Hydration and recognition of methylated CpG steps in DNA.

The analysis of the hydration pattern around methylated CpG steps in three high resolution (1.7, 2.15 and 2.2 A) crystal structures of A-DNA decamers reveals that the methyl groups of cytosine residues are well hydrated. In comparing the native structure with two structurally distinct forms of the decamer d(CCGCCGGCGG) fully methylated at its CpG steps, this study shows also that in certain structural and sequence contexts, the methylated cytosine base can be more hydrated that the unmodified one. These water molecules seem to be stabilized in front of the methyl group through the formation C-H...O interactions. In addition, these structures provide the first observation of magnesium cations bound to the major groove of A-DNA and reveal two distinct modes of metal binding in methylated and native duplexes. These findings suggest that methylated cytosine bases could be recognized by protein or DNA polar residues through their tightly bound water molecules.     

Monte-Carlo simulation of hydration of nucleic acid fragments

Monte-Carlo simulation of the systems containing a stack of 6 complementary base pairs and 180 water molecules has been performed. Characteristic of the hydration shell structure in major and minor grooves has been found for the stacks of repeating A : U and G : C base pairs as well as alternating (A : U, U : A) and (G : C, C : G) ones. Probabilities of the formation of bridges, formed by 1, 2 and 3 water molecules, between hydrophilic centres of the bases have been estimated. One water molecule forms an H-bonded bridge between two adjacent hydrophilic centres with high probability if N...N, N...O or O...O distance between these centres is close to 4.3 A. Hydration shell structure was found to depend significantly on the stack sequence and configuration, while global hydration characteristics (average energy, the number of water-water and water-base H-bonds) are only slightly dependent on the stack sequence and configuration. For the stacks in A conformation the number of water molecules forming more than one H-bonds with the bases is greater in comparison with the stacks in B-like conformation. This result is discussed in connection with the concept of hydration economy during B to A transition.     

Use of the parmbsc0 force field and trajectory analysis to study the binding of netropsin to the DNA fragment (5'CCAATTGG)(2) in the presence of excess NaCl salt in aqueous solution

The parmbsc0 force field was applied to study in detail the binding of netropsin, at a salt concentration of 0.28M Na(+), to the minor groove of an 8-mer (5'CCAATTGG)(2) DNA duplex forming a netropsin·DNA complex which previously has been characterized by X-ray crystallography, albeit with the use of closely related DNA duplexes. The X-ray structure revealed that the terminal guanidinium and amidinium groups of netropsin interact with the extreme ends of the palindromic AATT sequence of the receptor DNA. The parmbsc0 parameters of B-DNA and AMBER v9 parameters of netropsin generated a stable 6ns molecular dynamics (MD) trajectory for a 1:1 class I binding motif of this complex. Trajectory analysis for the salt and hydration effects on the binding of netropsin to the 8-mer DNA duplex revealed that 18 water molecules and 2 Na(+) are displaced from the DNA upon netropsin binding. A hydration density map of the complex parallels the X-ray data showing that two structured water molecules are localized near the netropsin guanidinium and amidinium groups forming H-bond bridges between the receptor and the ligand.     

Radiation-induced DNA damage as a function of hydration. I. Release of unaltered bases.

The release of unaltered bases from irradiated DNA, hydrated between 2.5 and 32.7 mol of water per mole of nucleotide (gamma), was investigated using HPLC. The objective of this study was to elucidate the yield of the four DNA bases as a function of dose, extent of hydration, and the presence or absence of oxygen. The increase in the yield of radiation-induced free bases was linear with dose up to 90 kGy, except for the DNA with gamma = 2.5, for which the increase was linear only to 10 kGy. The yield of free bases as a function of gamma was not constant in either the absence or the presence of oxygen over the range of hydration examined. For DNA with gamma between 2.5 and 15, the yield of free bases was nearly constant under nitrogen, but decreased under oxygen. However, for DNA with gamma greater than 15, the yield increased rapidly under both nitrogen and oxygen. The yield of free bases was described by a model that depended on two factors: 1) a change in the DNA conformation from a mixture of the A and C conformers in vacuum-dried DNA to predominantly the B conformer in the fully hydrated DNA, and 2) the proximity of the water molecules to the DNA. Irradiation of the inner water molecules (gamma less than 15) was less efficient than irradiation of the outer water molecules (gamma greater than 15), by a factor of approximately 3.3, in forming DNA lesions that resulted in the release of an unaltered base. This factor is similar to the previously published relative efficiency of 2.8 with which hydroxyl radicals and base cations induce DNA strand breaks. Our irradiation results are consistent with the hypothesis that the G value for the first 12-15 water molecules of the DNA hydration layer is the same as the G value for the form of DNA to which it is bound (i.e., the pseudo-C or the B form). Thus we suggest that the release of bases originating from irradiation of the hydration water is obtained predominantly: (1) by charge transfer from the direct ionization of the first 12-15 water molecules of the primary hydration layer and (2) by the attack of hydroxyl radicals generated in the outer, more loosely bound water molecules.     

Ordered water structure in an A-DNA octamer at 1.7 A resolution

The crystal structure of the deoxyoctamer d(G-G-Br U-A-BrU-A-C-C) was refined to a resolution of 1.7 A using combined diffractometer and synchrotron data. The analysis was carried out independently in two laboratories using different procedures. Although the final results are identical the comparison of the two approaches highlights potential problems in the refinement of oligonucleotides when only limited data are available. As part of the analysis the positions of 84 solvent molecules in the asymmetric unit were established. The DNA molecule is highly solvated, particularly the phosphate-sugar back-bone and the functional groups of the bases. The major groove contains, in the central BrU-A-BrU-A region, a ribbon of water molecules forming closed pentagons with shared edges. These water molecules are linked to the base O and N atoms and to the solvent chains connecting the O-1 phosphate oxygen atoms on each strand. The minor groove is also extensively hydrated with a continuous network in the central region and other networks at each end. The pattern of hydration is briefly compared with that observed in the structure of a B-dodecamer.     

Structure of the hydration shells of oligo(dA-dT).oligo(dA-dT) and oligo(dA).oligo(dT) tracts in B-type conformation on the basis of Monte Carlo calculations

Monte Carlo simulations [(N, V, T)-ensemble] were performed for the hydration shell of poly(dA-dT).poly(dA-dT) in canonical B form and for the hydration shell of poly(dA).poly(dT) in canonical B conformation and in a conformation with narrow minor groove, highly inclined bases, but with a nearly zero-inclined base pair plane (B' conformation). We introduced helical periodic boundary conditions with a rather small unit cell and a limited number of water molecules to reduce the dimensionality of the configuration space. The coordinates of local maxima of water density and the properties of one- and two-membered water bridges between polar groups of the DNA were obtained. The AT-alternating duplex hydration mirrors the dyad symmetry of polar group distribution. At the dApdT step, a water bridge between the two carbonyl oxygens O2 of thymines is formed as in the central base-pair step of Dickerson's dodecamer. In the major groove, 5-membered water chains along the tetranucleotide pattern d(TATA).d(TATA) are observed. The hydration geometry of poly(dA).poly(dT) in canonical B conformation is distinguished by autonomous primary hydration of the base-pair edges in both grooves. When this polymer adopts a conformation with highly inclined bases and narrow minor groove, the water density distribution in the minor groove is in excellent agreement with Dickerson's spine model. One local maximum per base pair of the first layer is located near the dyad axis between adjacent base pairs, and one local maximum per base pair in the second shell lies near the dyad axis of the base pair itself. The water bridge between the two strands formed within the first layer was observed with high probability. But the water molecules of the second layer do not have a statistically favored orientation necessary for bridging first layer waters. In the major groove, the hydration geometry of the (A.T) base-pair edge resembles the main features of the AT-pair hydration derived from other sequences for the canonical B form. The preference of the B' conformation for oligo(dA).oligo(dT) tracts may express the tendency to common hydration of base-pair edges of successive base pairs in the grooves of B-type DNA. The mean potential energy of hydration of canonical B-DNA was estimated to be -60 to -80 kJ/mole nucleotides in dependence on the (G.C) contents. Because of the small system size, this estimation is preliminary.     

Sequence Recognition of DNA by Protein-Induced Conformational Transitions

The binding of proteins to specific sequences of DNA is an important feature of virtually all DNA transactions. Proteins recognize specific DNA sequences using both direct readout (sensing types and positions of DNA functional groups) and indirect readout (sensing DNA conformation and deformability). Previously we showed that the P22 c2 repressor N-terminal domain (P22R NTD) forces the central non-contacted 5'-ATAT-3' sequence of the DNA operator into the B′ state, a state known to affect DNA hydration, rigidity and bending. Usually the B′ state, with a narrow minor groove and a spine of hydration, is reserved for A-tract DNA (TpA steps disrupt A-tracts). Here, we have co-crystallized P22R NTD with an operator containing a central 5′-ACGT-3′ sequence in the non-contacted region. C·G base pairs have not previously been observed in the B′ state and are thought to prevent it. However, P22R NTD induces a narrow minor groove and a spine of hydration to 5'-ACGT-3'. We observe that C·G base pairs have distinctive destabilizing and disordering effects on the spine of hydration. It appears that the reduced stability of the spine results in a higher energy cost for the B to B′ transition. The differential effect of DNA sequence on the barrier to this transition allows the protein to sense the non-contacted DNA sequence.     

Hydration of B-DNA: comparison between the water network around poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) on the basis of Monte Carlo computations

A computational method is elaborated for studying the water environment around regular polynucleotide duplexes; it allows rigorous structural information on the hydration shell of DNA to be obtained. The crucial aspect of this Monte Carlo simulation is the use of periodical boundary conditions. The output data consists of local maxima of water density in the space near the DNA molecule and the properties of one- and two-membered water bridges as function of pairs of polar groups of DNA. In the present paper the results for poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) are presented. The differences in their hydration shells are of a purely structural nature and are caused by the symmetry of the polar groups of the polymers under study, the symmetry being reflected by the hydration shell. The homopolymer duplex hydration shell mirrors the mononucleotide repeat. The water molecules contacting the polynucleotide in the minor groove are located nearly in the plane midway between the planes of successive base pairs. One water molecule per base pair forms a water bridge facing two polar groups of bases from adjacent base pairs and on different strands making a "spine"-like structure. In contrast, the major groove hydration is stabilized exclusively by two-membered water bridges; the water molecules deepest in the groove are concentrated near the plane of the corresponding base pair. The alternating polymer is characterized by a marked dyad symmetry of the hydration shell corresponding to the axis between two successive base pairs. The minor groove hydration of the dCpdG step resembles the characteristic features of the homopolymer, but the bridge between the O2 oxygens of the other base-stacking type is formed by two water molecules. The major groove hydration is characterized by high probability of one-membered water bridges and by localization of a water molecule on the dyad axis of the dGpdC step. The found structural elements are discussed as reasonable invariants of a dynamic hydration shell.     

Binding of the estrogen receptor to DNA. The role of waters.

Molecular dynamics simulations are carried out to investigate the binding of the estrogen receptor, a member of the nuclear hormone receptor family, to specific and non-specific DNA. Two systems have been simulated, each based on the crystallographic structure of a complex of a dimer of the estrogen receptor DNA binding domain with DNA. One structure includes the dimer and a consensus segment of DNA, ds(CCAGGTCACAGTGACCTGG); the other structure includes the dimer and a nonconsensus segment of DNA, ds(CCAGAACACAGTGACCTGG). The simulations involve an atomic model of the protein-DNA complex, counterions, and a sphere of explicit water with a radius of 45 A. The molecular dynamics package NAMD was used to obtain 100 ps of dynamics for each system with complete long-range electrostatic interactions. Analysis of the simulations revealed differences in the protein-DNA interactions for consensus and nonconsensus sequences, a bending and unwinding of the DNA, a slight rearrangement of several amino acid side chains, and inclusion of water molecules at the protein-DNA interface region. Our results indicate that binding specificity and stability is conferred by a network of direct and water mediated protein-DNA hydrogen bonds. For the consensus sequence, the network involves three water molecules, residues Glu-25, Lys-28, Lys-32, Arg-33, and bases of the DNA. The binding differs for the nonconsensus DNA sequence in which case the fluctuating network of hydrogen bonds allows water molecules to enter the protein-DNA interface. We conclude that water plays a role in furnishing DNA binding specificity to nuclear hormone receptors.     

Life: Self-Directing with Unlimited Variability on Self-Speeding

Abstract

The crystal structure of the deoxyoctamer d(G-G-^Br U-A-^BrU-A-C-C) was refined to a resolution of 1.7Å using combined diffractometer and synchrotron data. The analysis was carried out independently in two laboratories using different procedures. Although the final results are identical the comparison of the two approaches highlights potential problems in the refinement of oligonucleotides when only limited data are available.

As part of the analysis the positions of 84 solvent molecules in the asymmetric unit were established. The DNA molecule is highly solvated, particularly the phosphate-sugar backbone and the functional groups of the bases. The major groove contains, in the central ^BrU-A-^BrU-A region, a ribbon of water molecules forming closed pentagons with shared edges. These water molecules are linked to the base O and N atoms and to the solvent chains connecting the O-1 phosphate oxygen atoms on each strand. The minor groove is also extensively hydrated with a continuous network in the central region and other networks at each end. The pattern of hydration is briefly compared with that observed in the crystal structure of a B-dodecamer.     

Probing the role of interfacial waters in protein–DNA recognition using a hybrid implicit/explicit solvation model

When proteins bind to their DNA target sites, ordered water molecules are often present at the protein–DNA interface bridging protein and DNA through hydrogen bonds. What is the role of these ordered interfacial waters? Are they important determinants of the specificity of DNA sequence recognition, or do they act in binding in a primarily nonspecific manner, by improving packing of the interface, shielding unfavorable electrostatic interactions, and solvating unsatisfied polar groups that are inaccessible to bulk solvent? When modeling details of structure and binding preferences, can fully implicit solvent models be fruitfully applied to protein–DNA interfaces, or must the individualistic properties of these interfacial waters be accounted for? To address these questions, we have developed a hybrid implicit/explicit solvation model that specifically accounts for the locations and orientations of small numbers of DNA‐bound water molecules, while treating the majority of the solvent implicitly. Comparing the performance of this model with that of its fully implicit counterpart, we find that explicit treatment of interfacial waters results in a modest but significant improvement in protein side‐chain placement and DNA sequence recovery. Base‐by‐base comparison of the performance of the two models highlights DNA sequence positions whose recognition may be dependent on interfacial water. Our study offers large‐scale statistical evidence for the role of ordered water for protein–DNA recognition, together with detailed examination of several well‐characterized systems. In addition, our approach provides a template for modeling explicit water molecules at interfaces that should be extensible to other systems. Proteins 2013; 81:1318–1329. © 2013 Wiley Periodicals, Inc.     

Cations Form Sequence Selective Motifs within DNA Grooves via a Combination of Cation-Pi and Ion-Dipole/Hydrogen Bond Interactions

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl⁺) and the polarized first hydration shell waters of divalent cations (Mg²⁺, Ca²⁺) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves.     

Hydration of oligonucleotides in crystals

The solvent molecules found around crystallized oligonucleotides after X-ray refinement are analysed in terms of interaction sites to bases, phosphates and sugars in the three main forms of nucleic acid structures, the A-form, the B-form and the Z-form. The average numbers of contacts to nucleic acid atoms made by solvent molecules are identical in the three forms, but it appears that the average number of contacts solvent molecules make with each other depends on the resolution of the structure. The phosphate anionic oxygen atoms are the most hydrated, while the O(3′) and O(5′) backbone atoms and the ring oxygen atom O(4′) are the least hydrated. Among the hydrophilic atoms of the bases, there is a modulation of the relative water affinities with the nucleic acid form. Numerous hydration sites are such that water molecules can bridge hydrophilic atoms of the same residue, of adjacent residues on the same strand, of distant residues on the two strands, or belonging to symmetry-related residues. Through the helical periodicity of the nucleic acid structure, those bridges can lead to regular and striking hydration networks involving several water molecules and characteristic of the nucleic acid form. Solvent dynamics, as seen by temperature factor versus occupancy plots, seems intimately related to nucleic acid structure and dynamics, since they depend on hydration sites around the nucleic acids.     

Molecular dynamics analysis of the engrailed homeodomain-DNA recognition

Molecular dynamics (MD) simulations were performed for investigating the role of Gln50 in the engrailed homeodomain-DNA recognition. Employing the crystal structure of free engrailed homeodomain and homeodomain-DNA complex as a starting structure, we carried out MD simulations of: (i) the complex between engrailed homeodomain and a 20 base-pair DNA containing TAATTA core sequence; (ii) the free engrailed homeodomain. The simulations show that homeodomain flexibility does not depend on its ligation state. The engrailed homeodomain shows similar flexibility, and the recognition helix-3 shows very similar characteristic of high rigidity and limited conformational space in two complexation states. At the same time, DNA structure has also no obvious conformational fluctuations. These results preclude the possibility of the side chain of Gln50 forming direct hydrogen bonds to the core DNA bases. MD simulations confirm a few well-conserved sites for water-mediated hydrogen bonds from protein to DNA are occupied by water molecules, and Gln50 interacts with corresponding core DNA bases through water-mediated hydrogen bonds. So Gln50 plays a relatively modest role in determining the affinity and specificity of the engrailed homeodomain. In addition, the electrostatic interaction between homeodomain and phosphate backbone of the DNA is a main factor for N- and C-terminal arm becoming ordered upon DNA binding.     

Sequence-dependent DNA deformability studied using molecular dynamics simulations

Proteins recognize specific DNA sequences not only through direct contact between amino acids and bases, but also indirectly based on the sequence-dependent conformation and deformability of the DNA (indirect readout). We used molecular dynamics simulations to analyze the sequence-dependent DNA conformations of all 136 possible tetrameric sequences sandwiched between CGCG sequences. The deformability of dimeric steps obtained by the simulations is consistent with that by the crystal structures. The simulation results further showed that the conformation and deformability of the tetramers can highly depend on the flanking base pairs. The conformations of xATx tetramers show the most rigidity and are not affected by the flanking base pairs and the xYRx show by contrast the greatest flexibility and change their conformations depending on the base pairs at both ends, suggesting tetramers with the same central dimer can show different deformabilities. These results suggest that analysis of dimeric steps alone may overlook some conformational features of DNA and provide insight into the mechanism of indirect readout during protein–DNA recognition. Moreover, the sequence dependence of DNA conformation and deformability may be used to estimate the contribution of indirect readout to the specificity of protein–DNA recognition as well as nucleosome positioning and large-scale behavior of nucleic acids.     

Molecular dynamics simulations of DNA in solutions with different counter-ions

Molecular dynamics simulations of the [d(ATGCAGTCAG]2 fragment of DNA, in water and in the presence of three different counter-ions (Li+, Na+ and Cs+) are reported. Three-dimensional hydration structure and ion distribution have been calculated using spatial distribution functions for a detailed picture of local concentrations of ions and water molecules around DNA. According to the simulations, Cs+ ions bind directly to the bases in the minor groove, Na+ ions bind prevailing to the bases in the minor groove through one water molecule, whereas Li+ ions bind directly to the phosphate oxygens. The different behavior of the counter-ions is explained by specific hydration structures around the DNA and the ions. It is proposed how the observed differences in the ion binding to DNA may explain different conformational behavior of DNA. Calculated self-diffusion coefficients for the ions agree well with the available NMR data.     

DNA deformability changes of single base pair mutants within CDE binding sites in S. Cerevisiae centromere DNA correlate with measured chromosomal loss rates and CDE binding site symmetries

Background  

The centromeres in yeast (S. cerevisiae) are organized by short DNA sequences (125 bp) on each chromosome consisting of 2 conserved elements: CDEI and CDEIII spaced by a CDEII region. CDEI and CDEIII are critical sequence specific protein binding sites necessary for correct centromere formation and following assembly with proteins, are positioned near each other on a specialized nucleosome. Hegemann et al. BioEssays 1993, 15: 451–460 reported single base DNA mutants within the critical CDEI and CDEIII binding sites on the centromere of chromosome 6 and quantitated centromere loss of function, which they measured as loss rates for the different chromosome 6 mutants during cell division. Olson et al. Proc Natl Acad Sci USA 1998, 95: 11163–11168 reported the use of protein-DNA crystallography data to produce a DNA dinucleotide protein deformability energetic scale (PD-scale) that describes local DNA deformability by sequence specific binding proteins. We have used the PD-scale to investigate the DNA sequence dependence of the yeast chromosome 6 mutants' loss rate data. Each single base mutant changes 2 PD-scale values at that changed base position relative to the wild type. In this study, we have utilized these mutants to demonstrate a correlation between the change in DNA deformability of the CDEI and CDEIII core sites and the overall experimentally measured chromosome loss rates of the chromosome 6 mutants.     

The mechanism of the type III antifreeze protein action: a computational study

The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of differential distortions of the water-water hydrogen bonding angle in the first hydration shell. This method of hydration analysis is applied here to study the hydration of the wild type III thermal hysteresis protein from eel pout and three mutations at residue 16. Wild type and one mutant have full activity, the other two mutants have little or no anti-freeze (thermal hysteresis) activity. The analysis reveals significant differences in the hydration structure of the ice-binding site (centered on residue 16) among four proteins. For the A16T and A16Y mutants with reduced activity, polar groups have a typical polar-like hydration. For the wild type and mutant A16C with 100% of the wild type activity, polar groups have unusual, very apolar-like hydration. In the latter case, hydrating water molecules form a more ice-like pattern of hydrogen bonding on the ice-binding face, while in the former case water-water H-bonds are more distorted and more heterogenous. Overall, the binding surface of active protein strongly enhances the water tetrahedral structure, i.e. promotes ice-like hydration. It is concluded that the specific shape, residue size and clustering of both polar/apolar groups are essential for the binding surface to recognize, and preferentially interact with nascent ice crystals forming in liquid water.