[Opposite Effects of Histone H1 and HMGN5 Protein on Distant Interactions in Chromatin].

Transcriptional enhancers in the cell nuclei typically interact with the target promoters in cis over long stretches of chromatin, but the mechanism of this communication remains unknown. Previously we have developed a defined in vitro system for quantitative analysis of the rate of distant enhancer-promoter communication (EPC) and have shown that the chromatin fibers maintain efficient distant EPC in cis. Here we investigate the roles of linker histone H1 and HMGN5 protein in EPC. A considerable negative effect of histone H1 on EPC depending on its C- and N-tails was shown. Protein HMGN5 that affects chromatin compaction and is associated with active chromatin counteracts EPC inhibition by H1. The data suggest that the efficiency of the interaction between the enhancer and the promoter depends on the structure and dynamics of the chromatin fiber localized between them and can be regulated by proteins associated with chromatin.

Nucleosome positioning and composition modulate in silico chromatin flexibility.

The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes-the familiar assemblies of ∼150 DNA base pairs and eight histone proteins-found on chromatin fibers. Here we introduce a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs. We explore the effects of nucleosome positioning and the presence or absence of cationic N-terminal histone tails on the 'local' inter-nucleosomal interactions and the global deformations of the simulated chains. The correspondence between the predicted and observed effects of nucleosome composition and numbers on the long-range communication between the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We also extract effective nucleosome-nucleosome potentials from the simulations and implement the potentials in a larger-scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable effect of nucleosome spacing on chromatin flexibility, with small changes in DNA linker length significantly altering the interactions of nucleosomes and the dimensions of the fiber as a whole. In addition, we find that these changes in nucleosome positioning influence the statistical properties of long chromatin constructs. That is, simulated chromatin fibers with the same number of nucleosomes exhibit polymeric behaviors ranging from Gaussian to worm-like, depending upon nucleosome spacing. These findings suggest that the physical and mechanical properties of chromatin can span a wide range of behaviors, depending on nucleosome positioning, and that care must be taken in the choice of models used to interpret the experimental properties of long chromatin fibers.

Base sequence effects in bending induced by bulky carcinogen-DNA adducts: experimental and computational analysis.

The covalent binding of bulky mutagenic or carcinogenic compounds to DNA can lead to bending, which could significantly alter the interactions of DNA with critical replication and transcription proteins. The impact of adducts derived from the highly reactive bay region enantiomeric (+)- and (-)-anti-7,8-diol-9,10-epoxide derivatives of benzo[a]pyrene (BPDE) are of interest because the (+)-7R,8S,9S,10R-anti-BPDE enantiomer is highly tumorigenic in rodents, while the (-)-7S,8R,9R,10S-anti-BPDE enantiomer is not. Both (+)- and (-)-anti-BPDE bind covalently with DNA predominantly by trans addition at the exocyclic amino group of guanine to yield 10S (+)- and 10R (-)-trans-anti-[BP]-N(2)-dG adducts. We have synthesized a number of different oligonucleotides with single (+)- and (-)-trans-anti-[BP]-N(2)-dG adducts (G) in the base sequence context XG*Y, where X and Y are different DNA bases. The G* residues were positioned at or close to the center of 11 base pair ( approximately 1 helical turn) or 16 base pair ( approximately 1.5 turns) duplexes. All bases, except for X and Y and their partners, were identical. These sequences were self-ligated with T4 ligase to form multimers that yield a ladder of bands upon electrophoresis in native polyacrylamide gels. The extent of bending in each oligonucleotide was assessed by monitoring the decrease in gel mobilities of these linear, self-ligated oligomers, relative to unmodified oligonucleotides of the same base sequence. The extent of global bending was then estimated using a sequence-specific three-dimensional model from which the values of the base-pair step parameter roll adjacent to the lesion site could be extracted. We find that (+)-trans-anti-[BP]-N(2)-dG adducts are considerably more bent than the (-) isomers regardless of sequence and that A-T base pairs flanking the [BP]-N(2)-dG lesion site allow for local flexibility consistent with adduct conformational heterogeneity. Interestingly, the fit of computed versus observed gel mobilities using classical reptation treatments requires enhancement of unmodified DNA flexibility in gels, compared to aqueous salt solution. The differences in bending between the two stereoisomeric adduct duplexes and the observed base sequence context effects may play a significant role in the differential processing of these lesions by cellular replication, transcription, and repair enzymes.

Tightness of random knotting.

Long polymers in solution frequently adopt knotted configurations. To understand the physical properties of knotted polymers, it is important to find out whether the knots formed at thermodynamic equilibrium are spread over the whole polymer chain or rather are localized as tight knots. We present here a method to analyze the knottedness of short linear portions of simulated random chains. Using this method, we observe that knot-determining domains are usually very tight, so that, for example, the preferred size of the trefoil-determining portions of knotted polymer chains corresponds to just seven freely jointed segments.

A-form conformational motifs in ligand-bound DNA structures.

Recognition and biochemical processing of DNA requires that proteins and other ligands are able to distinguish their DNA binding sites from other parts of the molecule. In addition to the direct recognition elements embedded in the linear sequence of bases (i.e. hydrogen bonding sites), these molecular agents seemingly sense and/or induce an "indirect" conformational response in the DNA base-pairs that facilitates close intermolecular fitting. As part of an effort to decipher this sequence-dependent structural code, we have analyzed the extent of B-->A conformational conversion at individual base-pair steps in protein and drug-bound DNA crystal complexes. We take advantage of a novel structural parameter, the position of the phosphorus atom in the dimer reference frame, as well as other documented measures of local helical structure, e.g. torsion angles, base-pair step parameters. Our analysis pinpoints ligand-induced conformational changes that are difficult to detect from the global perspective used in other studies of DNA structure. The collective data provide new structural details on the conformational pathway connecting A and B-form DNA and illustrate how both proteins and drugs take advantage of the intrinsic conformational mechanics of the double helix. Significantly, the base-pair steps which exhibit pure A-DNA conformations in the crystal complexes follow the scale of A-forming tendencies exhibited by synthetic oligonucleotides in solution and the known polymorphism of synthetic DNA fibers. Moreover, most crystallographic examples of complete B-to-A deformations occur in complexes of DNA with enzymes that perform cutting or sealing operations at the (O3'-P) phosphodiester linkage. The B-->A transformation selectively exposes sugar-phosphate atoms, such as the 3'-oxygen atom, ordinarily buried within the chain backbone for enzymatic attack. The forced remodeling of DNA to the A-form also provides a mechanism for smoothly bending the double helix, for controlling the widths of the major and minor grooves, and for accessing the minor groove edges of individual base-pairs.

Human topoisomerase I poisoning by protoberberines: potential roles for both drug-DNA and drug-enzyme interactions.

Protoberberines represent a structural class of organic cations that induce topoisomerase I-mediated DNA cleavage, a behavior termed topoisomerase I poisoning. We have employed a broad range of biophysical, biochemical, and computer modeling techniques to characterize and cross-correlate the DNA-binding and topoisomerase poisoning properties of four protoberberine analogues that differ with respect to the substituents on their A- and/or D-rings. Our data reveal the following significant features: (i) The binding of the four protoberberines unwinds duplex DNA by approximately 11 degrees, an observation consistent with an intercalative mode of interaction. (ii) Enthalpically favorable interactions, such as stacking interactions between the intercalated ligand and the neighboring base pairs, provide <50% of the thermodynamic driving force for the complexation of the protoberberines to duplex DNA. Computer modeling studies on protoberberine-DNA complexes suggest that only rings C and D intercalate into the host DNA helix, while rings A and B protrude out of the helix interior into the minor groove. (iii) All four protoberberine analogues are topoisomerase I-specific poisons, exhibiting little or no topoisomerase II poisoning activity. (iv) Modifications of the D-ring influence both DNA binding and topoisomerase I poisoning properties. Specifically, transference of a methoxy substituent from the 11- to the 9-position diminishes both DNA binding affinity and topoisomerase I poisoning activity, an observation suggesting that DNA binding is important in the poisoning of topoisomerase I by protoberberines. (v) Modifications of the A-ring have a negligible impact on DNA binding affinity, while exerting a profound influence on topoisomerase I poisoning activity. Specifically, protoberberine analogues containing either 2,3-dimethoxy; 3,4-dimethoxy; or 3, 4-methylenedioxy substituents all bind DNA with a similar affinity. By contrast, these analogues exhibit markedly different topoisomerase I poisoning activities, with these activities following the hierarchy: 3,4-methylenedioxy > 2,3-dimethoxy >> 3, 4-dimethoxy. These differences in topoisomerase I poisoning activity may reflect the differing abilities of the analogues to interact with specific functionalities on the enzyme, thereby stabilizing the enzyme in its cleavable state. In the aggregate, our results are consistent with a mechanistic model in which both ligand-DNA and ligand-enzyme interactions are important for the poisoning of topoisomerase I by protoberberines, with the DNA-directed interactions involving ring D and the enzyme-directed interactions involving ring A. It is reasonable to suggest that the poisoning of topoisomerase I by a broad range of other naturally occurring and synthetic ligands may entail a similar mechanism.

Modeling DNA deformations.

Recent developments have been made in modeling double-helical DNA at four levels of three-dimensional structure: the all-atom level, whereby an oligonucleotide duplex is surrounded by a shroud of solvent molecules; the base-pair level, with explicit backbone atoms; the mesoscopic level, that is, a few hundred base pairs, with the local duplex conformation described by knowledge-based harmonic energy functions; and the scale of several thousand nucleotides, with the duplex described as an ideal elastic rod. Predictions of the sequence-dependent bending and twisting of the double helix, as well as solvent- and force-induced B-->A and over-stretching conformational transitions, are compared with experimental data. These subtle conformational changes are critical to the functioning of the double helix, including its packaging in the close confines of the cell, the mutual fit of DNA and protein in nucleoprotein complexes, and the effective recognition of base pairs in recombination and transcription.

Aminoglycoside binding in the major groove of duplex RNA: the thermodynamic and electrostatic forces that govern recognition.

We use a combination of spectroscopic, calorimetric, viscometric and computer modeling techniques to characterize the binding of the aminoglycoside antibiotic, tobramycin, to the polymeric RNA duplex, poly(rI).poly(rC), which exhibits the characteristic A-type conformation that is conserved among natural and synthetic double-helical RNA sequences. Our results reveal the following significant features: (i) CD-detected binding of tobramycin to poly(rI).poly(rC) reveals an apparent site size of four base-pairs per bound drug molecule; (ii) tobramycin binding enhances the thermal stability of the host poly(rI).poly(rC) duplex, the extent of which decreases upon increasing in Na(+) concentration and/or pH conditions; (iii) the enthalpy of tobramycin- poly(rI).poly(rC) complexation increases with increasing pH conditions, an observation consistent with binding-induced protonation of one or more drug amino groups; (iv) the affinity of tobramycin for poly(rI).poly(rC) is sensitive to both pH and Na(+) concentration, with increases in pH and/or Na(+) concentration resulting in a concomitant reduction in binding affinity. The salt dependence of the tobramycin binding affinity reveals that the drug binds to the host RNA duplex as trication. (v) The thermodynamic driving force for tobramycin- poly(rI).poly(rC) complexation depends on pH conditions. Specifically, at pH< or =6.0, tobramycin binding is entropy driven, but is enthalpy driven at pH > 6.0. (vi) Viscometric data reveal non-intercalative binding properties when tobramycin complexes with poly(rI).poly(rC), consistent with a major groove-directed mode of binding. These data also are consistent with a binding-induced reduction in the apparent molecular length of the host RNA duplex. (vii) Computer modeling studies reveal a tobramycin-poly(rI). poly(rC) complex in which the drug fits snugly at the base of the RNA major groove and is stabilized, at least in part, by an array of hydrogen bonding interactions with both base and backbone atoms of the host RNA. These studies also demonstrate an inability of tobramycin to form a stable low-energy complex with the minor groove of the poly(rI).poly(rC) duplex. In the aggregate, our results suggest that tobramycin-RNA recognition is dictated and controlled by a broad range of factors that include electrostatic interactions, hydrogen bonding interactions, drug protonation reactions, and binding-induced alterations in the structure of the host RNA. These modulatory effects on tobramycin-RNA complexation are discussed in terms of their potential importance for the selective recognition of specific RNA structural motifs, such as asymmetric internal loops or hairpin loop-stem junctions, by aminoglycoside antibiotics and their derivatives.

Pulling chromatin fibers: computer simulations of direct physical micromanipulations.

A low-resolution molecular model, which combines the known mechanical properties of protein-free DNA with the accumulating picture of chromatosome structure, has been developed to account for the stretching of single chromatin fibers by an imposed external force. Force-extension characteristics of sets of chains accumulated by Monte Carlo sampling are consistent with recently observed findings in the non-destructive regime (<20 pN imposed force), where the structure of the chromatosome remains intact. The correspondence between simulation and the relaxation phase of the experiment limits the equilibrium entry-exit angle of linker DNA on the chromatosome to W=50(+/-10) degrees and the effective DNA linker length to L(eff)=40(+/-5) bp. The computed force-extension characteristics are relatively insensitive to other parameters of the model, precluding their accurate estimation. The introduction of an attractive potential between closely spaced nucleosomes reproduces the added initial resistance of single fibers to extension at high salt conditions. The consideration of elastic linkers also improves the fitting of assorted classical measurements of unstressed chromatin structure in solution. The overall picture of chromatin that emerges is an irregular, fluctuating, three-dimensional, zig-zag structure with intact, mechanically stable chromatosome units and deformable linkers. The modeled fiber undergoes large-scale configurational rearrangements without significant perturbation of the constituent chromatosome beads, collapsing into a highly condensed form in response to small (<2kT) inter-nucleosomal attractions.

Protein-directed DNA structure. I. Raman spectroscopy of a high-mobility-group box with application to human sex reversal.

Protein-directed reorganization of DNA underlies mechanisms of transcription, replication, and recombination. A molecular model for DNA reorganization in the regulation of gene expression is provided by the sequence-specific high-mobility-group (HMG) box. Structures of HMG-box complexes with DNA are characterized by expansion of the minor groove, sharp bending toward the major groove, and local unwinding of the double helix. The Raman vibrational signature of such DNA reorganization has been identified in a study of the SRY HMG box, encoded by the human male-determining region of the Y chromosome. We observe in the human SRY-HMG:DNA complex extraordinarily large perturbations to Raman bands associated with vibrational modes of the DNA backbone and accompanying large increases in intensities of Raman bands attributable to base unstacking. In contrast, DNA major-groove binding, as occurs for the bZIP protein GCN4 [Benevides, J. M., Li, T., Lu, X.-J., Srinivasan, A. R., Olson, W. K., Weiss, M. A., and Thomas, G. J., Jr. (2000) Biochemistry 39, 548-556], perturbs the Raman signature of DNA only marginally. Raman markers of minor-groove recognition in the human SRY-HMG:DNA complex are due primarily to perturbation of specific vibrational modes of deoxyribose moieties and presumably reflect desolvation at the nonpolar interface of protein and DNA. These Raman markers may be diagnostic of protein-induced DNA bending and are proposed as a baseline for comparative analysis of mutations in SRY that cause human sex reversal.

Protein-directed DNA structure II. Raman spectroscopy of a leucine zipper bZIP complex.

Mechanisms of transcription may involve protein-directed changes in DNA structure and DNA-directed changes in protein structure. We have employed Raman spectroscopy to characterize vibrational signatures associated with such induced molecular fitting for two classes of transcription factors-the basic leucine-zipper (bZIP) motif and the high-mobility-group (HMG) box-each with a DNA target site. Results for bZIP are described here; findings for the HMG-box are reported in the preceding paper in this issue [Benevides, J. M., Chan, G., Lu, X.-J., Olson, W. K., Weiss, M. A., and Thomas, G. J., Jr. (2000) Biochemistry 39, 537-547]. The yeast activator GCN4 provides a well-studied example of bZIP recognition, wherein B-DNA serves essentially as a template for protein folding. Analysis of Raman spectra of the 57-residue GCN4 bZIP domain, its AP-1 binding site, and their specific complex confirms a DNA-induced increase in alpha-helicity, attributable to folding of GCN4 basic arms with virtually no change in B-DNA structure, consistent with previous X-ray and NMR structure determinations. The absence of DNA perturbations in the bZIP model contrasts sharply with the HMG box, where DNA structure perturbations predominate. The bZIP and HMG-box models represent two opposing extremes in a range of induced fits identifiable by Raman spectroscopy. Previously characterized lambda repressor/operator complexes [Benevides, J. M., Weiss, M. A., and Thomas, G. J. (1994) J. Biol. Chem. 269, 10869-10878] occupy an intermediate position within this range. A comprehensive tabulation of Raman markers proposed as diagnostic of different protein/DNA recognition motifs is presented. The results are analyzed in terms of available DNA crystal structures (Nucleic Acid Database) to identify details of DNA conformation that correlate with specific Raman recognition markers.

Influence of nucleosome structure on the three-dimensional folding of idealized minichromosomes.

BACKGROUND: The closed circular, multinucleosome-bound DNA comprising a minichromosome provides one of the best known examples of chromatin organization beyond the wrapping of the double helix around the core of histone proteins. This higher level of chain folding is governed by the topology of the constituent nucleosomes and the spatial disposition of the intervening protein-free DNA linkers. RESULTS: By simplifying the protein-DNA assembly to an alternating sequence of virtual bonds, the organization of a string of nucleosomes on the minichromosome can be treated by analogy to conventional chemical depictions of macromolecular folding in terms of the bond lengths, valence angles, and torsions of the chain. If the nucleosomes are evenly spaced and the linkers are sufficiently short, regular minichromosome structures can be identified from analytical expressions that relate the lengths and angles formed by the virtual bonds spanning the nucleosome-linker repeating units to the pitch and radius of the organized quaternary structures that they produce. CONCLUSIONS: The resulting models with 4-24 bound nucleosomes illustrate how a minichromosome can adopt the low-writhe folding motifs deduced from biochemical studies, and account for published images of the 30 nm chromatin fiber and the simian virus 40 (SV40) nucleohistone core. The marked sensitivity of global folding to the degree of protein-DNA interactions and the assumed nucleosomal shape suggest potential mechanisms for chromosome rearrangements upon histone modification.

The hydration of nucleic acid duplexes as assessed by a combination of volumetric and structural techniques.

Using high precision densimetric and ultrasonic measurements, we have determined, at 25 degrees C, the apparent molar volumes PhiV and the apparent molar compressibilities PhiK(S) of four nucleic acid duplexes-namely, the DNA duplex, poly(dIdC)poly(dIdC); the RNA duplex, poly(rA)poly(rU); and the two DNA/RNA hybrid duplexes, poly(rA)poly(dT) and poly(dA)poly(rU). Using available fiber diffraction data on these duplexes, we have calculated the molecular volumes as well as the solvent-accessible surface areas of the constituent charged, polar, and nonpolar atomic groups. We found that the hydration properties of these nucleic acid duplexes do not correlate with the extent and the chemical nature of the solvent-exposed surfaces, thereby suggesting a more specific set of duplex-water interactions beyond general solvation effects. A comparative analysis of our volumetric data on the four duplexes, in conjunction with available structural information, suggests the following features of duplex hydration: (a) The four duplexes exhibit different degrees of hydration, in the order poly(dIdC)poly(dIdC) > poly(dGdC)poly(dGdC) > poly(dAdT)poly(dAdT) approximately poly(dA)poly(dT). (b) Repetitive AT and IC sequences within a duplex are solvated beyond general effects by a spine of hydration in the minor groove, with this sequence-specific water network involving about 8 additional water molecules from the second and, perhaps, even the third hydration layers. (c) Repetitive GC and IC sequences within a duplex are solvated beyond general effects by a "patch of hydration" in the major groove, with this water network involving about 13 additional water molecules from the second and, perhaps, even the third hydration layers. (d) Random sequence, polymeric DNA duplexes, which statistically lack extended regions of repetitive AT, GC, or IC sequences, do not experience such specific enhancements of hydration. Consequently, consistent with our previous observations (T. V. Chalikian, A. P. Sarvazyan, G. E. Plum, and K. J. Breslauer, Biochemistry, 1994, Vol. 33, pp. 2394-2401), duplexes with approximately 50% AT content exhibit the weakest hydration, while an increase or decrease from this AT content causes enhancement of hydration, either due to stronger hydration of the minor groove (an increase in AT content) or due to stronger hydration of the major groove (an increase in GC content). (e) In dilute aqueous solutions, a B-DNA duplex is more hydrated than an A-DNA duplex, a volumetric-based conclusion that is in agreement with previous results obtained on crystals, fibers, and DNA solutions in organic solvent-water mixtures. (f) the A-like, RNA duplex poly(rA)poly(rU) and the structurally similar A-like, hybrid duplex poly(rA)poly(dT), exhibit similar hydration properties, while the structurally distinct A-like, hybrid duplex poly(rA)poly(dT) and non-A-like, hybrid duplex poly(dA)poly(rU) exhibit differential hydration properties, consistent with structural features dictating hydration characteristics. We discuss how volumetric characterizations, in conjunction with structural studies, can be used to describe, define, and resolve the general and sequence/conformation-specific hydration properties of nucleic acid duplexes.

DNA stretching and compression: large-scale simulations of double helical structures.

Computer-simulated elongation and compression of A - and B -DNA structures beyond the range of thermal fluctuations provide new insights into high energy "activated" forms of DNA implicated in biochemical processes, such as recombination and transcription. All-atom potential energy studies of regular poly(dG).poly(dC) and poly(dA).poly(dT) double helices, stretched from compressed states of 2.0 A per base-pair step to highly extended forms of 7.0 A per residue, uncover four different hyperfamilies of right-handed structures that differ in mutual base-pair orientation and sugar-phosphate backbone conformation. The optimized structures embrace all currently known right-handed forms of double-helical DNA identified in single crystals as well as non-canonical forms, such as the original "Watson-Crick" duplex with trans conformations about the P-O5' and C5'-C4' backbone bonds. The lowest energy minima correspond to canonical A and B -form duplexes. The calculations further reveal a number of unusual helical conformations that are energetically disfavored under equilibrium conditions but become favored when DNA is highly stretched or compressed. The variation of potential energy versus stretching provides a detailed picture of dramatic conformational changes that accompany the transitions between various families of double-helical forms. In particular, the interchanges between extended canonical and non-canonical states are reminiscent of the cooperative transitions identified by direct stretching experiments. The large-scale, concerted changes in base-pair inclination, brought about by changes in backbone and glycosyl torsion angles, could easily give rise to the observed sharp increase in force required to stretch single DNA molecules more than 1.6-1.65 times their canonical extension. Our extended duplexes also help to tie together a number of previously known structural features of the RecA-DNA complex and offer a self-consistent stereochemical model for the single-stranded/duplex DNA recognition brought in register by recombination proteins. The compression of model duplexes, by contrast, yields non-canonical structures resembling the deformed steps in crystal complexes of DNA with the TATA-box binding protein (TBP). The crystalline TBP-bound DNA steps follow the calculated compression-elongation pattern of an unusual "vertical" duplex with base planes highly inclined with respect to the helical axis, exposed into the minor groove, and accordingly accessible for recognition.Significantly, the double helix can be stretched by a factor of two and compressed roughly in half before its computed internal energy rises sharply. The energy profiles show that DNA extension-compression is related not only to the variation of base-pair Rise but also to concerted changes of Twist, Roll, and Slide. We suggest that the high energy "activated" forms calculated here are critical for DNA processing, e.g. nucleo-protein recognition, DNA/RNA synthesis, and strand exchange.

Overview of nucleic acid analysis programs.

We outline the mathematical distinctions among seven of the most popular computer programs currently used to analyze the spatial arrangements of bases and base pairs in nucleic acid helical structures. The schemes fall into three basic categories on the basis of their definitions of rotational parameters: matrix-based, projection-based, and combined matrix- and projection-based. The approaches also define and construct base and base-pair coordinate frames in a variety of ways. Despite these mathematical distinctions, the computed parameters from some programs are strongly correlated and directly comparable. By contrast, other programs which use identical methodologies sometimes yield very different results. The choice of reference frame rather than the mathematical formulation has the greater effect on calculated parameters. Any factor which influences the reference frame, such as fitting or not fitting standard bases to the experimentally derived coordinates, will have a noticeable effect on both complementary base pair and dimer step parameters.

Resolving the discrepancies among nucleic acid conformational analyses.

Growing interest in understanding the relationship between the global folding of nucleic acids and the sequence-dependent structure of individual base-pair steps has stimulated the development of new mathematical methods to define the geometry of the constituent base-pairs. Several approaches, designed to meet guidelines set by the nucleic acid community, permit rigorous comparative analyses of different three-dimensional structures, as well as allow for reconstruction of chain molecules at the base-pair level. The different computer programs, however, yield inconsistent descriptions of chain conformation. Here we report our own implementation of seven algorithms used to determine base-pair and dimer step parameters. Aside from reproducing the results of individual programs, we uncover the reasons why the different algorithms come to conflicting structural interpretations. The choice of mathematics has only a limited effect on the computed parameters, even in highly deformed duplexes. The results are much more sensitive to the choice of reference frame. The disparate schemes yield very similar conformational descriptions if the calculations are based on a common reference frame. The current positioning of reference frames at the inner and outer edges of complementary bases exaggerates the rise at distorted dimer steps, and points to the need for a carefully defined conformational standard.

Excess counterion binding and ionic stability of kinked and branched DNA.

We compute the excess number of counterions associated with kinked and branched DNA, and the ionic stabilities of these structures as a function of chain length and both sodium and magnesium salt concentration, using numerical counterion condensation theory. The DNA structures are modeled as two or more finite lines of phosphate charges radiating from the kink or junction center. The number of excess counterions around the (40-90 degrees) kinked duplex is very small (at most four). The geometries of large three- and four-way DNA junctions (with > 50 base pairs per arm) in solutions containing low to moderate NaCl concentrations, by contrast, accumulate a substantial number of excess sodium ions (> 20) but no more than 15 magnesium counterions. The excess number of counterions surrounding the kinked linear chain and the branched DNA structures either remains invariant or increases with chain length, tending to reach a plateau value. Open configurations, such as the planar Y-shaped three-way junction (with three 120 degrees inter-arm angles) and the 90 degrees cross-shaped four-way junction, are ionically more stable than compact geometries, such as pyramidal three-way junctions and X-shaped four-way junctions, over the entire range of salt concentration considered (10(-5)-10(-1) M NaCl or MgCl2). The ionic stabilities of the compact forms increase with increasing salt concentration and become comparable to those of the extended geometries at high salt (especially when magnesium is the supporting salt).

DNA sequence-dependent deformability deduced from protein-DNA crystal complexes.

The deformability of double helical DNA is critical for its packaging in the cell, recognition by other molecules, and transient opening during biochemically important processes. Here, a complete set of sequence-dependent empirical energy functions suitable for describing such behavior is extracted from the fluctuations and correlations of structural parameters in DNA-protein crystal complexes. These elastic functions provide useful stereochemical measures of the local base step movements operative in sequence-specific recognition and protein-induced deformations. In particular, the pyrimidine-purine dimers stand out as the most variable steps in the DNA-protein complexes, apparently acting as flexible "hinges" fitting the duplex to the protein surface. In addition to the angular parameters widely used to describe DNA deformations (i.e., the bend and twist angles), the translational parameters describing the displacements of base pairs along and across the helical axis are analyzed. The observed correlations of base pair bending and shearing motions are important for nonplanar folding of DNA in nucleosomes and other nucleoprotein complexes. The knowledge-based energies also offer realistic three-dimensional models for the study of long DNA polymers at the global level, incorporating structural features beyond the scope of conventional elastic rod treatments and adding a new dimension to literal analyses of genomic sequences.

Modeling chain folding in protein-constrained circular DNA.

An efficient method for sampling equilibrium configurations of DNA chains binding one or more DNA-bending proteins is presented. The technique is applied to obtain the tertiary structures of minimal bending energy for a selection of dinucleosomal minichromosomes that differ in degree of protein-DNA interaction, protein spacing along the DNA chain contour, and ring size. The protein-bound portions of the DNA chains are represented by tight, left-handed supercoils of fixed geometry. The protein-free regions are modeled individually as elastic rods. For each random spatial arrangement of the two nucleosomes assumed during a stochastic search for the global minimum, the paths of the flexible connecting DNA segments are determined through a numerical solution of the equations of equilibrium for torsionally relaxed elastic rods. The minimal energy forms reveal how protein binding and spacing and plasmid size differentially affect folding and offer new insights into experimental minichromosome systems.