Experimental mapping of DNA duplex shape enabled by global lineshape analyses of a nucleotide-independent nitroxide probe

Sequence-dependent variation in structure and dynamics of a DNA duplex, collectively referred to as ‘DNA shape’, critically impacts interactions between DNA and proteins. Here, a method based on the technique of site-directed spin labeling was developed to experimentally map shapes of two DNA duplexes that contain response elements of the p53 tumor suppressor. An R5a nitroxide spin label, which was covalently attached at a specific phosphate group, was scanned consecutively through the DNA duplex. X-band continuous-wave electron paramagnetic resonance spectroscopy was used to monitor rotational motions of R5a, which report on DNA structure and dynamics at the labeling site. An approach based on Pearson''s coefficient analysis was developed to collectively examine the degree of similarity among the ensemble of R5a spectra. The resulting Pearson''s coefficients were used to generate maps representing variation of R5a mobility along the DNA duplex. The R5a mobility maps were found to correlate with maps of certain DNA helical parameters, and were capable of revealing similarity and deviation in the shape of the two closely related DNA duplexes. Collectively, the R5a probe and the Pearson''s coefficient-based lineshape analysis scheme yielded a generalizable method for examining sequence-dependent DNA shapes.     

Molecular modeling and structural studies of 12-mer immobile four-way DNA junction in solution

Molecular modelling and structural studies of 12-mer immobile four-way DNA junction model is reported here. The DNA junction which was built and investigated, consisted of the following sequences 5''d(GGAAGGGGCTGG), 5''d(CCAGCCTGAGCC), 5''d(GGCTCAACTCGG) and 5''d(CCGAGTCCTTCC). The model was made in such a way that the junction may lack two-fold sequence symmetry at the crossover point. A new version of the AMBER force field has been used, in addition to the Particle Mesh Ewald (PME) method which deals with the refinement treatment of the long range interaction potentials, the well known limitation in MD protocol. After molecular dynamics simulation the backbone parameters and helical parameters of the DNA junction model is calculated and its dynamical pathway is discussed. A close observation near the junction point reveals the shifting in the orientation of some of the P-O bonds from the usual π3 turn for A- and B- DNA to either π1 or π2 type of turn in order to achieve conformational stability. With this study it seems possible to derivatize synthetic DNA molecules with special functional groups both on the bases and at the backbones as in the case of some natural processes by which drugs, particular proteins etc. recognizes and binds to the specific sites of DNA.     

Molecular Dynamics Simulations of a Nucleosome and Free DNA

Abstract

All atom molecular dynamics simulations (10ns) of a nucleosome and of its 146 basepairs of DNA free in solution have been conducted. DNA helical parameters (Roll, Tilt, Twist, Shift, Slide, Rise) were extracted from each trajectory to compare the conformation, effective force constants, persistence length measures, and fluctuations of nucleosomal DNA to free DNA. The conformation of DNA in the nucleosome, as determined by helical parameters, is found to be largely within the range of thermally accessible values obtained for free DNA. DNA is found to be less flexible on the nucleosome than when free in solution, however such measures are length scale dependent. A method for disassembling and reconstructing the conformation and dynamics of the nucleosome using Fourier analysis is presented. Long length variations in the conformation of nucleosomal DNA are identified other than those associated with helix repeat. These variations are required to create a proposed tetrasome conformation or to qualitatively reconstruct the 1.75 turns of the nucleosome's superhelix. Reconstruction of free DNA using selected long wavelength variations in conformation can produce either a left-handed or a right-handed superhelix. The long wavelength variations suggest 146 basepairs is a natural length of DNA to wrap around the histone core.     

Structural Properties of Hybrid Triplex of Polycation Deoxyribonucleic S-methylthiourea (DNmt) Strands with a Complementary DNA Strand,Probed by Nanosecond Molecular Dynamics

Abstract

The replacement of phosphodiester linkages of the polyanion DNA with S-methylthiourea linkers provides the polycation deoxyribonucleic S-methylthiourea (DNmt). Molecular dynamics studies to 1,220 ps of the hybrid triplex formed from octameric DNmt strands d(Tmt)₈ with a complementary DNA oligomer strand d(Ap)₈ have been carried out with explicit water solvent and Na counterions under periodic boundary conditions using the CHARMM force field and the Ewald summation method. The Watson-Crick and Hoogsteen hydrogen-bonding patterns of the A/T tracts remained intact without any structural restraints for triplex structures throughout the simulation. The duplex portion of the triplex structure equilibrated at a B-DNA conformation in terms of the helical rise and other helical parameters. The dynamic structures of the DNmt·DNA·DNmt triplex were determined by examining histograms from the last 800 ps of the dynamics run. These included the hydrogen-bonding pattern (sequence recognition), three-centered bifurcating occurrences, minor groove width variations, and bending of tracts for the hybrid triplex structures. Together with the Watson-Crick hydrogen-bondings, the strong Hoogsteen hydrogen-bondings, the partially maintained three-centered bifurcatings in the Watson-Crick pair, and the medium-strength three-centered bifurcatings in the Hoogsteen pair suggest that the hybrid triplex is energetically favorable as compared to a duplex with similar base stacking, van der Waals interactions, and helical parameters. This is in agreement with our previously reported thermody- namic study, in which only triplex structures were observed in solution. The bending angle measured between the local axis vectors of the first and last helical axis segments is about 20° for the Watson-Crick portion of the averaged structure. Propeller twist (associated with three-centered hydrogen-bonding) up to ?30°, native to DNA AT base pairing, was also observed for the triplex structure. The sugar pseudorotation phase angles and the ring rotation angles for the DNA strand are within the C3′-endo domain and C2′-endo domain for the DNmt strand. Water spines are observed in both minor and major grooves throughout the dynamics run. The molecular dynamics simulations of the structural properties of DNmt·DNA·DNmt hybrid triplex is compared to the DNG·DNA·DNG hybrid triplex (In DNG the -O-(PO₂-)-O- linkers in DNA is replaced by -NH-C(=N₂)-NH-).     

Molecular mechanics model of supercoiled DNA

We describe a pseudo-atomic model of supercoiled DNA. Each base-pair of the DNA is represented in the model by three particles placed in a plane. The particle triplets are stacked to model stacked base-pairs in double-helical DNA, and closed circular conformations are generated to investigate supercoiling. This model is less detailed than all-atom models, which are too computationally demanding to be used to study supercoiling. On the other hand, this model contains details at the base-pair level and is therefore more elaborate than elastomechanical models. A potential energy function is written in terms of a set of internal co-ordinates defined to resemble a limited number of helical parameters. The modeled helical parameters, helical twist, base-roll, tilt and rise, are the most important parameters of the global shape of DNA. Experimentally measured mechanical properties of DNA are used to define the forces holding the particles together. We then use a procedure incorporating energy minimization and molecular dynamics to locate low energy conformations of the model DNA. The model was found to behave very much like rubber-tubing and elastomechanical models. The conformations and the effects of supercoiling pressure (a number proportional to the degree to which the total twist of the DNA has been altered from its natural value) on these conformations are all very similar to those observed in the latter two models. We also used this model to examine the effects of supercoiling pressure, base-sequence and mechanical properties on the conformations and energies of five sequences. The sequences studied include models of naturally straight DNA and DNA with static or natural bends.     

Molecular dynamics simulations of a nucleosome and free DNA

All atom molecular dynamics simulations (10ns) of a nucleosome and of its 146 basepairs of DNA free in solution have been conducted. DNA helical parameters (Roll, Tilt, Twist, Shift, Slide, Rise) were extracted from each trajectory to compare the conformation, effective force constants, persistence length measures, and fluctuations of nucleosomal DNA to free DNA. The conformation of DNA in the nucleosome, as determined by helical parameters, is found to be largely within the range of thermally accessible values obtained for free DNA. DNA is found to be less flexible on the nucleosome than when free in solution, however such measures are length scale dependent. A method for disassembling and reconstructing the conformation and dynamics of the nucleosome using Fourier analysis is presented. Long length variations in the conformation of nucleosomal DNA are identified other than those associated with helix repeat. These variations are required to create a proposed tetrasome conformation or to qualitatively reconstruct the 1.75 turns of the nucleosome's superhelix. Reconstruction of free DNA using selected long wavelength variations in conformation can produce either a left-handed or a right-handed superhelix. The long wavelength variations suggest 146 basepairs is a natural length of DNA to wrap around the histone core.     

Structure and in silico simulations of a cold-active esterase reveals its prime cold-adaptation mechanism

Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7''s closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7''s cold adaptation rather than changes in dynamics.     

Molecular dynamics simulations of xDNA

xDNA is a modified DNA, which contains natural as well as expanded bases. Expanded bases are generated by the addition of a benzene spacer to the natural bases. A set of AMBER force‐field parameters were derived for the expanded bases and the structural dynamics of the xDNA decamer ( xT5 ′ G xT A xC xG C xA xG T3′ ) · ( xA5′ C T xG C G xT A xC A3′) was explored using a 22 ns molecular dynamics simulation in explicit solvent. During the simulation, the duplex retained its Watson‐Crick base‐pairing and double helical structure, with deviations from the starting B‐form geometry towards A‐form; the deviations are mainly in the backbone torsion angles and in the helical parameters. The sugar pucker of the residues were distributed among a variety of modes; C2′ endo, C1′ exo, O4′ endo, C4′ exo, C2′ exo, and C3′ endo. The enhanced stacking interactions on account of the modification in the bases could help to retain the duplex nature of the helix with minor deviations from the ideal geometry. In our simulation, the xDNA showed a reduced minor groove width and an enlarged major groove width in comparison with the NMR structure. Both the grooves are larger than that of standard B‐DNA, but major groove width is larger than that of A‐DNA with almost equal minor groove width. The enlarged groove widths and the possibility of additional hydration in the grooves makes xDNA a potential molecule for various applications. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 351–360, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Molecular dynamics simulation of hydrated d(CGGGTACCCG)4 as a four-way DNA Holliday junction and comparison with the crystallographic structure

Molecular dynamics (MD) simulation of decamer sequence (CGGGTACCCG)₄ as a four-way Holliday junction is reported here for 15.0 ns at three different temperatures 100, 200 and 300 K, respectively, using AMBER force field. Particle mesh Ewald method has been utilised to deal long-range interaction potentials. After MD simulation, various parameters of the junction model including backbone and helical parameters have been worked out and the dynamical pathway is discussed. Structural analysis and geometrical calculations were carried out through X3DNA. The computational results obtained are compared with the previously reported crystallographic outcomes. The width and depth of the major and minor grooves of the duplex of the four arms of the DNA junction have been calculated. The variations in the C1′–C1′ distances between the two complementary strands are discussed in detail. A close observation of the results reveals that the conformation of the average simulated structure at low temperature is of ‘B’ form and the structural integrity of the DNA junction having a twofold sequence symmetry is temperature dependent. It also seems that besides the other parameters (i.e. presence of ions, solvents, etc.), temperature may be playing a key role in preserving the structural integrity of the DNA junction.     

Relationship of DNA structure to internal dynamics: correlation of helical parameters from NOE-based NMR solution structures of d(GCGTACGC)(2) and d(CGCTAGCG)(2) with (13)C order parameters implies conformational coupling in dinucleotide units

The coupling between the conformational properties of double-stranded DNA and its internal dynamics has been examined. The solution structures of the isomeric DNA oligomers d(GCGTACGC)(2) (UM) and d(CGCTAGCG)(2) (CTSYM) were determined with (1)H NMR spectroscopy by utilizing distance restraints from total relaxation matrix analysis of NOESY cross-peak intensities in restrained molecular dynamics calculations. The root-mean-square deviation of the coordinates for the ensemble of structures was 0.13 A for UM and 0.49 A for CTSYM, with crystallographic equivalent R(c)=0.41 and 0.39 and sixth-root residual R(x)=0.11 and 0.10 for UM and CTSYM, respectively. Both UM and CTSYM are B-form with straight helical axes and show sequence-dependent variations in conformation. The internal dynamics of UM and CTSYM were previously determined by analysis of (13)C relaxation parameters in the context of the Lipari & Szabo model-free formalism. Helical parameters for the two DNA oligomers were examined for linear correlations with the order parameters (S(2)) of groups of (13)C spins in base-pairs and dinucleotide units of UM and CTSYM. Correlations were found for six interstrand base-pair parameters tip, y-displacement, inclination, buckle and stretch with various combinations of S(2) for atoms in Watson-Crick base-pairs and for two inter-base-pair parameters, rise and roll with various combinations of S(2) for atoms in dinucleotides. The correlations for the interstrand base-pair helical parameters indicate that the conformations of the deoxyribose residues of each strand are dynamically coupled. Also, the inter-base-pair separation has a profound effect on the local internal motions available to the DNA, supporting the idea that rise is a principal degree of freedom for DNA conformational variability. The correlations indicate collective atomic motions of spins that may represent specific motional modes in DNA, and that base sequence has a predictable effect on the relative order of groups of spins both in the bases and in the deoxyribose ring of the DNA backbone. These observations suggest that an important functional outcome of DNA base sequence is the modulation of both the conformation and dynamic behavior of the DNA backbone.     

CORGEN: A FORTRAN-77 generator of standard and non-standard DNA helices from the sequence

An analytical procedure CORGEN generates a variety of DNA double-strandedstructures from user-supplied sequence using a nucleic aciddatabase incorporated into a standard FORTRAN-77 program. Alternatively,the cylindrical polar coordinates of DNA components may be suppliedfrom the external table. An algorithm that performs intercalationsites in DNA is described. This procedure can be used to generatecomplexes of antibiotics with DNA. Non-standard DNA structurescan be built by alternating the global helical twist and globalhelical rise in the regular DNA helix. The procedures describedcan be used for computer generation of a variety of non-standardDNA structures which can be subjected to molecular mechanicsand dynamics simulations. Received on February 20, 1989; accepted on June 27, 1989     

Parallel Double Helices of DNA. Conformational Analysis of Regular Helices with the Second Order Symmetry Axis

Abstract

We have performed a conformational analysis of DNA double helices with parallel directed backbone strands connected with the second order symmetry axis being at the same time the helix axis. The calculations were made for homopolymers poly(dA) · poly(dA), poly(dC) · poly(dC), poly(dG) poly(dG), and poly(dT) · poly(dT). All possible variants of hydrogen bonding of base pairs of the same name were studied for each polymer. The maps of backbone chain geometrical existence were constructed. Conformational and helical parameters corresponding to local minima of conformational energy of “parallel” DNA helices, calculated at atom-atom approximation, were determined. The dependence of conformational energy on the base pair and on the hydrogen bond type was analysed. Two major conformational advantageous for “parallel” DNA's do not depend much on the hydrogen-bonded base pair type were indicated. One of them coincided with the conformational region typical for “antiparallel” DNA in particular for the B-form DNA Conformational energy of “parallel” DNA depends on the base pair type and for the most part is similar to the conformational energy of “antiparallel” B-DNA.     

A method for genome-wide analysis of DNA helical tension by means of psoralen–DNA photobinding

The helical tension of chromosomal DNA is one of the epigenetic landmarks most difficult to examine experimentally. The occurrence of DNA crosslinks mediated by psoralen photobinding (PB) stands as the only suitable probe for assessing this problem. PB is affected by chromatin structure when is done to saturation; but it is mainly determined by DNA helical tension when it is done to very low hit conditions. Hence, we developed a method for genome-wide analysis of DNA helical tension based on PB. We adjusted in vitro PB conditions that discern DNA helical tension and applied them to Saccharomyces cerevisiae cells. We selected the in vivo cross-linked DNA sequences and identified them on DNA arrays. The entire procedure was robust. Comparison of PB obtained in vivo with that obtained in vitro with naked DNA revealed that numerous chromosomal regions had deviated PB values. Similar analyses in yeast topoisomerase mutants uncovered further PB alterations across specific chromosomal domains. These results suggest that distinct chromosome compartments might confine different levels of DNA helical tension in yeast. Genome-wide analysis of psoralen–DNA PB can be, therefore, a useful approach to uncover a trait of the chromosome architecture not amenable to other techniques.     

Radical-induced purine lesion formation is dependent on DNA helical topology

Abstract

Herein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO^?) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5′,8-cyclo-2′-deoxyguanosine (5′,8-cdG) and 5′,8-cyclo-2′-deoxyadenosine (5′,8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT]₄ (TG4T), d[AGGG(TTAGGG)₃] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG)₃A] (mutTel24) were exposed to HO^? radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L?>?OC???SC indicating greater damage towards the extended B-DNA topology. Conversely, G-quadruplex sequences were significantly more resistant toward purine oxidation in their unfolded states as compared with G-tetrad folded topologies; this effect is confirmed upon comparative analysis of Tel22 (～50% solution folded) and mutTel24 (～90% solution folded). In an effort to identify the accessibly of hydroxyl radicals to quadruplex purine nucleobases, G-quadruplex solvent cavities were then modeled at 1.33?Å with evidence suggesting that folded G-tetrads may act as potential oxidant traps to protect against chromosomal DNA damage.     

Binding of Net-Fla,a Netropsin-Flavin Hybrid Molecule,to DNA: Molecular Mechanics and Dynamics Studies in vacuo and in Water Solution

Abstract

We have studied the binding of the hybrid netropsin-flavin (Net-Fla) molecule onto four sequences containing four A.T base pairs. Molecular mechanics minimizations in vacuo show numerous minimal conformations separated by one base pair. 400 ps molecular dynamics simulations in vacuo have been performed using the lowest minima as the starting conformations. During these simulations, the flavin moiety of the drug makes two hydrogen bonds with an amino group of a neighboring guanine. A 200 ps molecular dynamics simulation in explicit water solution suggests that the binding of Net-Fla upon the DNA substrate is enhanced by water bridges. A water molecule bridging the amidinium of Net-Fla to the N3 atom of an adenine seems to be stuck in the dmg-DNA complex during the whole simulation. The fluctuations of the DNA helical parameters and of the torsion angles of the sugar-phosphate backbone are very similar in the simulations in vacuo and in water. The time auto-correlation functions for the DNA helical parameters decrease rapidly in the picosecond range in vacuo. The same functions computed from the water solution molecular dynamics simulations seem to have two modes: the rapid mode is similar to the behavior in vacuo, and is followed by a slower mode in the 10 ps range.     

Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL₅) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL₅ ↔ FL₅ mechanism, in which the final helical state, FL₅, forms from IL₅ with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.     

Sampling techniques and the use of Tang's procedure in insect population dynamics studies

Some calculations were performed usingTang 's method as an aid in planning experiments for studying the population dynamics of the Jeffrey pine beetle. The population dynamics studies were aimed at detecting the importance of specific effects, e. g., tree diameter, tree height. TheTang procedure is a method of estimating the sample size required to detect effects of a given magnitude with analysis of variance tests. Using this procedure some sample calculations were performed which indicated the sample size needed, and the efficacy of different strategies of improving the results, e. g., increasing the number of trees sampled versus increasing the area of the tree sampled. The statistical parameters used in the calculations were estimated from some preliminary sampling data. Use of this procedure is recommended in insect population studies as a method of optimally planning experiments, and as a method of making precise conclusions about the significance of specific effects.     

Catalytic Analysis of APOBEC3G Involving Real-Time NMR Spectroscopy Reveals Nucleic Acid Determinants for Deamination

APOBEC3G (A3G) is a single-stranded DNA-specific cytidine deaminase that preferentially converts cytidine to uridine at the third position of triplet cytosine (CCC) hotspots. A3G restricts the infectivity of viruses, such as HIV-1, by targeting CCC hotspots scattered through minus DNA strands, reverse-transcribed from genomic RNA. Previously, we developed a real-time NMR method and elucidated the origin of the 3''→5'' polarity of deamination of DNA by the C-terminal domain of A3G (CD2), which is a phenomenon by which a hotspot located closer to the 5''-end is deaminated more effectively than one less close to the 5''-end, through quantitative analysis involving nonspecific binding to and sliding along DNA. In the present study we applied the real-time NMR method to analyze the catalytic activity of CD2 toward DNA oligonucleotides containing a nucleotide analog at a single or multiple positions. Analyses revealed the importance of the sugar and base moieties throughout the consecutive 5 nucleotides, the CCC hotspot being positioned at the center. It was also shown that the sugar or base moieties of the nucleotides outside this 5 nucleotide recognition sequence are also relevant as to CD2''s activity. Analyses involving DNA oligonucleotides having two CCC hotspots linked by a long sequence of either deoxyribonucleotides, ribonucleotides or abasic deoxyribonucleotides suggested that the phosphate backbone is required for CD2 to slide along the DNA strand and to exert the 3''→5'' polarity. Examination of the effects of different salt concentrations on the 3''→5'' polarity indicated that the higher the salt concentration, the less prominent the 3''→5'' polarity. This is most likely the result of alleviation of sliding due to a decrease in the affinity of CD2 with the phosphate backbone at high salt concentrations. We also investigated the reactivity of substrates containing 5-methylcytidine (5mC) or 5-hydroxymethylcytidine, and found that A3G exhibited low activity toward 5mC.