DNA Wrapping Research Articles

New insights into the helical structure of 30-nm chromatin fibers.

In eukaryotic cells, genomic DNA is highly packaged into chromatin to fit inside the nucleus. The accessibility of DNA is dependent on the packing density of chromatin fibers, which plays a critical role in transcriptional regulation and all other DNA-related biological processes. Understanding the structure of chromatin is key to illuminating the functions and molecular mechanisms of chromatin dynamics in epigenetic regulation. The structure of nucleosome—the basic unit of chromatin—has been defined by crystal X-ray studies at high resolution; it consists of 147 base pairs (bp) of DNA wrapping around an octamer of histones (two copies each of H2A, H2B, H3 and H4) approximately 1.7 times (Luger et al., 1997). Despite considerable efforts during the last three decades, however, how the nucleosomes interact with each other in a “beads-on-a-string” nucleosomal array to form a condensed 30-nm chromatin fiber—typically regarded as the secondary structure of chromatin—still remains controversial (Robinson and Rhodes, 2006). Based on the studies of native 30-nm fibers in nuclei or isolated from nuclei by electron microscopy, small angle X-ray/neutron scattering, optical dichroism and analytical centrifugation (Finch and Klug, 1976; Gerchman and Ramakrishnan, 1987; Ghirlando and Felsenfeld, 2008; Langmore and Paulson, 1983; Widom and Klug, 1985), two basic classes of structural models had been proposed in which nucleosomes are either arranged linearly in a one-start solenoid-type helix with bent linker DNA or zigzag back and forth in a two-start stack of nucleosomes connected by a relatively straight DNA linker (Widom and Klug, 1985; Williams et al., 1986; Woodcock et al., 1984). However, it is very difficult to resolve nucleosomes and linker DNA to trace the paths of nucleosomal arrays under conditions in nuclei or in isolated native chromatins in these early studies; in addition, the heterogeneous properties of nucleosomes in native chromatin with different DNA sequences/linker lengths and different histone compositions/modifications make it difficult to define the detailed structure of chromatin fibers. The problem has been partially addressed by reconstituting chromatin fibers in vitro on regular tandem repeats of unique nucleosome-positioning DNA sequences with purified histone proteins (Dorigo et al., 2004), which greatly improved the reproducibility and uniformity for structural analysis and allowed for a dissection of the contribution of individual factors, such as different NRLs, linker histones and histone variants/modifications. Using this system, the 3D cryo-EM structures of 30-nm chromatin fibers reconstituted in vitro from arrays of 12 nucleosomes in the presence of linker histone H1 have been recently determined at resolution of about 11 Å, which clearly show a histone H1-dependent left-handed twist of the repeating tetra-nucleosomal structural units (Song et al., 2014). The structures constitute the largest fragments of chromatin solved at this resolution which is high enough to allow clearly defining the spatial location of all individual nucleosomes and tracing the path of linker DNA, and provide new insights into the helical structure of 30-nm chromatin fibers.

Chiral symmetry breaking of a double-stranded helical chain through bend-writhe coupling.

This paper explores asymmetric elasticity of a double-stranded helical chain, which serves as a minimal model of biopolymers. The model consists of two elastic chains that mutually intertwine in a right-handed manner, forming a double-stranded helix. A simple numerical experiment for structural relaxation, which reduces the total elastic energy of the model monotonically without thermal fluctuations, reveals possible asymmetric elasticity inherent in the helical chain. It is first shown that a short segment of the double-stranded helical chain has a tendency to unwind when it is bent. It is also shown that a short segment of the helical chain has a tendency to writhe in the left direction upon bending. This tendency gives rise to a propensity for a longer segment of the chain to form a left-handed superhelix spontaneously upon bending. Finally, this propensity of the helical chain to form a left-handed superhelix is proposed to be a possible origin of the uniform left-handed wrapping of DNA around nucleosome core particles in nature. The results presented here could provide deeper insights into the roles and significance of helical chirality of biopolymers.

Minor groove geometry determines DNA exclusion from nucleosomes (929.1)

Like all eukaryotes, the meters of DNA in each of our cells must be compacted many thousands of times to fit inside the nucleus. The first level of compaction, spooling of DNA onto a nucleosome, involves the tight wrapping of DNA around eight histone proteins. Sharply bending DNA to wrap it into nucleosomes comes at an energetic cost related to the stiffness of the DNA. Nucleosomes prefer to form on DNA sequences that have a 10 bp periodicity between AA/AT/TA steps, with CC/CG/GC steps exactly out of phase. The sites of the AA/AT/TA and CC/CG/GC steps exactly correspond to locations of the highest DNA kinking, suggesting that these sequences represent anisotropically bendable sequences. Yet, inclusion of multiple AA steps, in the form of a poly(dA:dT) elements, results in exclusion of the DNA from nucleosomes in vivo. We seek to understand what unique chemical features of these poly‐dA elements are necessary for their exclusion from nucleosomes. Poly‐dA elements are known to adopt a unique structure; characterized crystallographically as a narrowed minor groove and by CD as a double peaked spectrum. Using CD spectroscopy and non‐natural nucleotides, we show that correct minor groove geometry at the base pair level is necessary for formation of the unique poly‐dA structure. Using in vitro competitive nucleosome reconstitution assays, we have further shown that non‐natural DNA sequences that adopt the unique poly‐dA structure are excluded from nucleosomes. Since poly‐dA like sequences are excluded from nucleosomes, this suggests that the unique structure formed by these sequences is energetically stiffer than generic sequence DNA.

Investigating the transcription initiation mechanism of E. coli RNAP by FRET (743.1)

E. coli RNA Polymerase (RNAP) recognizes the ‐10 and ‐35 upstream elements of the promoter DNA, which bends and wraps around RNAP on the back of the β subunit of the RNAP core. The recognition between RNAP and DNA promoter initiates the melting of about 13bp of DNA duplex surrounding the transcription start site in the active site of RNAP to form a promoter‐RNAP open complex (RPo). Even though transcription of DNA plays a critical role in gene expression and regulation, many important questions about the transcription initiation mechanism remains unanswered. To study the wrapping of DNA around RNAP, we have performed equilibrium FRET experiments on a strong promoter, λPr with Cy3 and Cy5 fluorophores on the upstream and downstream ends of the promoter DNA. We find that wrapping is present in closed complexes, as seen from complexes at 2°C and it persists in open complexes at 19°C. We further confirmed that the fluorophore‐labeled DNA complexed with RNAP is transcriptionally competent. Single‐labeled constructs also show permanganate reactivity, proving that fluorophore‐labeled promoter‐RNAP complexes are open. Further experiments are underway to study the kinetics of wrapping of promoter DNA around RNAP.Grant Funding Source: NIH GM103061

Complexes formed between DNA and poly(amido amine) dendrimers of different generations--modelling DNA wrapping and penetration.

This study deals with the build-up of biomaterials consisting of biopolymers, namely DNA, and soft particles, poly(amido amine) (PAMAM) dendrimers, and how to model their interactions. We adopted and applied an analytical model to provide further insight into the complexation between DNA (4331 bp) and positively charged PAMAM dendrimers of generations 1, 2, 4, 6 and 8, previously studied experimentally. The theoretical models applied describe the DNA as a semiflexible polyelectrolyte that interacts with dendrimers considered as either hard (impenetrable) spheres or as penetrable and soft spheres. We found that the number of DNA turns around one dendrimer, thus forming a complex, increases with the dendrimer size or generation. The DNA penetration required for the complex to become charge neutral depends on dendrimer generation, where lower generation dendrimers require little penetration to give charge neutral complexes. High generation dendrimers display charge inversion for all considered dendrimer sizes and degrees of penetration. Consistent with the morphologies observed experimentally for dendrimer/DNA aggregates, where highly ordered rods and toroids are found for low generation dendrimers, the DNA wraps less than one turn around the dendrimer. Disordered globular structures appear for high generation dendrimers, where the DNA wraps several turns around the dendrimer. Particularly noteworthy is that the dendrimer generation 4 complexes, where the DNA wraps about one turn around the dendrimers, are borderline cases and can form all types of morphologies. The net-charges of the aggregate have been estimated using zeta potential measurements and are discussed within the theoretical framework.

Correction: The Internal Organization of Mycobacterial Partition Assembly: Does the DNA Wrap a Protein Core?

As the result of an error that was introduced during the production process in the Materials and Methods section, there was an error in the equation contained in the 'Solution Neutron Scattering (SANS) with H/D-contrast Variation' section. The correct equation is: ∆λ/λ= 0.15.

Archaeal nucleosome positioning in vivo and in vitro is directed by primary sequence motifs

BackgroundHistone wrapping of DNA into nucleosomes almost certainly evolved in the Archaea, and predates Eukaryotes. In Eukaryotes, nucleosome positioning plays a central role in regulating gene expression and is directed by primary sequence motifs that together form a nucleosome positioning code. The experiments reported were undertaken to determine if archaeal histone assembly conforms to the nucleosome positioning code.ResultsEukaryotic nucleosome positioning is favored and directed by phased helical repeats of AA/TT/AT/TA and CC/GG/CG/GC dinucleotides, and disfavored by longer AT-rich oligonucleotides. Deep sequencing of genomic DNA protected from micrococcal nuclease digestion by assembly into archaeal nucleosomes has established that archaeal nucleosome assembly is also directed and positioned by these sequence motifs, both in vivo in Methanothermobacter thermautotrophicus and Thermococcus kodakarensis and in vitro in reaction mixtures containing only one purified archaeal histone and genomic DNA. Archaeal nucleosomes assembled at the same locations in vivo and in vitro, with much reduced assembly immediately upstream of open reading frames and throughout the ribosomal rDNA operons. Providing further support for a common positioning code, archaeal histones assembled into nucleosomes on eukaryotic DNA and eukaryotic histones into nucleosomes on archaeal DNA at the same locations. T. kodakarensis has two histones, designated HTkA and HTkB, and strains with either but not both histones deleted grow normally but do exhibit transcriptome differences. Comparisons of the archaeal nucleosome profiles in the intergenic regions immediately upstream of genes that exhibited increased or decreased transcription in the absence of HTkA or HTkB revealed substantial differences but no consistent pattern of changes that would correlate directly with archaeal nucleosome positioning inhibiting or stimulating transcription.ConclusionsThe results obtained establish that an archaeal histone and a genome sequence together are sufficient to determine where archaeal nucleosomes preferentially assemble and where they avoid assembly. We confirm that the same nucleosome positioning code operates in Archaea as in Eukaryotes and presumably therefore evolved with the histone-fold mechanism of DNA binding and compaction early in the archaeal lineage, before the divergence of Eukaryotes.

Charge transport and photoresponses in a single-stranded DNA/single-walled carbon nanotube composite film

Electrical conductivity and photoresponse measurements have been carried out on a single-stranded DNA (ssDNA)/single-walled carbon nanotube (SWNT) composite film in comparison to those of a SWNT film. While the temperature-dependent electrical conductivity of the pristine SWNT film was described well by the combined mechanism of a three-dimensional variable-range hopping and hopping conduction, that of the ssDNA/SWNT composite film followed a fluctuation-induced tunneling model. Besides, competition of photoexcited charge carrier generation and oxygen adsorption/photodesorption in the photoresponses of the films was observed and discussed in view of the role of the DNA wrapping. Thus, the biopolymer coating of the SWNTs is shown to play a significant role in modifying the charge dynamics of the composite system.

Single molecule analysis of DNA wrapping and looping by a circular 14mer wheel of the bacteriophage 186 CI repressor.

The lytic–lysogenic decision in bacteriophage 186 is governed by the 186 CI repressor protein in a unique way. The 186 CI is proposed to form a wheel-like oligomer that can mediate either wrapped or looped nucleoprotein complexes to provide the cooperative and competitive interactions needed for regulation. Although consistent with structural, biochemical and gene expression data, many aspects of this model are based on inference. Here, we use atomic force microscopy (AFM) to reveal the various predicted wrapped and looped species, and new ones, for CI regulation of lytic and lysogenic transcription. Automated AFM analysis showed CI particles of the predicted dimensions on the DNA, with CI multimerization favoured by DNA binding. Measurement of the length of the wrapped DNA segments indicated that CI may move on the DNA, wrapping or releasing DNA on either side of the wheel. Tethered particle motion experiments were consistent with wrapping and looping of DNA by CI in solution, where in contrast to λ repressor, the looped species were exceptionally stable. The CI regulatory system provides an intriguing comparison with that of nucleosomes, which share the ability to wrap and release similar sized segments of DNA.

The Nucleosome Revisited: Combined Small-Angle X-Ray/Neutron Scattering and Molecular Dynamics Applied to Reconstituted Mononucleosomes

Nucleosomes are the fundamental structural units of chromatin. A mononucleosome is comprised of 147 base pairs of DNA wrapped around a heterooctameric core of histone proteins that include H2A, H2B, H3, and H4, and other naturally occurring functional variants. In the mid-1970s, the SANS technique was key to the determination of the composition, structure, and conformation of the DNA within this basic unit, fifteen years before the first x-ray diffraction results were available. Small-angle scattering is a long-established technique that is well-suited for the study of macromolecules in vitro, as they exist in solution. Small-angle neutron scattering (SANS) is ideally suited to study nucleoprotein complexes, since the neutron scattering strengths of the different macromolecules differ with respect to each other. By employing contrast variation techniques, where the D2O:H2O ratio of the solutions is systematically varied, the gross structure and distribution of the individual components of a composite particle can be resolved in a model-independent fashion.We will present work which revisits these decades-old canonical measurements with a modern perspective. Experimental neutrons from beam line CG-3 at the ORNL High-Flux Isotope Reactor (HFIR) were successfully utilized to study reconstituted mononucleosomes by contrast variation. Our work marries this method with analytical centrifugation and the atomistic modeling approaches implemented in the program SASSIE. This approach allows us to directly reconcile available atomic structures with their solution properties, resulting in complete single and ensemble solution models. As a result of this work, we will present new insights into the distribution of the protein and DNA components of the mononucleosome in solution, including the properties of histone tails in solution and DNA wrapping. The methods presented provide an experimental framework for future examination of variant nucleosome and nucleosome-derived higher order assemblies.

Single-Molecule Assessment of DNA Supercoiling and Relaxation by S. Typhimurium and E. Coli DNA Gyrases

Type II topoisomerases maintain DNA topology by regulating the level of supercoiling of chromosomes. DNA gyrase is a unique and highly conserved bacterial Type II topoisomerase which is able to introduce negative supercoils into the genome. Using magnetic-tweezers, we assessed the supercoil generation and relaxation activities of Escherichia coli and Salmonella typhimurium DNA gyrases. Our results indicate that under single-enzyme conditions and 0.6-pN tension, both enzymes relax DNA at similar rates, but Salmonella gyrase pauses more often. At high enzyme concentration and lower tensions, Salmonella gyrase introduced negative supercoils faster and to a larger extent than E. coli gyrase. Sequence and structure analyses show that most of the differences between the two enzymes are in the C-terminal domain, involved in DNA wrapping. Our ongoing single-molecule experiments to assess the DNA wrapping activities of both enzymes may reveal differences in their mechanistic properties.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Carbon nanotube decorated magnetic microspheres as an affinity matrix for biomolecules.

Carbon nanotube (CNT) decorated magnetic microspheres were fabricated to develop a multimodal platform that utilizes non-covalent molecular interactions of CNTs to magnetically separate biomolecules. Hybrid CNT-microspheres prepared by a feasible method reported herein had a well-defined structure as characterized by Raman spectroscopy and scanning electron microscopy. Binding interactions of resulting magnetic CNT-microspheres with DNA oligonucleotides were studied to demonstrate that single stranded DNA (ssDNA) in a solution can be effectively recovered by magnetic CNT-microspheres through strong physical wrapping of DNA around CNTs' walls. The magnetic character of these CNT-microspheres combined with their capability to bind other molecules including DNA allows their use as an affinity matrix that can be utilized in affinity separation of biomolecules, and also as a platform to monitor non-covalent binding interactions of CNTs with other biomolecules. As a proof of concept, we report on the use of these CNT-microspheres in in vitro selection of ssDNA aptamers against carcinoembryonic antigen (CEA), a cancer biomarker, by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). ssDNA aptamer candidates that have strong affinity towards CEA were successfully separated magnetically from a pool of ssDNA (∼1014 molecules). Our results demonstrate that CNT-microspheres can serve as strong tools for affinity separation methodologies and can be utilized for various affinity pairs in solution.

Bio-functionalization of multi-walled carbon nanotubes

Here we present a hybrid approach to functionalize multi-walled carbon nanotubes in aqueous solution, exploring a non-covalent binding strategy. We focus on formation of hybrid complexes consisting of carbon nanotubes decorated by single stranded DNA, non-covalently attached using surfactants as intermediate layers. Unlike single walled carbon nanotubes, revealing easy side wall wrapping of DNA, we observe that wrapping of nucleic acids around multi-walled carbon nanotubes is diameter dependent.

Nucleosome Rewrapping Facilitates Transcription Factor Exchange

Transcription factors (TFs) play a central role in regulating the transcriptional state of gene. However, TFs are sterically occluded from their target sequences when it is wrapped around the histone octamer into a nucleosome. The combination of nucleosomal DNA unwrapping fluctuations and the high DNA sequence specificity of TFs facilitate TF to binding within nucleosomes. TFs achieve their high DNA sequence specificity by having resident times at their recognition sites that are much longer than minutes. This dramatically limits the rate at which a gene can switch between transcriptionally active and repressed states. We investigated with single-molecule Fluorescence Resonance Energy Transfer(FRET) measurements the influence of nucleosomal DNA wrapping fluctuations on TF binding and release from its target sequence within a mononucleosome and/or a nucleosome array. As expected, we find that nucleosomes inhibit the binding rate of a TF to its target sequence within a nucleosome. In contrast,we observe that the rate of TF dissociation from a nucleosome is increase by over a 100-fold as compared to dissociation from duplex DNA. This result suggests that the nucleosome promotes TF dissociation to facilitate the exchange of TFs at their target sequences and enable rapid regulation of gene expression.

Two Lysine Residues Enhance the Flexibility of DNA Wrapping around CENP-A Containing Nucleosome Core Particle

The structural dynamics of two nucleosomes, one was H3 containing and other was CENP-A containing nucleosome, were characterized with molecular dynamics simulations and the findings were experimentally confirmed. The simulations showed that histone proteins of both, which is the core structure of nucleosome, are structurally stable and maintain the structure determined by x-ray crystallography, while the wrapped DNAs are highly flexible at the entry or exit region and largely deviate from the crystal structures. In particular, about 20-25 bp DNAs of entry or exit of the CENP-A containing nucleosomes showed several times of open and close conformational changes within 100ns simulations, which was not observed on the H3 containing nucleosomes. The detailed analysis clarifies that this dynamics difference is due to the difference in two basic amino acids at the α-N helix, two Arg residues of H3 are mutated to Lys residue at the corresponding sites. The difference in ability of forming hydrogen-bond to the DNA controls the flexibility of the nucleosomal DNA at entry or exit region. This increase in flexibility was confirmed with a nuclease susceptibility assay of a nucleosome that contains H3 mutant with the two Arg residues replaced with Lys.

Effect of DNA Methylation on the Assembly of Nucleosomes

We monitored nucleosome assembly real-time in a time-resolved manner at a single molecule level using FRET. Our data indicate that the mechanical property of nucleosomal DNA altered upon CpG methylation impact on the kinetics of DNA wrapping during nucleosome assembly. Based on the results, we propose paradoxical roles of CpG methylation in gene regulation.

Asymmetric Unwrapping of Nucleosome Revealed by Single Molecule Fluorescence-Force Spectroscopy

The nucleosome is the fundamental unit of eukaryotic chromatin structure which compacts DNA in the nucleus. It is a stable yet dynamic complex which is significant to transcriptional regulation. In cells, DNA is likely to be under tension to varying extents. For example enzymes such as RNA polymerase and chromatin remodelers can generate force on nucleosomes. Thus it is important to understand the dynamics of nucleosomes under mechanical constraints. We are utilizing a cutting-edge single-molecule optomechanical technology which combines fluorescence with optical tweezers (aka ‘fleezers') to probe the conformational transitions of nucleosomes under force. Our hybrid fleezers instrument provides fluorescence readouts with nm resolution under pN levels of force applied in order to locally monitor conformational dynamics of nucleosome at various coordinates. Here, we report the first observation of force-dependent gradual unwrapping and rewrapping of the nucleosome using fluorescence reporters. Moreover, we observed force-induced two-state hopping corresponding to the opening and closing of the outer DNA wrap as well as more complex transitions for the inner wrap. Interestingly, our fluorescence probes at various locations report significant asymmetric unwrapping behavior. In our pulling experiments, it takes less than 6 pN to unwrap the outer DNA at the 3' end of the 601 positioning sequence but takes up to ∼15-17 pN to unwrap the DNA from the 5' end. Asymmetric unwrapping may have implications for how enzymes such as chromatin remodelers interact with nucleosome substrates.

Impact of DNA Twist Accumulation on Progressive Helical Wrapping of Torsionally Constrained DNA

DNA wrapping is an important mechanism for chromosomal DNA packaging in cells and viruses. Previous studies of DNA wrapping have been performed mostly on torsionally unconstrained DNA, while in vivo DNA is often under torsional constraint. In this study, we extend a previously proposed theoretical model for wrapping of torsionally unconstrained DNA to a new model including the contribution of DNA twist energy, which influences DNA wrapping drastically. In particular, due to accumulation of twist energy during DNA wrapping, it predicts a finite amount of DNA that can be wrapped on a helical spool. The predictions of the new model are tested by single-molecule study of DNA wrapping under torsional constraint using magnetic tweezers. The theoretical predictions and the experimental results are consistent with each other and their implications are discussed.

The GyrA-box determines the geometry of DNA bound to gyrase and couples DNA binding to the nucleotide cycle

DNA gyrase catalyses the adenosine triphosphate-dependent introduction of negative supercoils into DNA. The enzyme binds a DNA-segment at the so-called DNA-gate and cleaves both DNA strands. DNA extending from the DNA-gate is bound at the perimeter of the cylindrical C-terminal domains (CTDs) of the GyrA subunit. The CTDs are believed to contribute to the wrapping of DNA around gyrase in a positive node as a prerequisite for strand passage towards negative supercoiling. A conserved seven amino acid sequence motif in the CTD, the so-called GyrA-box, has been identified as a hallmark feature of gyrases. Mutations of the GyrA-box abolish supercoiling. We show here that the GyrA-box marginally stabilizes the CTDs. Although it does not contribute to DNA binding, it is required for DNA bending and wrapping, and it determines the geometry of the bound DNA. Mutations of the GyrA-box abrogate a DNA-induced conformational change of the gyrase N-gate and uncouple DNA binding and adenosine triphosphate hydrolysis. Our results implicate the GyrA-box in coordinating DNA binding and the nucleotide cycle.

Chiral selection in supercoiling and wrapping of DNA

This paper gives an account of our recent studies on the mechanisms for chiral selection in super-coiling and wrapping of DNA. We first present a compact model of double-stranded DNA (Model 1), which consists of two elastic chains that mutually intertwine in a right-handed manner to form a double-stranded helix. Numerical analysis of this model suggests an intrinsic propensity of DNA to writhe in the left direction upon bending. Based on this asymmetric elasticity of DNA, we present a further simplified model of DNA (Model 2), which is a single-chained homopolymer with the propensity to writhe in the left direction upon bending. This simplified model is incorporated into a Langevin dynamics study to explore the origin of the uniform left-handed wrapping of DNA around a nucleosome core particle in nature. We finally show that the propensity of DNA to writhe in the left direction upon bending gives rise to the selective left-handed wrapping, provided that the size of the core particle is appropriate. This result suggests the fundamental significance of the asymmetric elasticity of helical biopolymers in their structural dynamics and functions.