological constraints placed on short fragments of DNA change the disorder found in chain molecules randomly decorated by nonspecific, architectural proteins into tightly organized 3D structures. The bacterial heat-unstable (HU) protein builds up, counter to expectations, in greater quantities and at particular sites along simulated DNA minicircles and loops. Moreover, the placement of HU along loops with the "wild-type" spacing found in the Escherichia coli lactose (lac) and galactose (gal) operons precludes access to key recognition elements on DNA. The HU protein introduces a unique spatial pathway in the DNA upon closure. The many ways in which the protein induces nearly the same closed circular configuration point to the statistical advantage of its nonspecificity. The rotational settings imposed on DNA by the repressor proteins, by contrast, introduce sequential specificity in HU placement, with the nonspecific protein accumulating at particular loci on the constrained duplex. Thus, an architectural protein with no discernible DNA sequence-recognizing features becomes site-specific and potentially assumes a functional role upon loop formation. The locations of HU on the closed DNA reflect long-range mechanical correlations. The protein responds to DNA shape and deformability­the stiff, naturally straight double-helical structure­rather than to the unique features of the constituent base pairs. The structures of the simulated loops suggest that HU architecture, like nucleosomal architecture, which modulates the ability of regulatory proteins to recognize their binding sites in the context of chromatin, may influence repressor-operator interactions in the context of the bacterial nucleoid.

DNA simulation benchmarks revealed with the accumulation of high-resolution structures.

DNA carries more than the list of biochemical instructions that drive the basic functions of living systems. The sequence of base pairs includes a multitude of structural and energetic signals that determine the degree to which the long, threadlike molecule moves and how it responds to proteins and other molecules involved in its processing and packaging. The arrangements of successive base pairs in high-resolution protein-DNA crystal structures provide useful benchmarks for atomic-level simulations of double-helical DNA as well as information potentially useful in interpreting the properties of specific DNA sequences. The set of currently available structures has enough examples to characterize the conformational preferences of the DNA base-pair steps within the context of their immediate neighbors, i.e., in the context of tetramers, and reveals surprising effects of certain neighbors on local chain properties. The proteins in contact with DNA present various microenvironments that sense and/or induce the observed spatial forms. The cumulative buildup of amino-acid atoms in different protein-DNA complexes produces a binding cloud around the double helix with subtle sequence-dependent features. While the microenvironment presented by each protein to DNA is highly unique, the overall composition of amino-acid atoms within close range of DNA in a broad collection of structures is fairly uniform. The buildup of protein atoms of different types around the DNA provides new information for the improvement of nucleic acid force fields and fresh ideas for the exploration of the properties of DNA in solution.

Structural insights into the role of architectural proteins in DNA looping deduced from computer simulations.

Bacterial gene expression is regulated by DNA elements that often lie far apart along the genomic sequence, but come close together during genetic processing. The intervening residues form loops, which are organized by the binding of various proteins. For example, the Escherichia coli Lac repressor protein binds DNA operators, separated by 92 or 401 bp, and suppresses the formation of gene products involved in the metabolism of lactose. The system also includes several highly abundant architectural proteins, such as the histone-like (heat-unstable) HU protein, which severely deform the double helix upon binding. In order to gain a better understanding of how the naturally stiff DNA double helix forms the short loops detected in vivo, we have developed new computational methods to study the effects of various non-specific binding proteins on the three-dimensional configurational properties of DNA sequences. The present article surveys the approach that we use to generate ensembles of spatially constrained protein-decorated DNA structures (minicircles and Lac repressor-mediated loops) and presents some of the insights gained from the correspondence between computation and experiment about the potential contributions of architectural and regulatory proteins to DNA looping and gene expression.

Molecular simulations of aromatase reveal new insights into the mechanism of ligand binding.

CYP19A1, also known as aromatase or estrogen synthetase, is the rate-limiting enzyme in the biosynthesis of estrogens from their corresponding androgens. Several clinically used breast cancer therapies target aromatase. In this work, explicitly solvated all-atom molecular dynamics simulations of aromatase with a model of the lipid bilayer and the transmembrane helix are performed. The dynamics of aromatase and the role of titration of an important amino acid residue involved in aromatization of androgens are investigated via two 250-ns long simulations. One simulation treats the protonated form of the catalytic aspartate 309, which appears more consistent with crystallographic data for the active site, while the simulation of the deprotonated form shows some notable conformational shifts. Ensemble-based computational solvent mapping experiments indicate possible novel druggable binding sites that could be utilized by next-generation inhibitors. In addition, the effects of protonation on the ligand positioning and channel dynamics are investigated using geometrical models that estimate the opening width of critical channels. Significant differences in channel dynamics between the protonated and deprotonated trajectories are exhibited, suggesting that the mechanism for substrate and product entry and the aromatization process may be coupled to a "locking" mechanism and channel opening. Our results may be particularly relevant in the design of novel drugs, which may be useful therapeutic treatments of cancers such as those of the breast and prostate.

Revisiting DNA Sequence-Dependent Deformability in High-Resolution Structures: Effects of Flanking Base Pairs on Dinucleotide Morphology and Global Chain Configuration.

DNA carries more than the list of biochemical ingredients that drive the basic functions of living systems. The sequence of base pairs includes a multitude of structural and energetic signals, which determine the degree to which the long, threadlike molecule moves and how it responds to proteins and other molecules that control its processing and govern its packaging. The chemical composition of base pairs directs the spatial disposition and fluctuations of successive residues. The observed arrangements of these moieties in high-resolution protein-DNA crystal structures provide one of the best available estimates of the natural, sequence-dependent structure and deformability of the double-helical molecule. Here, we update the set of knowledge-based elastic potentials designed to describe the observed equilibrium structures and configurational fluctuations of the ten unique base-pair steps. The large number of currently available structures makes it possible to characterize the configurational preferences of the DNA base-pair steps within the context of their immediate neighbors, i.e., tetrameric context. Use of these knowledge-based potentials shows promise in accounting for known effects of sequence in long chain molecules, e.g., the degree of curvature reported in classic gel mobility studies and the recently reported sequence-dependent responses of supercoiled minicircles to nuclease cleavage.

How stiff is DNA?

The natural stiffness of DNA, which contributes to the interactions of the many proteins involved in its biological processing and packaging, also plays an important role in modern nanotechnology. Here we report new Monte-Carlo simulations of deformable DNA molecules of potential utility in understanding the behavior of the long, double-helical polymer in the tight confines of a cell and in the design of novel nanomaterials and molecular devices. We directly determine the fluctuations in end-to-end extension associated with the conventional elastic-rod representation of DNA and with more realistic models that take account of the precise deformability of the constituent base-pair steps. Notably, the variance of end-to-end distance shows a quadratic increase with chain length in short chains of both types. We also consider the contributions to chain extension from the chemical linkages used to attach small molecular probes to DNA. The distribution of computed distances is sensitive to the intrinsic structure and allowed deformations of the tether. Surprisingly, the enhancement in end-to-end variance associated with the presence of the probe depends upon chain length, even when the probe is rigidly connected to DNA. We find that the elastic rod model of DNA in combination with a slightly fluctuating tether accounts satisfactorily for the distributions of end-to-end distances extracted from the small-angle X-ray scattering of gold nanocrystals covalently linked to the ends of short DNAs. There is no need to introduce additional structural fluctuations to reproduce the measured uptake in end-to-end fluctuations with chain length.

Effect of confinement on DNA, solvent and counterion dynamics in a model biological nanopore.

The application of recent advances in nanopore technology to high-throughput DNA sequencing requires a more detailed understanding of solvent, ion and DNA interactions occurring within these pores. Here we present a combination of atomistic and coarse-grained modeling studies of the dynamics of short single-stranded DNA (ssDNA) homopolymers within the alpha-hemolysin pore, for the two single-stranded homopolymers poly(dA)40 and poly(dC)40. Analysis of atomistic simulations along with the per-residue decomposition of protein-DNA interactions in these simulations gives new insight into the very complex issues that have yet to be fully addressed with detailed MD simulations. We discuss a modification of the solvent properties and ion distribution around DNA within nanopore confinement and put it into the general framework of counterion condensation theory. There is a reasonable agreement in computed properties from our all-atom simulations and the resulting predictions from analytical theories with experimental data, and our equilibrium results here support the conclusions from our previous non-equilibrium Brownian dynamics studies with a recently developed BROMOC protocol that cations are the primary charge carriers through alpha-hemolysin nanopores under an applied voltage in the presence of ssDNA. Clustering analysis led to an identification of distinct conformational states of captured polymer and depth of the current blockade. Therefore, our data suggest that confined polymer may act as a flickering gate, thus contributing to excess noise phenomena. We also discuss the extent of water structuring due to nanopore confinement and the relationship between the conformational dynamics of a captured polymer and the distribution of blocked current.

Interplay of protein and DNA structure revealed in simulations of the lac operon.

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.

BROMOC-D: Brownian Dynamics/Monte-Carlo Program Suite to Study Ion and DNA Permeation in Nanopores.

A theoretical framework is presented to model ion and DNA translocation across a nanopore confinement under an applied electric field. A combined Grand Canonical Monte Carlo Brownian Dynamics (GCMC/BD) algorithm offers a general approach to study ion permeation through wide molecular pores with a direct account of ion-ion and ion-DNA correlations. This work extends previously developed theory by incorporating the recently developed coarse-grain polymer model of DNA by de Pablo and colleagues [Knotts, T. A.; Rathore, N.; Schwartz, D. C.; de Pablo, J. J. J. Chem. Phys. 2007, 126] with explicit ions for simulations of polymer dynamics. Atomistic MD simulations were used to guide model developments. The power of the developed scheme is illustrated with studies of single-stranded DNA (ss-DNA) oligomer translocation in two model cases: a cylindrical pore with a varying radius and a well-studied experimental system, the staphylococcal α-hemolysin channel. The developed model shows good agreement with experimental data for model studies of two homopolymers: ss-poly(dA)(n) and ss-poly(dC)(n). The developed protocol allows for direct evaluation of different factors (charge distribution and pore shape and size) controlling DNA translocation in a variety of nanopores.

Understanding apparent DNA flexibility enhancement by HU and HMGB architectural proteins.

Understanding and predicting the mechanical properties of protein/DNA complexes are challenging problems in biophysics. Certain architectural proteins bind DNA without sequence specificity and strongly distort the double helix. These proteins rapidly bind and unbind, seemingly enhancing the flexibility of DNA as measured by cyclization kinetics. The ability of architectural proteins to overcome DNA stiffness has important biological consequences, but the detailed mechanism of apparent DNA flexibility enhancement by these proteins has not been clear. Here, we apply a novel Monte Carlo approach that incorporates the precise effects of protein on DNA structure to interpret new experimental data for the bacterial histone-like HU protein and two eukaryotic high-mobility group class B (HMGB) proteins binding to ∼200-bp DNA molecules. These data (experimental measurement of protein-induced increase in DNA cyclization) are compared with simulated cyclization propensities to deduce the global structure and binding characteristics of the closed protein/DNA assemblies. The simulations account for all observed (chain length and concentration dependent) effects of protein on DNA behavior, including how the experimental cyclization maxima, observed at DNA lengths that are not an integral helical repeat, reflect the deformation of DNA by the architectural proteins and how random DNA binding by different proteins enhances DNA cyclization to different levels. This combination of experiment and simulation provides a powerful new approach to resolve a long-standing problem in the biophysics of protein/DNA interactions.

Effects of the nucleoid protein HU on the structure, flexibility, and ring-closure properties of DNA deduced from Monte Carlo simulations.

The histone-like HU (heat unstable) protein plays a key role in the organization and regulation of the Escherichia coli genome. The nonspecific nature of HU binding to DNA complicates analysis of the mechanism by which the protein contributes to the looping of DNA. Conventional models of the looping of HU-bound duplexes attribute the changes in biophysical properties of DNA brought about by the random binding of protein to changes in the effective parameters of an ideal helical wormlike chain. Here, we introduce a novel Monte Carlo approach to study the effects of nonspecific HU binding on the configurational properties of DNA directly. We randomly decorated segments of an ideal double-helical DNA with HU molecules that induce the bends and other structural distortions of the double helix find in currently available X-ray structures. We find that the presence of HU at levels approximating those found in the cell reduces the persistence length by roughly threefold compared with that of naked DNA. The binding of protein has particularly striking effects on the cyclization properties of short duplexes, altering the dependence of ring closure on chain length in a way that cannot be mimicked by a simple wormlike model and accumulating at higher-than-expected levels on successfully closed chains. Moreover, the uptake of protein on small minicircles depends on chain length, taking advantage of the HU-induced deformations of DNA structure to facilitate ligation. Circular duplexes with bound HU show much greater propensity than protein-free DNA to exist as negatively supercoiled topoisomers, suggesting a potential role of HU in organizing the bacterial nucleoid. The local bending and undertwisting of DNA by HU, in combination with the number of bound proteins, provide a structural rationale for the condensation of DNA and the observed expression levels of reporter genes in vivo.

Sequence-Dependent Effects in the Cyclization of Short DNA.

A new, computationally efficient Monte Carlo approach has been developed to estimate the ring-closure properties of short, realistically modeled DNA chains. The double helix is treated at the level of base-pair steps using an elastic potential that accounts for the sequence-dependent variability in the intrinsic structure and elastic moduli of the base-pair steps, including the known coupling of conformational variables. Rather than using traditional Metropolis-Monte Carlo techniques to generate representative configurations, a Gaussian sampling method is introduced to construct three-dimensional structures from linear combinations of the rigid-body parameters defining the relative orientation and displacement of successive base pairs. The computation of the J factor, the well-known ratio of the equilibrium constants for cyclization vs bimolecular association of a linear molecule, takes into account restrictions on the displacement and directions of the base pairs joined in ring closure, including the probability that the end-to-end vector is null and the terminal base pairs coincide. The increased sample sizes needed to assess the likelihood that very short chains satisfy these criteria are attained using the Alexandrowicz half-chain sampling enhancement technique in combination with selective linkage of the two-half-chain segments. The method is used to investigate the cyclization properties of arbitrary-length DNA with greatly enhanced sampling sizes, i.e., O(10(14)) configurations, and to estimate J factors lower than 0.1 pM with high accuracy. The methodology has been checked against classic theoretical predictions of the cyclization properties of an ideal, inextensible, naturally straight, DNA elastic rod and then applied to investigate the extent to which one can account for the unexpectedly large J factors of short DNA chains without the need to invoke significant distortions of double helical structure. Several well-known structural features of DNA [Formula: see text] including the presence of intrinsic curvature, roll-twist coupling, or enhanced pyrimidine-purine deformability [Formula: see text] bring the computed J factors in line with the observed data. Moreover, periodically distributed roll-twist coupling reduces the magnitude of oscillations in J, seen in plots of J vs chain length, to the extent found experimentally.