BII Conformation Research Articles

How acidic amino acid residues facilitate DNA target site selection

Despite the negative charge of the DNA backbone, acidic residues (Asp/Glu) commonly participate in the base readout, with a strong preference for cytosine. In fact, in the solved DNA/protein structures, cytosine is recognized almost exclusively by Asp/Glu through a direct hydrogen bond, while at the same time, adenine, regardless of its amino group, shows no propensity for Asp/Glu. Here, we analyzed the contribution of Asp/Glu to sequence-specific DNA binding using classical and ab initio simulations of selected transcription factors and found that it is governed by a fine balance between the repulsion from backbone phosphates and attractive interactions with cytosine. Specifically, Asp/Glu lower the affinity for noncytosine sites and thus act as negative selectors preventing off-target binding. At cytosine-containing sites, the favorable contribution does not merely rely on the formation of a single H-bond but usually requires the presence of positive potential generated by multiple cytosines, consistently with the observed excess of cytosine in the target sites. Finally, we show that the preference of Asp/Glu for cytosine over adenine is a result of the repulsion from the adenine imidazole ring and a tendency of purine-purine dinucleotides to adopt the BII conformation.

DNA Backbone BI/BII Distribution and Dynamics in E2 Protein-Bound Environment Determined by Molecular Dynamics Simulations.

BI and BII conformational substates in the DNA backbone typify canonical B-form DNA. The BI and BII substates are important for structural variation of DNA and have been implicated in protein-nucleic acid recognition mechanisms. Recent refinements have been made to nucleic acid force fields employed in molecular dynamics simulations that demonstrate a better ability to model the BI and BII states, leading to overall improved modeling of DNA structure and dynamics. These force field improvements have yet to be significantly demonstrated in the context of a protein-DNA system extended to long time scales. Our plan was to run molecular dynamics simulations of a well-studied protein-DNA system (E2-DNA) into the microsecond time scale and determine the ability of the force field to populate BII states in the DNA backbone consistent with dinucleotide steps crystallized in the BII conformation. The results showed that the dinucleotide steps in the E2-DNA complex with the highest BII populations from simulation trajectories corresponded to the dinucleotide steps crystallized in the BII state and that decoy BI and BII states converge to the same results within approximately one microsecond.

Optimization of the CHARMM additive force field for DNA: Improved treatment of the BI/BII conformational equilibrium.

The B-form of DNA can populate two different backbone conformations: BI and BII, defined by the difference between the torsion angles ε and ζ (BI = ε-ζ < 0 and BII = ε-ζ > 0). BI is the most populated state, but the population of the BII state, which is sequence dependent, is significant and accumulating evidence shows that BII affects the overall structure of DNA, and thus influences protein-DNA recognition. This work presents a reparametrization of the CHARMM27 additive nucleic acid force field to increase the sampling of the BII form in MD simulations of DNA. In addition, minor modifications of sugar puckering were introduced to facilitate sampling of the A form of DNA under the appropriate environmental conditions. Parameter optimization was guided by quantum mechanical data on model compounds, followed by calculations on several DNA duplexes in the condensed phase. The selected optimized parameters were then validated against a number of DNA duplexes, with the most extensive tests performed on the EcoRI dodecamer, including comparative calculations using the Amber Parm99bsc0 force field. The new CHARMM model better reproduces experimentally observed sampling of the BII conformation, including sampling as a function of sequence. In addition, the model reproduces the A form of the 1ZF1 duplex in 75 % ethanol, and yields a stable Z-DNA conformation of duplex (GTACGTAC) in its crystal environment. The resulting model, in combination with a recent reoptimization of the CHARMM27 force field for RNA, will be referred to as CHARMM36.

DFT study of polymorphism of the DNA double helix at the level of dinucleoside monophosphates

AbstractWe apply DFT calculations to deoxydinucleoside monophosphates (dDMPs) which represent minimal fragments of the DNA chain to study the molecular basis of stability of the DNA duplex, the origin of its polymorphism and conformational heterogeneity. In this work, we continue our previous studies of dDMPs where we detected internal energy minima corresponding to the “classical” B conformation (BI‐form), which is the dominant form in the crystals of oligonucleotide duplexes. We obtained BI local energy minima for all existing base sequences of dDMPs. In the present study, we extend our analysis to other families of DNA conformations, successfully identifying A, BI, and BII energy minima for all dDMP sequences. These conformations demonstrate distinct differences in sugar ring puckering, but similar sequence‐dependent base arrangements. Internal energies of BI and BII conformers are close to each other for nearly all the base sequences. The dGpdG, dTpdG, and dCpdA dDMPs slightly favor the BII conformation, which agrees with these sequences being more frequently experimentally encountered in the BII form. We have found BII‐like structures of dDMPs for the base sequences both existing in crystals in BII conformation and those not yet encountered in crystals till now. On the other hand, we failed to obtain dDMP energy minima corresponding to the Z family of DNA conformations, thus giving us the ground to conclude that these conformations are stabilized in both crystals and solutions by external factors, presumably by interactions with various components of the media. Overall the accumulated computational data demonstrate that the A, BI, and BII families of DNA conformations originate from the corresponding local energy minimum conformations of dDMPs, thus determining structural stability of a single DNA strand during the processes of unwinding and rewinding of DNA. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem 110:2548–2559, 2010

Contribution of the intrinsic mechanical energy of the phosphodiester linkage to the relative stability of the A, BI, and BII forms of duplex DNA.

Canonical forms of duplex DNA are known to sample well-defined regions of the alpha, beta, gamma, epsilon, and zeta dihedral angles that define the conformation of the phosphodiester linkage in the backbone of oligonucleotides. While extensive studies of base composition and base sequence dependent effects on the sampling of the A, B1, and BII canonical forms of duplex DNA have been presented, our understanding of the intrinsic contribution of the five dihedral degrees of freedom associated with the phosphodiester linkage to the conformational properties of duplex DNA is still limited. To better understand this contribution, ab initio quantum mechanical (QM) calculations were performed on a model compound representative of the phosphodiester backbone to systematically sample the energetics about the alpha, beta, gamma, epsilon, and zeta dihedral angles relevant to the conformational properties of duplex DNA. Low-energy regions of dihedral potential energy surfaces are shown to correlate with the regions of dihedral space sampled in experimental crystal structures of the canonical forms of DNA, validating the utility of the model compound and emphasizing the contribution of the intrinsic mechanical properties of the phosphodiester backbone to the conformational properties of duplex DNA. Those contributions include the relative stability of the A, BI, and BII conformations of duplex DNA, where the gas-phase energetics favor the BI form over the A and BII forms. In addition, subtle features of the potential energy surfaces mimic changes in the probability distributions of alpha, beta, gamma, epsilon, and zeta dihedral angles in A, BI, and BII forms of DNA as well as with conformations sampled in single-stranded DNA. These results show that the intrinsic mechanical properties of the phosphodiester backbone make a significant contribution to conformational properties of duplex DNA observed in the condensed phase and allow for the prediction that single-stranded DNA primarily samples folded conformations thereby possibly lowering the entropic barrier to the formation of duplex DNA.

Understanding the Chemistry of Cytosine Methylation Effects

Proteins bind to DNA to initiate several important biological processes including DNA replication and transcription of genes, these processes are also highly regulated. A common method of bioregulation is cytosine methylation. Recent research suggests that cytosine methylation quenches DNA dynamics, preventing the DNA deformation often necessary for efficient protein-DNA complex formation. Using 31P NMR, we have shown that methylation induced rigidity may stem from conformational changes in the backbone from BI to BII conformation. To explore the mechanism of these conformational changes, we have turned to the study of cytosine fluorination. Through comparison of FTIR and NMR spectra, respectively, one can qualitatively and quantitatively observe the effects of methylation and fluorination on DNA structure and dynamics. Preliminary results demonstrate that these different covalent modifications of the cytosine base affect the structure and dynamics in different ways. Both modifications alter the BI/BII ratio but fluorination does not appear to affect the dynamics as significantly as methylation. We attribute the enhanced quenching of backbone dynamics by methylation to the formation of a strong dipolar interaction between the negatively charged backbone oxygen with the hydrogens of the methyl group whose proximity to the backbone is closer in BII than BI. Comparisons of fluorination and methylation effects on sequences containing both the EcoRI and Cre binding sites, as well as their modulation due to sequence context, will be presented.

31P NMR investigation of backbone dynamics in DNA binding sites.

The backbone conformation of DNA plays an important role in the indirect readout mechanisms for protein--DNA recognition events. Thus, investigating the backbone dynamics of each step in DNA binding sequences provides useful information necessary for the characterization of these interactions. Here, we use 31P dynamic NMR to characterize the backbone conformation and dynamics in the Dickerson dodecamer, a sequence containing the EcoRI binding site, and confirm solid-state 2H NMR results showing that the C3pG4 and C9pG10 steps experience unique dynamics and that these dynamics are quenched upon cytosine methylation. In addition, we show that cytosine methylation affects the conformation and dynamics of neighboring nucleotide steps, but this effect is localized to only near neighbors and base-pairing partners. Last, we have been able to characterize the percent BII in each backbone step and illustrate that the C3pG4 and C9pG10 favor the noncanonical BII conformation, even at low temperatures. Our results demonstrate that 31P dynamic NMR provides a robust and efficient method for characterizing the backbone dynamics in DNA. This allows simple, rapid determination of sequence-dependent dynamical information, providing a useful method for studying trends in protein-DNA recognition events.

Insertion of a Bulky Rhodium Complex into a DNA Cytosine−Cytosine Mismatch: An NMR Solution Study

The bulky octahedral complex Rh(bpy)2chrysi3+ (chrysi = 5,6-chrysenequinonediimine) binds single-base mismatches in a DNA duplex with micromolar binding affinities and high selectivity. Here we present an NMR solution study to characterize the binding mode of this bulky metal complex with its target CC mismatch in the oligonucleotide duplex (5'-CGGACTCCG-3')2. Both NOESY and COSY studies indicate that Rh(bpy)2chrysi3+ inserts deeply in the DNA at the mismatch site via the minor groove and with ejection of both destabilized cytosines into the opposite major groove. The insertion only minimally distorts the conformation of the oligonucleotide local to the binding site. Both flanking, well-matched base pairs remain tightly hydrogen-bonded to each other, and 2D DQF-COSY experiments indicate that all sugars maintain their original C2'-endo conformation. Remarkably, 31P NMR reveals that opening of the phosphate angles from a BI to a BII conformation is sufficient for insertion of the bulky metal complex. These results corroborate those obtained crystallographically and, importantly, provide structural evidence for this specific insertion mode in solution.

Sequence Preference for BI/BII Conformations in DNA: MD and Crystal Structure Data Analysis

Deciphering sequence information from sugar-phosphate backbone is finely tuned through the conformational substates of DNA. BII conformation, one of the conformational substates of B-DNA, is known to play a key role in DNA-protein recognition. BI and BII are identified by the ϵ-ζ difference, which is negative in BI and positive in BII. Our analysis of MD and ciystal structures shows that BII conformation is sequence specific and dinucleotides GC, CG, CA, TG, TA show high preference to take up BII conformation, while TT, TC, CT, CC dinucleotides rarely take up this conformation. Significant changes were observed in the dinucleotide parameters viz. twist, roll, and slide for the steps having BII conformation. Interestingly, the magnitude of variation in the dinucleotide parameters is seen to depend mainly on two factors, the magnitude of ϵ-ζ difference and the presence or absence of BII conformation in the second strand, across the WC base-paired dinucleotide step. Based on these two factors, the conformational substate of a dinucleotide step can be further classified as BI.BI (BI conformation in both strands), BI.BII (BI conformation in one strand and BII conformation in the other), and BII.BII (BII conformation in both strands). The occurrence of BII in both strands was found to be quite rare and thus, it can be concluded that BI.BI and BI.BII hybrid steps are more favorable than a BII.BII step. In conformity with the sequence preference seen for dinucleotides in each strand, BII.BII combination of backbone conformation was observed only for GC, CG, CA, and TG containing dinucleotide steps. We further classified BII.BII step as strong BII and weak BII depending on the magnitude of the average ϵ-ζ difference. The dinucleotide steps which belong to the categoiy of strong BII, have large twist, high positive slide and negative roll values, while those in the weak BII group have roll, twist, and slide values similar to that of hybrid BI.BII steps. This conformational property could be contributing to the groove opening/closing and thus can modulate protein-DNA interaction.

The Role of DNA Structure and Dynamics in the Recognition of Bovine Papillomavirus E2 Protein Target Sequences

The papillomavirus E2 transcription and replication factors bind to the DNA consensus ACCGN4CGGT sequence (E2-BS), through both direct and indirect readout mechanisms. The two symmetric half-sites ACCG·CGGT are highly conserved in the genomes and are hydrogen bound with E2. Although E2 does not contact the N4 spacer, the affinities are modulated by the base composition of this DNA part. Nevertheless, the origin of either the global recognition mechanism or the spacer effect remains unclear, particularly in the case of the bovine papillomavirus type 1 E2 (BPV-1-E2) system, used as model to study the papillomaviruses. We present, herein, studies carried out on oligomers differently recognized by the BPV-1-E2 protein and based on molecular dynamic simulations including counterions and water. The sequences contain the conserved half-sites but three different spacers (CCAT, ACGT and AAAC), resulting in very high, high and low affinity targets for BPV-1-E2. In order to estimate how much the free DNAs resemble the bound conformations, comparisons are made with two DNAs extracted from E2-BS-BPV-1 crystallographic complexes, representative of high and moderate affinity structures. The analysis of 15 ns trajectories reveals that the ACCG/CGGT half-sites, whatever the spacer, have the same behavior and adopt average stable base-pair parameters very close to those of the bound conformations. In contrast, the three different free spacers strongly differ in their BI↔BII backbone dynamics. The low affinity AAAC spacer exhibits stable BI backbone conformations, the high affinity ACGT spacer is characterized by a dramatic instability of the CpG phosphate groups, and the CpA and GpG backbones in the very high affinity CCAT·ATGG spacer are trapped in BII conformations. All resemble more of the moderate affinity complex DNA than the high affinity one. Nevertheless, the particular behavior of the CCAT and ACGT backbones allows the emergence of BII-rich spacers, a configuration reproducing both local and global helical features of the bound DNA conformation of the high affinity complex and favoring the minor groove curvature required in the complex. In particular, the CCAT-containing site spends almost half of the time in this form that well mimics the bound one. Thus, we propose that the E2 protein could take advantage of the invariant favorable structures of the half-sites to form a pre-complex, but would require a specific spacer intrinsic malleability to lock the interaction. Finally, the backbone conformational states, by their ability to translate information coded in the sequence into structural properties, provide insight into the mechanisms that contribute to fine binding site selection and specific nucleic acid ligand recognition.

The Role of DNA Structure and Dynamics in the Recognition of Bovine Papillomavirus E2 Protein Target Sequences

The papillomavirus E2 transcription and replication factors bind to the DNA consensus ACCGN4CGGT sequence (E2-BS), through both direct and indirect readout mechanisms. The two symmetric half-sites ACCG·CGGT are highly conserved in the genomes and are hydrogen bound with E2. Although E2 does not contact the N4 spacer, the affinities are modulated by the base composition of this DNA part. Nevertheless, the origin of either the global recognition mechanism or the spacer effect remains unclear, particularly in the case of the bovine papillomavirus type 1 E2 (BPV-1-E2) system, used as model to study the papillomaviruses. We present, herein, studies carried out on oligomers differently recognized by the BPV-1-E2 protein and based on molecular dynamic simulations including counterions and water. The sequences contain the conserved half-sites but three different spacers (CCAT, ACGT and AAAC), resulting in very high, high and low affinity targets for BPV-1-E2. In order to estimate how much the free DNAs resemble the bound conformations, comparisons are made with two DNAs extracted from E2-BS-BPV-1 crystallographic complexes, representative of high and moderate affinity structures. The analysis of 15ns trajectories reveals that the ACCG/CGGT half-sites, whatever the spacer, have the same behavior and adopt average stable base-pair parameters very close to those of the bound conformations. In contrast, the three different free spacers strongly differ in their BI↔BII backbone dynamics. The low affinity AAAC spacer exhibits stable BI backbone conformations, the high affinity ACGT spacer is characterized by a dramatic instability of the CpG phosphate groups, and the CpA and GpG backbones in the very high affinity CCAT·ATGG spacer are trapped in BII conformations. All resemble more of the moderate affinity complex DNA than the high affinity one. Nevertheless, the particular behavior of the CCAT and ACGT backbones allows the emergence of BII-rich spacers, a configuration reproducing both local and global helical features of the bound DNA conformation of the high affinity complex and favoring the minor groove curvature required in the complex. In particular, the CCAT-containing site spends almost half of the time in this form that well mimics the bound one. Thus, we propose that the E2 protein could take advantage of the invariant favorable structures of the half-sites to form a pre-complex, but would require a specific spacer intrinsic malleability to lock the interaction. Finally, the backbone conformational states, by their ability to translate information coded in the sequence into structural properties, provide insight into the mechanisms that contribute to fine binding site selection and specific nucleic acid ligand recognition.

Molecular Dynamics Simulation of 7, 8-dihydro-8-Oxoguanine DNA

To elucidate the effect of guanine lesion produced by the oxidative damage on DNA, 1 nanosecond molecular dynamics simulations of native and oxidized DNA were performed. The target DNA molecules are dodecamer duplex d(CGCGAATTCGCG)2 and its derivative duplex d(C1G2C3(8-oxoG)4A5A6T7T8C9G10C11G12)·d(C13G14C15G16A17A18T19T20C21G22C23G24), which has one oxidized guanine, 7, 8-dihydro-8-oxoguanine (8-oxoG), at the fourth position. The local structural change due to the lesion of 8-oxoG and the global dynamic structure of the 8-oxoG DNA were studied. It was found that the 8-oxoG DNA remained structurally stable during the simulation due to newly produced hydrogen bonds around the (8-oxoG)4 residue. However, there were distinguishable differences in structural parameters and dynamic property in the 8-oxoG DNA. The conformation around the (8- oxoG)4 residue departed from the usual conformation of native DNA and took an unique conformation of ϵ-ζ in BII conformation and χ in high anti orientation at the (8-oxoG)4 residue, and adopted a very low helical twist angle at the C3:G22—(8-oxoG)4:C21 step. Further analysis by principal component analysis indicated that the formation of the hydrogen bonds around the (8-oxoG)4 residue plays a role as a trigger for the conformational transition of the 8-oxoG DNA in the conformational space.

Impact of CpG methylation on structure, dynamics and solvation of cAMP DNA responsive element.

Methylation of CpG motifs in DNA is involved in the control of gene expression and in several other epigenic effects. It suppresses also the immuno-stimulation properties of bacterial or viral DNAs that contain CPGS: However, effects of methylation on the DNA structure and dynamics are not clear. Here we carried out a 10 ns MD simulation, confronted to an NMR analysis, of a hexadecanucleotide with the cAMP responsive element (CRE) DNA methylated at its center: d(GAGATGAmCGTCATCTC)(2) (CREmet). Methylation does not introduce significant structure modification but reduces the dynamics. Molecular mechanics and generalized Born solvation energy calculations showed that the stiffness of CREmet arises from both a restriction of the conformational space by the bulky methyl groups and a folding of DNA around the hydrophobic methyls. The latter effect is favored when the GpA steps belonging to the TGA binding half-sites adopt the BII conformation. The inability of the methylated DNAs to interact with their protein partners-either transcription factors for gene regulation or a Toll-like receptor for immunostimulation-could result from both the obstacle created by methyls, preventing crucial interactions, and the loss of DNA flexibility, reducing its adaptability. Results are discussed in the light of NMR and crystallographic data.

BII Nucleotides in the B and C Forms of Natural-sequence Polymeric DNA: A New Model for the C Form of DNA

A combination of solid-state 31P and 13C NMR, X-ray diffraction, and model building is used to show that the B and C forms of fibrous macromolecular DNA consist of two distinct nucleotide conformations, which correspond closely to the BI and BII nucleotide conformations known from oligonucleotide crystals. The proportion of the BII conformation is higher in the C form than in the B form. We show structural models for a 10 1 double helix involving BI nucleotides and a 9 1 double helix involving BII nucleotides. The 10 1 BI model is similar to a previous model of B-form DNA, while the 9 1 BII model is novel. The BII model has a very deep and narrow minor groove, a shallow and wide major groove, and highly inclined bases. This work shows that the B to C transition in fibers corresponds to BI to BII conformational changes of the individual nucleotides.

Contribution of the Phosphodiester Backbone and Glycosyl Linkage Intrinsic Torsional Energetics to DNA Structure and Dynamics

The intrinsic conformational energetics of macromolecules are an important contribution to their structure and dynamics. This information, however, is lacking for most dihedrals about rotatable bonds in nucleic acids. To bridge this gap, high-level ab initio quantum mechanical calculations have been performed on a number of furanose-based model compounds designed to model the intrinsic torsional energetics of the dihedrals ε, γ, β, and χ in deoxyribonucleic acid (DNA). Energy profiles have been obtained with furanose in both the C3‘ endo and C2‘ endo conformations, to model the torsions in the context of the two conformational ranges populated by the sugar in DNA. The resulting energy profiles are compared to the corresponding crystallographically determined population distributions, with which they can be reconciled to a large extent. This suggests that the models used to derive these energy surfaces are relevant to DNA. Torsion ε is found to be intrinsically very flexible with the low-energy regions encompassing the BI and BII substates. The BI internal energy is found to be on the order of 1.5 kcal/mol more stable than the BII conformation. The torsional energy barrier between BI and BII is relatively low, on the order of 2.1 kcal/mol. Estimates of other torsional energy barriers suggest that the paths of lowest energy between γ = g+ and γ = g- and between the χ anti and syn orientations are through γ = 0° and χ = 120°, respectively. The χ energy surfaces offer insights into the contribution of base−furanose interactions to the corresponding distributions in DNA and its components, in terms of purines versus pyrimidines, and point to the unique properties of cytosine. These surfaces also indicate that a syn base interacts more favorably with a north furanose than with a south furanose, in contrast with a widely accepted view.

Solution structure of a non-palindromic 16 base-pair DNA related to the HIV-1 κb site: evidence for BI-BII equilibrium inducing a global dynamic curvature of the duplex

1H and 31P NMR spectroscopy have been used together with molecular modelling to determine the fine structure of a non-palindromic 16 bp DNA containing the NF-κB binding site. Much emphasis has been placed upon NMR optimization of both two-dimensional 31P NMR techniques to extract structural information defining the phosphodiester backbone conformation and selective homonuclear 2D COSY experiments to determine sugar conformations. NMR data show evidence for a dynamic behaviour of steps flanking the ten base-pairs of the NF-κB binding site. A BI-BII equilibrium at these steps is demonstrated and two models for each extreme conformation are proposed in agreement with NMR data. In the refined BII structures, the NF-κB binding site exhibits an intrinsic curvature towards the major groove that is magnified by the four flanking steps in the BII conformation. Furthermore, the base-pairs are translated into the major groove. Thus, we present a novel mode of dynamic intrinsic curvature compatible with the DNA curvature observed in the X-ray structure of the p50-DNA complex.

Solution structure of the CpG containing d(CTTCGAAG)2 oligonucleotide: NMR data and energy calculations are compatible with a BI/BII equilibrium at CpG.

We report the analysis of the solution structure of the DNA duplex d(CTTCGAAG)2 compared to that of d(CATCGATG)2, the two oligonucleotides being related by the permutation of residues 2 and 7. An earlier study has demonstrated the malleability of CpG in the tetrad TCGA of d(CATCGATG)2 [Lefebvre et al. (1995) Biochemistry 34, 12019-12028]. Conformations of d(CTTCGAAG)2 were evaluated by (a) two-dimensional NMR, including proton and phosphorus experiments, (b) adiabatic mapping of the conformational space, (c) restrained molecular mechanics undertaken with sugar phase angle, epsilon-zeta difference angle, and NOE distances as input, and (d) back-calculation-refinement against NOE spectra at various mixing times. d(CTTCGAAG)2 like d(CATCGATG)2 exhibits a B-DNA conformation. However, significant differences are noted between the two oligonucleotides, extending up to the central CpG step, although this step resides in the same TCGA tetrad in both sequences. In structures obtained with refined NMR data, CpG adopts, for instance, a greater twist and a higher guanine phase within d(CTTCGAAG)2 compared to d(CATCGATG)2. In the former oligonucleotide, the structure of CpG resembles strikingly that found in the ACGT tetrad of the cAMP responsive element [Mauffret et al. (1992) J. Mol. Biol. 227, 852-875]. Moreover, two conformers with CpG either in the BII state (epsilon, zeta = g-, t) or in the BI state (epsilon, zeta = t, g-) are found equally stable for d(CTTCGAAG)2. The energy barrier from BI to BII comes to only 5.7 kcal/mol, and the path of the transition is very short. When calculations on d(CTTCGAAG)2 are performed taking the BI/BII equilibrium into account, the agreement with both the 1H and 31P data is found better than in the case with a single conformation taken alone. The BI/BII equilibrium may also occur in d(CATCGATG)2, but the amount of BII conformer is now found weaker compared to its analogue. The ability of the CpG phosphate groups to adopt the BII conformation could provide a satisfying explanation for the high mutation rates observed at these sites.

Molecular dynamics simulations of chlorambucil/DNA adducts. A structural basis for the 5'-GNC interstrand DNA crosslink formed by nitrogen mustards.

The alkylation of DNA by chlorambucil has been studied using a computational approach. Molecular dynamics simulations were performed on the fully solvated non-covalent complex, two monoadducts and a crosslinked diadduct of chlorambucil with the d(CGG3G2CGC).-d(GCG1CCCG) duplex, in which the N7 atoms of G1, G2 and G3 are potential alkylation sites. The results provide a structural basis for the preference of nitrogen mustards to crosslink DNA duplexes at a 5'-GNC site (a 1,3 crosslink, G1-G3) rather than at a 5'-GC sites (a 1,2 crosslink, G1-G2). In the non-covalent complex simulation the drug reoriented from a non-interstrand crosslinking location to a position favorable for G1-G3 diadduct formation. It proved possible to construct a G1-G3 diadduct from a structure from the non-covalent simulation, and continue the molecular dynamics calculation without further disruption of the DNA structure. A crosslinked diadduct developed with four BII conformations on the 3' side of each alkylated guanine and of their respective complementary cytosine. In the first monoadduct simulation the starting point was the same DNA conformation used in the crosslinked diadduct simulation with alkylation at G1. In this simulation the DNA deformation was reduced, with the helix returning to a more canonical form. A second monoadduct simulation was started from a canonical DNA conformation alkylated at G3. Here, no significant motion towards a potential crosslinking conformation occurred. Collectively, the results suggest that crosslink formation is dependent upon the drug orientation prior to alkylation and the required deformation of the DNA to permit 1,3 crosslinking can largely be achieved in the non-covalent complex.

The relationship between mutation rates for the (C-G)-->(T-A) transition and features of T-G mispair structures in different neighbor environments, determined by free energy molecular mechanics.

The results of this theoretical study combining sequence analysis and minimization with integral equation liquid structural methods indicate that the local sequence context of a T-G wobble mismatch influences the local conformation of the helix, and that conformational alterations are correlated with mutational activity. Studies on the mismatch in four different 5' and 3' neighbor contexts indicate that the nature of the 5' base to the thymine of the mispair is probably the single most critical factor in determining the structural features that facilitate or discourage mutations. When cytosine is the 5' neighbor, the helix adopts a mostly BII conformation, whereas a 5' guanine preserves the canonical BI. Structures that vary little from the BI structure on the incorporation of the mismatch have sequences that correspond to lower rates of transition, whereas those with mostly BII conformations, have sequences with high mutation rates. Subtle variations in stacking patterns around the mismatch precipitate a structural Domino-effect, with a variety of changes in conformation. The helix opens at the mismatch with increased roll angle and propeller twist, causing the thymine to migrate into the major groove and the guanine into the minor groove, exposing the heteroatomic groups to the solvent in the major and minor grooves, respectively, and allowing for some unusual hydrogen bonds. These alterations show a tentative correlation with mutation rates, implying that stacking and structure around the mismatch are important features in the discrimination by proofreading activities of canonical W-C and wobble mismatch base pairs during replication-repair. Variations in the C1'-C1' distances, high propeller twists, changes in the electrostatic complementarity leading to unusual hydrogen bonding patterns probably all correlate with detectability.

A molecular dynamics simulation of a polyamine-induced conformational change of DNA. A possible mechanism for the B to Z transition.

A 75ps molecular dynamics simulation has been performed on a fully solvated complex of spermine with the B DNA decamer (dGdC)5.(dGdC)5. The simulation indicates a possible mechanism by which polyamines might induce the formation of a left-handed helix, the B to Z transition. Spermine was initially located in the major groove, hydrogen bonded to the helix. During the simulation the ligand migrates deeper into the DNA, maintaining strong hydrogen bonding to the central guanine bases and destroying the Watson-Crick base pairing with their respective cytosines. Significant rotation of these and other cytosine bases was observed, in part due to interactions of the helix with the aminopropyl chains of spermine. An intermediate BII conformation might be of importance in this process.