Modeling high-resolution hydration patterns in correlation with DNA sequence and conformation

Hydration around the DNA fragment d(C5T5).(A5G5) is presented from two molecular dynamics simulations of 10 and 12 ns total simulation time. The DNA has been simulated as a flexible molecule with both the CHARMM and AMBER force fields in explicit solvent including counterions and 0.8 M additional NaCl salt. From the previous analysis of the DNA structure B-DNA conformations were found with the AMBER force-field and A-DNA conformations with CHARMM parameters. High-resolution hydration patterns are compared between the two conformations and between C.G and T.A base-pairs from the homopolymeric parts of the simulated sequence. Crystallographic results from a statistical analysis of hydration sites around DNA crystal structures compare very well with the simulation results. Differences between the crystal sites and our data are explained by variations in conformation, sequence, and limitations in the resolution of water sites by crystal diffraction. Hydration layers are defined from radial distribution functions and compared with experimental results. Excellent agreement is found when the measured experimental quantities are compared with the equivalent distribution of water molecules in the first hydration shell. The number of water molecules bound to DNA was found smaller around T.A base-pairs and around A-DNA as compared to B-DNA. This is partially offset by a larger number of water molecules in hydrophobic contact with DNA around T.A base-pairs and around A-DNA. The numbers of water molecules in minor and major grooves have been correlated with helical roll, twist, and inclination angles. The data more fully explain the observed B-->A transition at low humidity.     

Sodium and chlorine ions as part of the DNA solvation shell.

The distribution of sodium and chlorine ions around DNA is presented from two molecular dynamics simulations of the DNA fragment d(C(5)T(5)). (A(5)G(5)) in explicit solvent with 0.8 M additional NaCl salt. One simulation was carried out for 10 ns with the CHARMM force field that keeps the DNA structure close to A-DNA, the other for 12 ns with the AMBER force field that preferentially stabilizes B-DNA conformations (, Biophys. J. 75:134-149). From radial distributions of sodium and chlorine ions a primary ion shell is defined. The ion counts and residence times of ions within this shell are compared between conformations and with experiment. Ordered sodium ion sites were found in minor and major grooves around both A and B-DNA conformations. Changes in the surrounding hydration structure are analyzed and implications for the stabilization of A-DNA and B-DNA conformations are discussed.     

Computational analysis of DNA gyrase action

DNA gyrase introduces negative supercoiling into circular DNA by catalyzing the passage of one DNA segment through another. The efficiency of the reaction is many times higher than that of other topological transformations. We analyze, by a computer simulation, the reaction selectivity for a model of DNA gyrase action that assumes existence of a free loop between the G- and T- DNA segments participating in the reaction. A popular model of this type assumed that the selectivity can be provided by the conformation of the DNA segment wrapped around the enzyme into the right-handed helix turn (G-segment). We simulated the distribution of the reaction products for this model. Equilibrium sets of DNA conformations with one segment of the double helix wrapped around the enzyme were constructed. From these sets we selected conformations that had a second segment properly juxtaposed with the first one. Assuming that the juxtapositions result in the strand-passing reaction, we calculated the reaction products for all these conformations. The results show that different products have to be formed if the enzyme acts according to the model. This conclusion can be extended for any model with a free loop between the G- and T-segments. An alternative model that is consistent with the major experimental observations and the computational analysis, is suggested.     

Solvent bridging sites in A- and B-DNA helices

Nucleotide hydration is important for the understanding of the stability of and the transitions between the different helical conformations of DNA. We have used energy minimization and geometric criteria in order to look for possible sites for solvent which can bridge more than one polar or charged atomic group on a nucleotide. Such bridging sites between phosphate groups have been seen experimentally and used to explain the A to B transition. We show that these phosphate bridging sites occur at energy minima around A-DNA but do not occur around B-DNA. We also find that there are further low energy bridging sites which depend on sequence and which enable the more economical hydration of the A form.     

The N-terminal Tails of Histones H2A and H2B Adopt Two Distinct Conformations in the Nucleosome with Contact and Reduced Contact to DNA

The nucleosome comprises two histone dimers of H2A-H2B and one histone tetramer of (H3-H4)₂, wrapped around by ~145 bp of DNA. Detailed core structures of nucleosomes have been established by X-ray and cryo-EM, however, histone tails have not been visualized. Here, we have examined the dynamic structures of the H2A and H2B tails in 145-bp and 193-bp nucleosomes using NMR, and have compared them with those of the H2A and H2B tail peptides unbound and bound to DNA. Whereas the H2A C-tail adopts a single but different conformation in both nucleosomes, the N-tails of H2A and H2B adopt two distinct conformations in each nucleosome. To clarify these conformations, we conducted molecular dynamics (MD) simulations, which suggest that the H2A N-tail can locate stably in either the major or minor grooves of nucleosomal DNA. While the H2B N-tail, which sticks out between two DNA gyres in the nucleosome, was considered to adopt two different orientations, one toward the entry/exit side and one on the opposite side. Then, the H2A N-tail minor groove conformation was obtained in the H2B opposite side and the H2B N-tail interacts with DNA similarly in both sides, though more varied conformations are obtained in the entry/exit side. Collectively, the NMR findings and MD simulations suggest that the minor groove conformer of the H2A N-tail is likely to contact DNA more strongly than the major groove conformer, and the H2A N-tail reduces contact with DNA in the major groove when the H2B N-tail is located in the entry/exit side.     

Choreography for nucleosomes: the conformational freedom of the nucleosomal filament and its limitations

Eukaryotic DNA is organized into nucleosomes by coiling around core particles of histones, forming a nucleosomal filament. The significance for the conformation of the filament of the DNA entry/exit angle (α) at the nucleosome, the angle of rotation (β) of nucleosomes around their interconnecting DNA (linker DNA) and the length of the linker DNA, has been studied by means of wire models with straight linkers. It is shown that variations in α and β endow the filament with an outstanding conformational freedom when α is increased beyond 60–90°, owing to the ability of the filament to change between forward right-handed and backward left-handed coiling. A wealth of different helical and looped conformations are formed in response to repeated β sequences, and helical conformations are shown to be able to contract to a high density and to associate pairwise into different types of double fibers. Filaments with random β sequences are characterized by relatively stable loop clusters connected by segments of higher flexibility. Displacement of core particles along the DNA in such fibers, combined with limited twisting of the linkers, can generate the β sequence necessary for compaction into a regular helix, thus providing a model for heterochromatinization.     

Structures of Oligonucleotides Containing 5-(methoxymethyl)-2′-deoxyuridine Determined by NMR Spectroscopy

Abstract

Base pairing of 5-(methoxymethyl)-2′-deoxyuridine (MMdU) opposite either adenine or guanine in a seven base pair oligonucleotide duplex has been studied by NMR spectroscopy. When paired with A, we observe that the MMdU. Abase pair adopts Watson-Crick geometry. The methoxymethyl substituent is not held in a fixed conformation and may rotate around the C5-CH₂ and CH₂?O bonds. Examination of the potential energy as a function of rotation around these bonds indicates the presence of four low energy conformations. No hydrogen bonding is indicated for the methoxymethyl substituent, and the four potential minima result from reduced steric clash. For the MMdU. G base pair, the two bases adopt a wobble geometry which does not change with increasing solvent pH. Similarly, we find four low energy conformations for the methoxymethyl substituent in the major groove of the DNA helix.     

A molecular simulation picture of DNA hydration around A- and B-DNA

Recent results from molecular dynamics (MD) simulations on hydration of DNA with respect to conformation are reviewed and compared with experimental data. MD simulations of explicit solvent around DNA can now give a detailed model of DNA that not only matches well with the experimental data but provides additional insight beyond current experimental limitations. Such simulation results are analyzed with a focus on differential hydration properties between A- and B-DNA and between C/G and A/T base pairs. The extent of hydration is determined from the number of waters in the primary shell and compared to experimental numbers from different measurements. High-resolution hydration patterns around the whole DNA are shown and correlated with the conformations. The role of ions associating with DNA is discussed with respect to changes in the hydration structure correlating with DNA conformation.     

Flexibility of duplex DNA on the submicrosecond timescale

Using a site-specific, Electron Paramagnetic Resonance (EPR)-active spin probe that is more rigidly locked to the DNA than any previously reported, the internal dynamics of duplex DNAs in solution were studied. EPR spectra of linear duplex DNAs containing 14-100 base pairs were acquired and simulated by the stochastic Liouville equation for anisotropic rotational diffusion using the diffusion tensor for a right circular cylinder. Internal motions have previously been assumed to be on a rapid enough time scale that they caused an averaging of the spin interactions. This assumption, however, was found to be inconsistent with the experimental data. The weakly bending rod model is modified to take into account the finite relaxation times of the internal modes and applied to analyze the EPR spectra. With this modification, the dependence of the oscillation amplitude of the probe on position along the DNA was in good agreement with the predictions of the weakly bending rod theory. From the length and position dependence of the internal flexibility of the DNA, a submicrosecond dynamic bending persistence length of around 1500 to 1700 A was found. Schellman and Harvey (Biophys. Chem. 55:95-114, 1995) have estimated that, out of the total persistence length of duplex DNA, believed to be about 500 A, approximately 1500 A is accounted for by static bends and 750 A by fluctuating bends. A measured dynamic persistence length of around 1500 A leads to the suggestion that there are additional conformations of the DNA that relax on a longer time scale than that accessible by linear CW-EPR. These measurements are the first direct determination of the dynamic flexibility of duplex DNA in 0.1 M salt.     

Interpretation of DNA vibration modes: III--The behaviour of the sugar pucker vibration modes as a function of its pseudorotation parameters

A systematic study of the sugar pucker characteristic vibration modes as a function of its geometrical conformations, has been performed. The present investigation is based on the Wilson GF method and a non-redundant valence force field. The calculated results allow to assign the modes arising mainly from the sugar motions and present in quasi whole vibrational spectra related to the right or left-handed double-helices (i.e., 1050 cm-1, 960 cm-1 and 890 cm-1). Moreover, the conformation dependent modes as those at 860 cm-1 and around 810 cm-1 (A form) as well as the one located around 830 cm-1 (B form) are interpreted by the present investigation. The possibility of the interaction of the latter modes with the phosphate group motions along the DNA double-helical chains are also discussed.     

Human TFIID Binds to Core Promoter DNA in a Reorganized Structural State

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Highlights? TFIID has distinct conformations with different positions of the TBP-containing lobe ? A rearranged state of TFIID interacts with promoter DNA in a TFIIA-dependent manner ? TFIID induces multiple topological changes on promoter DNA around the start site ? The different conformations of TFIID may serve as targets for regulatory factors     

Interaction of DNA with lysine-rich polypeptides and proteins. The influence of polypeptide composition and secondary structure

Using X-ray diffraction we have studied fibres obtained from complexes of DNA with lysine-rich polypeptides and with proteins that have different conformations, to ascertain whether the conformations of the polypeptides and the DNA are maintained upon interaction. Substances investigated include N-acetyl-Lys-Ala-Tyr-Ala-Lys-ethylamide, random poly(Leu50, Lys50), sequential poly(Leu-Lys), poly(Val-Lys), poly(Ala-Lys), poly(Lys-Ala-Ala-Lys), poly(Lys-Ala-Ala), poly(Lys-Leu-Ala), poly(Lys-Ala-Gly), protein phi 0 from sea cucumber spermatozoa, histone H1 and two fragments of this protein obtained by chemical cleavage. In general, the B form of DNA with ten base-pairs per helical turn is maintained upon interaction at high levels of humidity. The A form is never observed; it appears to be forbidden in a protein environment. No evidence for transition into any novel DNA conformation has been observed, although the B form is altered in some cases, in particular upon dehydration. Such alteration occurs always in the sense of tightening the double helix, so that the number of base-pairs per helical turn diminishes. The polypeptides may interact with DNA in both the alpha and beta conformations. We have found different types of complexes in which either a monolayer or a double layer of beta-pleated sheets is intercalated between layers of DNA molecules. Alternatively, the polypeptide chain may be wrapped around the DNA, following one of the grooves. The polypeptide conformation may be either maintained or changed upon interaction. The charge density of the polypeptide is an important parameter of the interaction. When it matches the charge density of the DNA, the polypeptide conformation is maintained in most cases; otherwise it is modified. The globular part of histone H1 gives a unique X-ray pattern upon interaction, indicative of a loss of order of DNA in the complex. On the other hand, the C-terminal part of histone H1 gives a very well-ordered complex, similar to a nucleoprotamine, in spite of its lower charge density.     

An FT-IR study of DNA and RNA conformational transitions at low temperatures

Fourier Transform Infrared (FT-IR) spectra of solid samples of DNA and RNA obtained from freeze-drying at solid CO2 and liquid nitrogen temperatures, have been recorded and correlation between the conformational transitions and spectral changes is proposed. It is concluded that an equilibrium exists between A, B and Z conformations at low temperatures for the DNA molecule, which is temperature dependent, whereas the RNA molecule exhibits only the A conformation. The results have been compared with the metal-adducts of DNA and RNA, where one of the conformations is predominant. Marker infrared bands for the B conformer have been found to be the strong band at 825 cm-1 (sugar conformer mode) and a band with medium intensity at 690 cm-1 (guanine breathing mode). The A conformation showed characteristic bands at 810 and 675 cm-1. The B to Z conformational transition was characterized by the strong absorption bands near 820-810 cm-1 and at 665-600 cm-1.     

Sequence dependent hydration of DNA

The transitions between the different helical conformations of DNA depend on the base sequence and the ambient conditions such as humidity and counter-ion concentration. In this study energy minimization techniques have been used to locate water molecule sites around nucleotides especially those which form hydrogen bonds between two or more nucleotide atoms and thus form solvent mediated bridges. We have studied several sequences and find that those which are known not to exist in the low hydration ‘A’ form have very similar number of bridging sites in both ‘A’ and ‘B’ conformations. Those sequences which are found in the ‘A’ conformation have considerably more bridging sites in this low hydration form than in the ‘B’ conformation. Sequence related solvent effects for a given conformation have also been analysed.     

Photochemical determination of different DNA structures

The various conformations of DNA are thought to have important biological roles. Investigation of the local DNA conformational changes associated with biological events is therefore essential to an understanding of the functions of DNA. We have reported the photoreactivities of 5-halouracil in the five characteristic local DNA structures: the A, B and Z forms, protein-induced DNA kinks and the G-quadruplex form. These studies demonstrate the detailed relationships between the local DNA structures and the photochemical products of photoinduced hydrogen abstraction by the resulting uracil-5-yl radicals, and show that this photochemical method can be used to detect DNA structures. Here, we describe in detail procedures that have been developed in our laboratory for probing DNA conformations by product analysis of photoirradiated 5-halouracil-containing DNA. The protocol includes the preparation of 5-halouracil-containing DNA and the characterization of the photoproducts, and it can be completed in 2 weeks.     

Non-B DNA conformations analysis through molecular dynamics simulations

BackgroundNon-B DNA conformations are molecular structures that do not follow the canonical DNA double helix. Mutagenetic instability in nuclear and mitochondrial DNA (mtDNA) genomes has been associated with simple non-B DNA conformations, as hairpins or more complex structures, as G-quadruplexes. One of these structures is Structure A, a cloverleaf-like non-B conformation predicted for a 93-nt (nucleotide) stretch of the mtDNA control region 5′-peripheral domain. Structure A is embedded in a hot spot for the 3′ end of human mtDNA deletions revealing its importance in influencing the mutational instability of the mtDNA genome.MethodsTo better characterize Structure A, we predicted its 3D conformation using state-of-art methods and algorithms. The methodologic workflow consisted in the prediction of non-B conformations using molecular dynamics simulations. The conservation scores of alignments of the Structure A region in humans, primates, and mammals, was also calculated.ResultsOur results show that these computational methods are able to measure the stability of non-B conformations by using the level of base pairing during molecular dynamics. Structure A showed high stability and low flexibility correlated with high conservation scores in mammalian, more specifically in primate lineages.ConclusionsWe showed that 3D non-B conformations can be predicted and characterized by our methodology. This allowed the in-depth analysis of the structure A, and the main results showed the structure remains stable during the simulations.General significanceThe fine-scale atomic molecular determination of this type of non-B conformation opens the way to perform computational molecular studies that can show their involvement in mtDNA cellular mechanisms.     

Comparative conformational analysis of cholesterol and ergosterol by molecular mechanics

A comparative conformational analysis of cholesterol and ergosterol has been carried out using molecular mechanics methods. These studies are aimed at giving a better understanding of the molecular nature of the interaction of these sterols with polyene macrolide antibiotics. Structures of cholesterol and ergosterol determined by X-ray methods have been used as initial geometries of these molecules for force field calculations. The calculation of steric energy has also been made for conformations which do not appear in the crystal. The latter conformers have different conformations of the side chain as well as different conformations of rings A and D. The rotational barriers around bonds C17–C20 and C20–C22 have also been calculated. The results obtained on differences and similarities in the conformations of cholesterol and ergosterol allow us to postulate a mechanism for differential interaction with the antibiotics. The relatively rigid side chain of ergosterol (stretched molecule) in comparison with the flexible side chain of cholesterol (bent molecule), allows better intermolecular contact of the first sterol molecule with a polyene macrolide and in consequence facilitates complex formation involving Van der Waal's forces.     

Effect of DNA supercoiling on the geometry of holliday junctions

Unusual DNA conformations including cruciforms play an important role in gene regulation and various DNA transactions. Cruciforms are also the models for Holliday junctions, the transient DNA conformations critically involved in DNA homologous and site-specific recombination, repair, and replication. Although the conformations of immobile Holliday junctions in linear DNA molecules have been analyzed with the use of various techniques, the role of DNA supercoiling has not been studied systematically. We utilized atomic force microscopy (AFM) to visualize cruciform geometry in plasmid DNA with different superhelical densities at various ionic conditions. Both folded and unfolded conformations of the cruciform were identified, and the data showed that DNA supercoiling shifts the equilibrium between folded and unfolded conformations of the cruciform toward the folded one. In topoisomers with low superhelical density, the population of the folded conformation is 50-80%, depending upon the ionic strength of the buffer and a type of cation added, whereas in the sample with high superhelical density, this population is as high as 98-100%. The time-lapse studies in aqueous solutions allowed us to observe the conformational transition of the cruciform directly. The time-dependent dynamics of the cruciform correlates with the structural changes revealed by the ensemble-averaged analysis of dry samples. Altogether, the data obtained show directly that DNA supercoiling is the major factor determining the Holliday junction conformation.     

Theoretical model of the B-Z transition in DNA with an arbitrary sequence

A statistical-mechanical model is suggested that makes it possible to describe the B-Z transition in DNA with an arbitrary sequence of nucleotides. The key point consists in allowance for the fact that each base pair can assume one of the two states with different energies. One of these states corresponds to the standard Z-form with purines in the syn conformation and pyrimidines in the anti conformation. However, in natural DNA sequences such standard base-pair conformations should be interrupted by energetically unfavorable conformations (syn for pyrimidines and anti for purines). Open regions and cruciform structures are also allowed for in the model. The probabilities of formation of the Z-form stretches, open regions and cruciform structures have been calculated for different values of parameters for pBR322 and pAO3 DNA.