Minor Groove Width Research Articles

Coarse-grained modeling of DNA curvature.

The interaction of DNA with proteins occurs over a wide range of length scales, and depends critically on its local structure. In particular, recent experimental work suggests that the intrinsic curvature of DNA plays a significant role on its protein-binding properties. In this work, we present a coarse grained model of DNA that is capable of describing base-pairing, hybridization, major and minor groove widths, and local curvature. The model represents an extension of the recently proposed 3SPN.2 description of DNA [D. M. Hinckley, G. S. Freeman, J. K. Whitmer, and J. J. de Pablo, J. Chem. Phys. 139, 144903 (2013)], into which sequence-dependent shape and mechanical properties are incorporated. The proposed model is validated against experimental data including melting temperatures, local flexibilities, dsDNA persistence lengths, and minor groove width profiles.

GBshape: a genome browser database for DNA shape annotations.

Many regulatory mechanisms require a high degree of specificity in protein-DNA binding. Nucleotide sequence does not provide an answer to the question of why a protein binds only to a small subset of the many putative binding sites in the genome that share the same core motif. Whereas higher-order effects, such as chromatin accessibility, cooperativity and cofactors, have been described, DNA shape recently gained attention as another feature that fine-tunes the DNA binding specificities of some transcription factor families. Our Genome Browser for DNA shape annotations (GBshape; freely available at http://rohslab.cmb.usc.edu/GBshape/) provides minor groove width, propeller twist, roll, helix twist and hydroxyl radical cleavage predictions for the entire genomes of 94 organisms. Additional genomes can easily be added using the GBshape framework. GBshape can be used to visualize DNA shape annotations qualitatively in a genome browser track format, and to download quantitative values of DNA shape features as a function of genomic position at nucleotide resolution. As biological applications, we illustrate the periodicity of DNA shape features that are present in nucleosome-occupied sequences from human, fly and worm, and we demonstrate structural similarities between transcription start sites in the genomes of four Drosophila species.

DNA shape dominates sequence affinity in nucleosome formation.

Nucleosomes provide the basic unit of compaction in eukaryotic genomes, and the mechanisms that dictate their position at specific locations along a DNA sequence are of central importance to genetics. In this Letter, we employ molecular models of DNA and proteins to elucidate various aspects of nucleosome positioning. In particular, we show how DNA's histone affinity is encoded in its sequence-dependent shape, including subtle deviations from the ideal straight B-DNA form and local variations of minor groove width. By relying on high-precision simulations of the free energy of nucleosome complexes, we also demonstrate that, depending on DNA's intrinsic curvature, histone binding can be dominated by bending interactions or electrostatic interactions. More generally, the results presented here explain how sequence, manifested as the shape of the DNA molecule, dominates molecular recognition in the problem of nucleosome positioning.

Synonymous codon bias and functional constraint on GC3-related DNA backbone dynamics in the prokaryotic nucleoid.

While mRNA stability has been demonstrated to control rates of translation, generating both global and local synonymous codon biases in many unicellular organisms, this explanation cannot adequately explain why codon bias strongly tracks neighboring intergene GC content; suggesting that structural dynamics of DNA might also influence codon choice. Because minor groove width is highly governed by 3-base periodicity in GC, the existence of triplet-based codons might imply a functional role for the optimization of local DNA molecular dynamics via GC content at synonymous sites (≈GC3). We confirm a strong association between GC3-related intrinsic DNA flexibility and codon bias across 24 different prokaryotic multiple whole-genome alignments. We develop a novel test of natural selection targeting synonymous sites and demonstrate that GC3-related DNA backbone dynamics have been subject to moderate selective pressure, perhaps contributing to our observation that many genes possess extreme DNA backbone dynamics for their given protein space. This dual function of codons may impose universal functional constraints affecting the evolution of synonymous and non-synonymous sites. We propose that synonymous sites may have evolved as an ‘accessory’ during an early expansion of a primordial genetic code, allowing for multiplexed protein coding and structural dynamic information within the same molecular context.

NMR studies of DNA support the role of pre-existing minor groove variations in nucleosome indirect readout.

We investigated how the intrinsic sequence-dependent properties probed via the phosphate linkages (BI ↔ BII equilibrium) influence the preferred shape of free DNA, and how this affects the nucleosome formation. First, this exploits NMR solution studies of four B-DNA dodecamers that together cover 39 base pairs of the 5' half of the sequence 601, of special interest for nucleosome formation. The results validate our previous prediction of a systematic, general sequence effect on the intrinsic backbone BII propensities. NMR provides new evidence that the backbone behavior is intimately coupled to the minor groove width. Second, application of the backbone behavior predictions to the full sequence 601 and other relevant sequences demonstrates that alternation of intrinsic low and high BII propensities, coupled to intrinsic narrow and wide minor grooves, largely coincides with the sinusoidal variations of the DNA minor groove width observed in crystallographic structures of the nucleosome. This correspondence is much poorer with low affinity sequences. Overall, the results indicate that nucleosome formation involves an indirect readout process implicating pre-existing DNA minor groove conformations. It also illustrates how the prediction of the intrinsic structural DNA behavior offers a powerful framework to gain explanatory insight on how proteins read DNA.

Correction: Role of Minor Groove Width and Hydration Pattern on Amsacrine Interaction with DNA

The incorrect Figures 3 and 4 were erroneously included in the article. Please see the correct Figures 3 and 4 here: Figure 3: Figure 4:

DNA conformational flexibility study using phosphate backbone neutralization model.

Due to the critical role of DNA in the processes of the cell cycle, the structural and physicochemical properties of DNA have long been of concern. In the present work, the effect of interplay between the DNA duplex and metal ions in solution on the DNA structure and conformational flexibility is studied by comparing the structure and dynamic conformational behavior of a duplex in a normal form and its “null isomer” using molecular dynamics methods. It was found that the phosphate neutralization changes the cation atmosphere around the DNA duplex greatly, increases the major groove width, decreases the minor groove width, and reduces the global bending direction preference. We also noted that the probability of BI phosphate linkages increases significantly because of the charge reduction in the backbone phosphate groups. More importantly, we found that the electrostatic effect on the DNA conformational flexibility is dependent on the sequence; that is, the phosphate backbone neutralization induces the global dynamic bending to be less flexible for GC-rich sequences but more flexible for AT-rich sequences.

TFBSshape: a motif database for DNA shape features of transcription factor binding sites

Transcription factor binding sites (TFBSs) are most commonly characterized by the nucleotide preferences at each position of the DNA target. Whereas these sequence motifs are quite accurate descriptions of DNA binding specificities of transcription factors (TFs), proteins recognize DNA as a three-dimensional object. DNA structural features refine the description of TF binding specificities and provide mechanistic insights into protein–DNA recognition. Existing motif databases contain extensive nucleotide sequences identified in binding experiments based on their selection by a TF. To utilize DNA shape information when analysing the DNA binding specificities of TFs, we developed a new tool, the TFBSshape database (available at http://rohslab.cmb.usc.edu/TFBSshape/), for calculating DNA structural features from nucleotide sequences provided by motif databases. The TFBSshape database can be used to generate heat maps and quantitative data for DNA structural features (i.e., minor groove width, roll, propeller twist and helix twist) for 739 TF datasets from 23 different species derived from the motif databases JASPAR and UniPROBE. As demonstrated for the basic helix-loop-helix and homeodomain TF families, our TFBSshape database can be used to compare, qualitatively and quantitatively, the DNA binding specificities of closely related TFs and, thus, uncover differential DNA binding specificities that are not apparent from nucleotide sequence alone.

Role of Minor Groove Width and Hydration Pattern on Amsacrine Interaction with DNA

Amsacrine is an anilinoacridine derivative anticancer drug, used to treat a wide variety of malignancies. In cells, amsacrine poisons topoisomerase 2 by stabilizing DNA-drug-enzyme ternary complex. Presence of amsacrine increases the steady-state concentration of these ternary complexes which in turn hampers DNA replication and results in subsequent cell death. Due to reversible binding and rapid slip-out of amsacrine from DNA duplex, structural data is not available on amsacrine-DNA complexes. In the present work, we designed five oligonucleotide duplexes, differing in their minor groove widths and hydration pattern, and examined their binding with amsacrine using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Complexes of amsacrine with calf thymus DNA were also evaluated for a comparison. Our results demonstrate for the first time that amsacrine is not a simple intercalator; rather mixed type of DNA binding (intercalation and minor groove) takes place between amsacrine and DNA. Further, this binding is highly sensitive towards the geometries and hydration patterns of different minor grooves present in the DNA. This study shows that ligand binding to DNA could be very sensitive to DNA base composition and DNA groove structures. Results demonstrated here could have implication for understanding cytotoxic mechanism of aminoacridine based anticancer drugs and provide directions to modify these drugs for better efficacy and few side-effects.

DBSI: DNA-binding site identifier

In this study, we present the DNA-Binding Site Identifier (DBSI), a new structure-based method for predicting protein interaction sites for DNA binding. DBSI was trained and validated on a data set of 263 proteins (TRAIN-263), tested on an independent set of protein-DNA complexes (TEST-206) and data sets of 29 unbound (APO-29) and 30 bound (HOLO-30) protein structures distinct from the training data. We computed 480 candidate features for identifying protein residues that bind DNA, including new features that capture the electrostatic microenvironment within shells near the protein surface. Our iterative feature selection process identified features important in other models, as well as features unique to the DBSI model, such as a banded electrostatic feature with spatial separation comparable with the canonical width of the DNA minor groove. Validations and comparisons with established methods using a range of performance metrics clearly demonstrate the predictive advantage of DBSI, and its comparable performance on unbound (APO-29) and bound (HOLO-30) conformations demonstrates robustness to binding-induced protein conformational changes. Finally, we offer our feature data table to others for integration into their own models or for testing improved feature selection and model training strategies based on DBSI.

Conformational modulation of DNA by polyamide binding: structural effects of f‐Im‐Py‐Im based derivatives on 5′‐ACGCGT‐3′

The DNA sequence 5'-ACGCGT-3' is in the core site of the Mlu 1 cell-cycle box, a transcriptional element in the promoter region of human Dbf4 gene that is highly correlated with a large number of aggressive solid cancers. The polyamide formamido-imidazole-pyrrole-imidazole-amine(+) (f-Im-Py-Im-Am(+) ) can target the minor groove of 5'-ACGCGT-3' as an antiparallel stacked dimer and has shown good activity in inhibiting transcription factor binding. Recently, f-Im-Py-Im-Am(+) derivatives that involve different orthogonally positioned substituents were synthesized to target the same binding site, and some of them have displayed improved binding and pharmacological properties. In this study, the gel electrophoresis-ligation ladders assay was used to evaluate the conformational effects of f-Im-Py-Im-Am(+) and derivatives on the target DNA, an essential factor for establishing the molecular basis of polyamide-DNA complexes and their transcription factor inhibition. The results show that the ACGCGT site in DNA has a relatively wide minor groove and a B-form like overall structure. After binding with f-Im-Py-Im-Am(+) derivatives, the DNA conformation is changed as indicated by the different mobilities in the gel. These conformational effects on DNA will at least help to point to the mechanism for the observed Mlu 1 inhibition activity of these polyamides. Therefore, modulating DNA transcription by locking the DNA shape or altering the minor groove geometry to affect the binding affinity of certain transcription factors is an attractive possible therapeutic mechanism for polyamides. Some of the substituents are charged with electrostatic interactions with DNA phosphate groups, and their charge effects on DNA gel mobility have been observed.

DNAshape: a method for the high-throughput prediction of DNA structural features on a genomic scale

We present a method and web server for predicting DNA structural features in a high-throughput (HT) manner for massive sequence data. This approach provides the framework for the integration of DNA sequence and shape analyses in genome-wide studies. The HT methodology uses a sliding-window approach to mine DNA structural information obtained from Monte Carlo simulations. It requires only nucleotide sequence as input and instantly predicts multiple structural features of DNA (minor groove width, roll, propeller twist and helix twist). The results of rigorous validations of the HT predictions based on DNA structures solved by X-ray crystallography and NMR spectroscopy, hydroxyl radical cleavage data, statistical analysis and cross-validation, and molecular dynamics simulations provide strong confidence in this approach. The DNAshape web server is freely available at http://rohslab.cmb.usc.edu/DNAshape/.

Control of DNA minor groove width and Fis protein binding by the purine 2-amino group

The width of the DNA minor groove varies with sequence and can be a major determinant of DNA shape recognition by proteins. For example, the minor groove within the center of the Fis–DNA complex narrows to about half the mean minor groove width of canonical B-form DNA to fit onto the protein surface. G/C base pairs within this segment, which is not contacted by the Fis protein, reduce binding affinities up to 2000-fold over A/T-rich sequences. We show here through multiple X-ray structures and binding properties of Fis–DNA complexes containing base analogs that the 2-amino group on guanine is the primary molecular determinant controlling minor groove widths. Molecular dynamics simulations of free-DNA targets with canonical and modified bases further demonstrate that sequence-dependent narrowing of minor groove widths is modulated almost entirely by the presence of purine 2-amino groups. We also provide evidence that protein-mediated phosphate neutralization facilitates minor groove compression and is particularly important for binding to non-optimally shaped DNA duplexes.

Abstract 4549: Use of molecular dynamic simulations to explore changes in conformation and sequence preference for DNA-binding biaryl polyamide structures.

Abstract As a class, minor-groove non-covalent binding small molecules generally show A/T selectivity. The lack of G/C selectivity is thought to relate to the wider minor groove in G/C regions and the presence of the exocyclic 2-amino groups of guanines which project into the minor groove and prevent multiple close van der Waals contacts with the G/C-rich minor groove. AT-selective structures such as distamycin and netropsin orientate in the minor groove in an “NH down” conformation, with their NH groups pointing into the minor groove to enhance the isohelicity of the structures and produce favourable van der Waal interactions with the groove floor below. Using a distamycin scaffold as a starting point we introduced biaryl building blocks in place of pyrroles to change the curvature and van der Waals interaction potential of this novel class of polyamides. We started by designing a library of fragments to span two DNA base pairs based on simple phenyl-substituted heterocycles of sufficient length. With each library member containing one heterocyclic and one carbocyclic component, sufficient diversification was achieved through modifications to, and substitutions within, both components. This allowed the production of libraries of building blocks possessing different curvatures and hydrogen-bonding abilities with the potential to complement functional groups and hydrogen bond donors and acceptors in the walls and floor of the minor groove. A fluorescent intercalator displacement (FID) assay was then used to select molecules with a preference for GC-rather than AT-rich sequences which identified the 4-(1-methyl-1H-pyrrol-3-yl)benzenamine (MPB) motif. Molecular dynamics simulations were undertaken using the AMBER v11 software in implicit solvent over a time-scale of 20ns with the structures in both a “carbonyl-in” and “NH-in” conformation. This demonstrated that the MPB component within the biaryl polyamides undergoes a switch in orientation, with a “carbonyl down” conformation preferred which induces GC specificity. As well as occupying an idealised hydrogen-bonding distance between two side-by-side guanine residues, MPB units contained within the MPB biaryl polyamide structures showed superior hydrogen bonding interactions with GC compared to AT sequences, with each carbonyl of the MPB unit hydrogen bonding to a guanine residue. This was further supported by free-energy of binding calculations undertaken using AMBER-MMPBSA, where the MPB units showed a distinct preference for GC sequences (a difference of -13kcal/mol in favour of “carbonyl -in” over “NH-in” structures). This discovery has been applied to the design and synthesis of more-complex MPB-containing DNA minor-groove binding ligands with a high level of GC-selectivity and significant in vitro and in vivo antitumour activity. Citation Format: Paul J M Jackson, Colin M. James, Khondaker M. Rahman, David E. Thurston. Use of molecular dynamic simulations to explore changes in conformation and sequence preference for DNA-binding biaryl polyamide structures. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4549. doi:10.1158/1538-7445.AM2013-4549

Pack it up, Pack it in: Unraveling H-NS Mediated Genome Packaging

Pack it up, Pack it in: Unraveling H-NS Mediated Genome Packaging

Computational Studies on the Effects of DNA Methylation on its Structure and Dynamics

DNA methylation occurs in CpG dinucleotides when a methyl group is added at the carbon 5 position of the C base ring, and it has an important role in regulating gene expression in higher eukaryotes. It is known that when cytosine bases are methylated, gene is silenced, but the precise role of methylated DNA in this process is still debated, and the mechanism by which methylation affects gene expression remains poorly understood.One hypothesis is that the methylation state of a DNA segment can alter the positions of nucleosomes, which in turn, determine gene expression. Several studies point to a strong link between CpG methylation and nucleosome positioning, but these effects might be indirect because methylation influences the binding of other factors, which could in turn trigger nucleosome repositioning. Because methylation chemically modifies DNA, the methylation status of a DNA sequence could influence its flexibility and, thus, its affinity to the nucleosome. Recent experimental studies suggest that CpG methylation can decrease the ability of DNA to bend into the major groove at the methylated site, and can thereby influence nucleosome positioning.To test the idea that methylation affects the ability of DNA to bend, we performed MD simulations of short DNA segments using an all atom force field. DNA conformations were characterized using the six rigid body base-pair step parameters, the minor groove width and the hybridization state. These properties were measured and compared for dsDNA segments in both the unmethylated and methylated states. In addition, all atom trajectories for both the cases were analyzed to extract a new set of parameters for a coarse grain model for DNA that can now be used to study the effects of methylation in larger DNA systems such as nucleosomes.

68 Deconvoluting the recognition of DNA shape from DNA sequence

In previous work (Slattery et al., 2011; Joshi et al., 2007), we described the importance of the 3-D structure of the DNA double helix – DNA shape – in the recognition of DNA binding sites by the Hox family of transcription factors. In particular, we found that local minima in the width of the DNA minor groove create electronegative pockets that are binding sites for amino acids with positively-charged side chains such as arginine. Moreover, our data argued that DNA shape is a consequence of DNA sequence, and thus led to the idea that the recognition of specific DNA sequences by DNA binding proteins is mediated by both base-readout, typically in the major groove, and shape-readout (Rohs et al., 2010). The relationship between DNA shape and DNA sequence leads to a logical loop that is difficult to tease apart: if shape is a consequence of sequence, then is the recognition of a binding site by a transcription factor mediated by the sequence of base pairs or by the resulting shape of the DNA molecule? One prediction of the shape recognition model is that if the shape-detecting amino acids are mutated, the shape of the preferred binding sites would become less important. We tested this prediction by mutating the basic residues of Hox proteins known to insert into narrow regions of the DNA minor groove and carrying out in vitro SELEX-seq experiments. Consistent with the importance of DNA shape recognition by this family of homeodomain proteins, we found that DNA molecules selected to bind these mutants had a much smaller propensity to have local minor groove width minima compared with DNA molecules selected to bind the wild type proteins. Further, we were able to transfer the shape recognition properties of one Hox protein to another Hox protein by introducing residues that bind to narrow minor grooves. Together, these findings argue that the recognition of DNA shape is a key aspect of binding site selection by this family of DNA binding proteins.

104 Probing DNA shape on molecular and genomic scales

Linear DNA sequence encodes its three-dimensional structure. The resulting DNA shape gives DNA unique physical and chemical properties that are explored by DNA-binding proteins (Rohs et al., 2009). Probing DNA shape, therefore, helps to reveal the origin of specificity in DNA-protein recognition. We have developed a Monte Carlo (MC) method for all-atom DNA structure prediction (Joshi et al., 2007). With key components such as parallelization and functionality to handle chemical modifications added, the revamped MC method can efficiently recover the DNA shape of any DNA sequence. To embrace the genomic era, we have trained a high-throughput (HT) prediction method on 2121 independent MC simulations (Slattery et al., 2011). Applying this method on whole genome DNA sequences of 66 Drosophila individuals, we found that among single nucleotide polymorphisms (SNPs) within the immediate vicinity of in vivo transcription factor (TF) binding sites, low-frequency SNPs tend to change the DNA minor groove width more than high-frequency SNPs. This indicates that shape-changing mutations are detrimental to TF binding and in vivo function. We also examined the dependency of DNase I cleavage events on DNA shape using our HT method. Our results show that the rate of DNase I cleavage closely tracks the width of the minor groove. MC predictions for chemically modified bases further demonstrate that cytosine methylation enhances cleavage directly adjacent to CpG dinucleotides through narrowing of the minor groove. In summary, with tools on hand to address biological questions on both molecular and genomic scales, our work provides new insights into gene regulation and evolution.

The DNA binding site specificity and antiproliferative property of ternary Pt(ii) and Zn(ii) complexes of phenanthroline and N,N′-ethylenediaminediacetic acid

The binding site specificity of the ternary complexes, [M(II)(phen)(edda)] (M(II) = Pt(2+) and Zn(2+); phen = 1,10-phenanthroline; edda = N,N'-ethylenediaminediacetic acid), for the self-complementary oligonucleotides (ODNs), ds(C(1)G(2)C(3)G(4)A(5)A(6)T(7)T(8)C(9)G(10)C(11)G(12))(2) (ODN1) and ds(C(1)G(2)C(3)G(4)T(5)A(6)T(7)A(8)C(9)G(10)C(11)G(12))(2) (ODN2), was studied by NMR measurements. The results indicated that [Pt(ii)(phen)(edda)] was partially intercalated between C(3)/G(10) and G(4)/C(9) base pairs of ODN1 and ODN2 in the major grooves, whereas [Zn(II)(phen)(edda)] was bound specifically to the TATA region of ODN2 in the minor groove and to the terminal G(2)/C(11) base pair of ODN1 in the major groove. The preference for the TATA sequence over the AATT sequence in the binding of [Zn(phen)(edda)] was attributed to the wider minor groove width of the TATA sequence. The bindings of the complexes to ct-DNA were also studied by UV, CD, and fluorescence spectroscopy. Additionally, the antiproliferative property of [Pt(II)(phen)(edda)] towards MCF7 breast cancer cells and normal MCF10-A cells was compared with that of [Zn(II)(phen)(edda)].

Molecular dynamics study of the role of the spine of hydration in DNA A-tracts in determining nucleosome occupancy.

A-tracts in DNA are generally associated with reduced nucleosome occupancy relative to other sequences, such that the longer the A-tract, the less likely that nucleosomes are found. In this paper, we use molecular dynamics methods to study the structural properties of A-tracts, and in particular the role that the spine of hydration in A-tracts plays in allowing DNA to distort to the highly bent structure needed to form nucleosomes. This study includes a careful assessment of the ability of the Amber (parmbsc0), CHARMM27, and BMS force fields to describe these structural waters for the AAATTT sequence (here capped with CGC and GCG), including comparisons with X-ray results. All three force fields show a spine of hydration, but BMS and Amber show better correlation with measured properties, such as in narrowing of the minor groove width associated with the A-tract. We have used Amber to study the spine properties for several 6 and 14 base-pair A-tracts (all capped with CGC and GCG). These calculations show that the structural waters are tightly bound for "pure" A-tracts that allow for A-water-T links, and for AT steps that allow for a T-water-T link, but other sequences disfavor structural water, especially those that lead to A-water-A, G-water-G, and C-water-A structures. In addition, we show that pure A-tracts favor roll values close to the Watson-Crick value for linear DNA, while A-tract sequences containing embedded T's, C's, or G's that are less favorable to structural water are more flexible. This implies the essential role of the spine of hydration in disfavoring nucleosome formation.