Bare DNA Research Articles

Nucleosome Kinetics Regulates the Binding Timescales of Non-Histone Proteins to DNA Sites

Nucleosome organization in the promoter region of genes is thought to be a crucial factor in determining the accessibility of target DNA sites of regulatory non-histone proteins (e.g. transcription factors (TF), TATA- binding proteins (TBP) and RNA polymerase). Binding of most of these regulatory proteins needs access to bare DNA which depends on the “kinetic” rearrangement of nucleosomes in that region. Most of the works done in the past, focus only on static nucleosome occupancy and not on its stochastic kinetics. We go beyond this static picture and using a simple model for nucleosome kinetics address the kinetic problem of TF/TBP binding by posing it as a “first passage time (FPT)” problem. We have solved this model analytically (with some approximations) and supported our theory numerically via Monte Carlo simulations. In our model, we account for binding and dissociation of nucleosomes, action of ATP-dependent remodelers, sequence dependent histone-DNA interaction potential, and explicit competition between TF/TBP and nucleosomes for site accessibility. We find that even if the nucleosome occupancy is the same, differences in kinetics of nucleosomes can cause differences in kinetic histories of TF/TBP binding. We show that the first passage time of TF/TBP binding could be very different, depending upon the initial nucleosome organization and the genomic sequence. The first passage timescales of TF/TBP binding (in competition with nucleosomes) may also be indirectly indicated from calculations of exposure timescales with kinetic nucleosomes alone. Our analytical method provides an easy way to compute the first passage time of TF/TBP binding. We apply this method to a genome-wide study of TBP binding timescales at TATA sites for the genes of Saccharomyces cerevisiae.

Relevance of the Drag Force during Controlled Translocation of a DNA-Protein Complex through a Glass Nanocapillary.

Combination of glass nanocapillaries with optical tweezers allowed us to detect DNA-protein complexes in physiological conditions. In this system, a protein bound to DNA is characterized by a simultaneous change of the force and ionic current signals from the level observed for the bare DNA. Controlled displacement of the protein away from the nanocapillary opening revealed decay in the values of the force and ionic current. Negatively charged proteins EcoRI, RecA, and RNA polymerase formed complexes with DNA that experienced electrophoretic force lower than the bare DNA inside nanocapillaries. Force profiles obtained for DNA-RecA in our system were different than those in the system with nanopores in membranes and optical tweezers. We suggest that such behavior is due to the dominant impact of the drag force comparing to the electrostatic force acting on a DNA-protein complex inside nanocapillaries. We explained our results using a stochastic model taking into account the conical shape of glass nanocapillaries.

DNA Elasticity from Short DNA to Nucleosomal DNA.

Active biological processes like transcription, replication, recombination, DNA repair, and DNA packaging encounter bent DNA. Machineries associated with these processes interact with the DNA at short length (<100 base pair) scale. Thus, the study of elasticity of DNA at such length scale is very important. We use fully atomistic molecular dynamics (MD) simulations along with various theoretical methods to determine elastic properties of dsDNA of different lengths and base sequences. We also study DNA elasticity in nucleosome core particle (NCP) both in the presence and the absence of salt. We determine stretch modulus and persistence length of short dsDNA and nucleosomal DNA from contour length distribution and bend angle distribution, respectively. For short dsDNA, we find that stretch modulus increases with ionic strength while persistence length decreases. Calculated values of stretch modulus and persistence length for DNA are in quantitative agreement with available experimental data. The trend is opposite for NCP DNA. We find that the presence of histone core makes the DNA stiffer and thus making the persistence length 3-4 times higher than the bare DNA. Similarly, we also find an increase in the stretch modulus for the NCP DNA. Our study for the first time reports the elastic properties of DNA when it is wrapped around the histone core in NCP. We further show that the WLC model is inadequate to describe DNA elasticity at short length scale. Our results provide a deeper understanding of DNA mechanics and the methods are applicable to most protein-DNA complexes.

Identifying the Location of a Single Protein along the DNA Strand Using Solid-State Nanopores.

Solid-state nanopore has been widely studied as an effective tool to detect and analyze small biomolecules, such as DNA, RNA, and proteins, at a single molecule level. In this study, we demonstrate a rapid identification of the location of zinc finger protein (ZFP), which is bound to a specific locus along the length of a double-stranded DNA (dsDNA) to a single protein resolution using a low noise solid-state nanopore. When ZFP labeled DNAs were driven through a nanopore by an externally applied electric field, characteristic ionic current signals arising from the passage of the DNA/ZFP complex and bare DNA were detected, which enabled us to identify the locations of ZFP binding site. We examined two DNAs with ZFP binding sites at different positions and found that the location of the additional current drop derived from the DNA/ZFP complex is well-matched with a theoretical one along the length of the DNA molecule. These results suggest that the protein binding site on DNA can be mapped or that genetic information can be read at a single molecule level using solid-state nanopores.

Influence of DNA Binding Dyes on Bare DNA Structure Studied with Atomic Force Microscopy

Fluorescent dyes are widely used for staining and visualization of DNA in optical microscopy based methods. Even though for some dyes the mechanism of binding is known, how this binding affects DNA remains poorly understood. Here we present a novel experimental study of the influence of staining dyes on DNA properties. We use atomic force microscopy which allows quantification and measurement of structural properties of stained DNA with nanometer resolution. We studied the influence of dyes on the persistence length, the total contour length, and the morphology of individual DNA molecules. We tested three widely used dyes known to differently bind DNA molecules, namely PicoGreen, Dapi, and DRAQ5. Based on our measurements, when imaged at typical concentrations (manufacturer suggested concentrations used for cell imaging), PicoGreen dye showed little effect, Dapi dye decreased the DNA persistence length, and DRAQ5 decreased the persistence length and elongated the DNA. When used at high concentrations, all of the dyes induced drastic changes in the DNA morphology. Our study clearly shows that DNA-binding dyes, irrespective of their DNA binding mechanisms, strongly influence the physical properties of DNA. These changes are strongly dose and dye type dependent and therefore should be taken into consideration when conducting experiments with DNA.

Liquid crystals of self-assembled DNA bottlebrushes.

Early theories for bottlebrush polymers have suggested that the so-called main-chain stiffening effect caused by the presence of a dense corona of side chains along a central main chain should lead to an increased ratio of effective persistence length (lp,eff) over the effective thickness (Deff) and, hence, ultimately to lyotropic liquid crystalline behavior. More recent theories and simulations suggest that lp,eff ∼ Deff, such that no liquid crystalline behavior is induced by bottlebrushes. In this paper we investigate experimentally how lyotropic liquid crystalline behavior of a semiflexible polymer is affected by a dense coating of side chains. We use semiflexible DNA as the main chain. A genetically engineered diblock protein polymer C4K12 is used to physically adsorb long side chains on the DNA. The C4K12 protein polymer consists of a positively charged binding block (12 lysines, K12) and a hydrophilic random coil block of 400 amino acids (C4). From light scattering we find that, at low ionic strength (10 mM Tris-HCl), the thickness of the self-assembled DNA bottlebrushes is on the order of 30 nm and the effective grafting density is 1 side chain per 2.7 nm of DNA main chain. We find these self-assembled DNA bottlebrushes form birefringent lyotropic liquid crystalline phases at DNA concentrations as low as 8 mg/mL, roughly 1 order of magnitude lower than for bare DNA. Using small-angle X-ray scattering (SAXS) we show that, at DNA concentrations of 12 mg/mL, there is a transition to a hexagonal phase. We also show that, while the effective persistence length increases due to the bottlebrush coating, the effective thickness of the bottlebrush increases even more, such that in our case the bottlebrush coating reduces the effective aspect ratio of the DNA. This is in agreement with theoretical estimates that show that, in most cases of practical interest, a bottlebrush coating will lead to a decrease of the effective aspect ratio, whereas, only for bottlebrushes with extremely long side chains at very high grafting densities, a bottlebrush coating may be expected to lead to an increase of the effective aspect ratio.

Enhanced Electrostatic Force Microscopy Imaging Reveals Mechanism of TRF2 Mediated DNA Compaction

T-loop formation at telomeres is proposed to play an important role in telomere protection through the sequestration of the 3’ single-stranded overhang. Previous studies indicated that shelterin protein TRF2 modulates telomere structure by promoting DNA compaction and T-loop formation. To further understand the mechanism underlying TRF2-mediated DNA compaction, we applied Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM), a recently developed technique capable of high-resolution imaging of weak electrostatic potentials. DREEM images of nucleosomes clearly reveal DNA strands wrapped around histone proteins in nucleosomal arrays. In contrast, DREEM imaging shows DNA compacted inside TRF2 complexes through a 3-dimensional stacking of TRF2 dimers mediated by collective actions of multiple copies of TRF2 proteins. TRF2-mediated DNA compaction leads to electric potential gradients across the complexes. Surprisingly, while DNA wrapped around histones displays similar electrostatic potential signals compared to bare DNA, TRF2 DNA compaction leads to significant differences in the electrical potential signals inside multi-oligomeric TRF2 complexes compared to protein or DNA alone. These results clearly demonstrate the electrostatic changes in TRF2-DNA complexes upon oligomerizaton of proteins and DNA compaction, and underscore the importance of developing new electrostatic force microscopy imaging techniques for studying biological systems.

Quantitative DNA Binding, Looping, and Compaction Properties of the HIV-1 Viral Protein R

Human immunodeficiency virus type 1 (HIV-1) Viral protein R (Vpr) is packaged into virions (∼200 molecules) and is essential for viral replication. While several in vivo functions have been attributed to Vpr, one primary function is believed to be transport of the HIV-1 pre-integration complex into the nucleus. Because the nuclear pore diameter is approximately 25 nm, the DNA must be highly compact in order to enter the nucleus. To understand the mechanism by which Vpr may facilitate this nuclear transport, we combine single molecule stretching and atomic force microscopy (AFM). We measure the DNA binding affinity of Vpr for the first time. We then investigate the ability of Vpr to both bind and compact DNA. To do this, we hold DNA at low force for fixed times, allowing it to form loops and other compact structures. We find that the timescale required for the formation of large loops due to protein-DNA bridging is several minutes. In contrast, by holding the DNA molecule at a constant force of 15 pN and measuring its length change, Vpr can also actively compact DNA on the timescale of tens of seconds (15 ± 2 s), representing a different compaction process involving DNA shortening likely due to Vpr binding alone. The persistence length determined from AFM at a low concentration is much longer than that of bare DNA, suggesting that Vpr forms shorter but more rigid structures upon binding DNA. AFM images also demonstrate DNA bridging between strands, consistent with the looping observed in optical tweezers experiments. These results support a model in which Vpr translocates the viral DNA into the nucleus by forming compact structures mediated by protein-DNA and protein-protein interactions.

Self-Healing Biomaterials: Entangled DNA Networks

The material that the cell uses to store genetic information actually becomes self-healing material when it must be segregated, transported across the length of the cell even in the face of its entanglements with itself, or condensed to prepare for this large scale transport. This is a naturally-occuring soft material that is capable of self-repair. Moreover, there is evidence that the material first senses the possibility of a stress-inducing, potentially fatal (to genome stability) topological entanglement, and quickly fortifies itself to limit or prevent the damage. The issue of how topology is conserved (controlled) by this type of biomaterial is itself an interesting mystery. I will present our experimental results using a bottom-up design approach, borrowing the naturally-occurring materials from the cell to study the design principles of this behavior. We incorporated micron-sized particles in lambda-DNA entangled networks in the presence of the topoisomerase II motor that performs the strand passage, at controllable ratio of enzyme units per average DNA entanglement and ATP concentration. We used bright-field microscopy to directly track the movement of the particles, which couple to the DNA fluctuating movement. Our observed scaling behavior suggests entangled dynamics in the bare DNA system, and nonentangled Rouse dynamics, with enzyme performing topological constraint relaxation, in the DNA+topo II + ATP system. These very time-dependent scaling behaviors are all predicted theoretically for entangled polymers with inclusion of a constrained release process in the case of presence of active topo II. The material self-heals to such a degree that, at saturating topoisomerase II motor and ATP concentrations, the long DNA polymer molecules do not even “feel” one another despite being entangled. We compare our experimental results to predictions of the constraint release model, measuring the “healing rate” at different dynamical length scales.

Chiral electronic transitions in fluorescent silver clusters stabilized by DNA.

Fluorescent, DNA-stabilized silver clusters are receiving much attention for sequence-selected colors and high quantum yields. However, limited knowledge of cluster structure is constraining further development of these "AgN-DNA" nanomaterials. We report the structurally sensitive, chiroptical activity of four pure AgN-DNA with wide ranging colors. Ubiquitous features in circular dichroism (CD) spectra include a positive dichroic peak overlying the lowest energy absorbance peak and highly anisotropic, negative dichroic peaks at energies well below DNA transitions. Quantum chemical calculations for bare chains of silver atoms with nonplanar curvature also exhibit these striking features, indicating electron flow along a chiral, filamentary metallic path as the origin for low-energy AgN-DNA transitions. Relative to the bare DNA, marked UV changes in CD spectra of AgN-DNA and silver cation-DNA solutions indicate that ionic silver content constrains nucleobase conformation. Changes in solvent composition alone can reorganize cluster structure, reconfiguring chiroptical properties and fluorescence.

Probing transient protein-mediated DNA linkages using nanoconfinement.

We present an analytic technique for probing protein-catalyzed transient DNA loops that is based on nanofluidic channels. In these nanochannels, DNA is forced in a linear configuration that makes loops appear as folds whose size can easily be quantified. Using this technique, we study the interaction between T4 DNA ligase and DNA. We find that T4 DNA ligase binding changes the physical characteristics of the DNApolymer, in particular persistence length and effective width. We find that the rate of DNA fold unrolling is significantly reduced when T4 DNA ligase and ATP are applied to bare DNA. Together with evidence of T4 DNA ligase bridging two different segments of DNA based on AFM imaging, we thus conclude that ligase can transiently stabilize folded DNA configurations by coordinating genetically distant DNA stretches.

On the Effects of Intercalators in DNA Condensation: A Force Spectroscopy and Gel Electrophoresis Study

In this work we have characterized the effects of the intercalator ethidium bromide (EtBr) on the DNA condensation process by using force spectroscopy and gel electrophoresis. We have tested two condensing agents: spermine (spm(4+)), a tetravalent cationic amine which promotes cation-induced DNA condensation, and poly(ethylene glycol) (PEG), a neutral polymer which promotes DNA ψ-condensation. Two different types of experiments were performed. In the first type, bare DNA molecules disperse in solution are first treated with EtBr for intercalation, and then the condensing agent is added to the sample with the purpose of verifying the effects of the intercalator in hindering DNA condensation. In the second experiment type, the bare DNA molecules are first condensed, and then the intercalator is added to the sample in order to verify its influence on the previously condensed DNA. The results obtained with the two different experimental techniques used agree very well, indicating that previously intercalated EtBr can hinder both cation-induced and ψ-condensation, being more efficient in the first case. On the other hand, EtBr has little effect on the previously formed cation-induced condensates, but is efficient in unfolding the ψ-condensates.

Virus-Encapsulated DNA Origami Nanostructures for Cellular Delivery

DNA origami structures can be programmed into arbitrary shapes with nanometer scale precision, which opens up numerous attractive opportunities to engineer novel functional materials. One intriguing possibility is to use DNA origamis for fully tunable, targeted, and triggered drug delivery. In this work, we demonstrate the coating of DNA origami nanostructures with virus capsid proteins for enhancing cellular delivery. Our approach utilizes purified cowpea chlorotic mottle virus capsid proteins that can bind and self-assemble on the origami surface through electrostatic interactions and further pack the origami nanostructures inside the viral capsid. Confocal microscopy imaging and transfection studies with a human HEK293 cell line indicate that protein coating improves cellular attachment and delivery of origamis into the cells by 13-fold compared to bare DNA origamis. The presented method could readily find applications not only in sophisticated drug delivery applications but also in organizing intracellular reactions by origami-based templates.

Transcription factors IIS and IIF enhance transcription efficiency by differentially modifying RNA polymerase pausing dynamics

Transcription factors IIS (TFIIS) and IIF (TFIIF) are known to stimulate transcription elongation. Here, we use a single-molecule transcription elongation assay to study the effects of both factors. We find that these transcription factors enhance overall transcription elongation by reducing the lifetime of transcriptional pauses and that TFIIF also decreases the probability of pause entry. Furthermore, we observe that both factors enhance the processivity of RNA polymerase II through the nucleosomal barrier. The effects of TFIIS and TFIIF are quantitatively described using the linear Brownian ratchet kinetic model for transcription elongation and the backtracking model for transcriptional pauses, modified to account for the effects of the transcription factors. Our findings help elucidate the molecular mechanisms by which transcription factors modulate gene expression.

Dynamics of NAP1-Assisted Nucleosome Assembly Imaged with High-Speed Atomic Force Microscopy

Nucleosome assembly is a vital part of chromatin maintenance for all eukaryotic cells. The histone chaperone NAP1 is one of the proteins involved in this process, guiding histones into the nucleus and assembling them into nucleosomes without use of ATP or other energy sources. It is known that NAP1 first brings H3H4 histone tetramers to the DNA to form a tetrasome, and in a second step H2a and H2B histones are added, but mechanistic insight into this process is still lacking. We use high-speed AFM to image NAP-1 assembled tetrasomes in vitro with spatial and temporal resolutions of the order of 1 nm and 1 s. We observe a rich palette of dynamical processes, among which are spontaneous tetrasome dissociation, cluster formation and a transient association between bare DNA and NAP1. One intriguing observation is the hopping of a tetrasome between two stable positions spaced only a few nanometers apart. We believe this motion can be ascribed to a re-orientation of the DNA around the histones, a phenomenon that we also observed in magnetic tweezer single molecule assays.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Friction and Interactions between Bare DNA Molecules: The Role of DNA Handedness

Cellular DNA is packaged through a range of re-modeling proteins. As a result, disparate regions of the genome may find themselves in close contact with one another. Here, we address an important and emerging issue resulting from this fact: what happens when DNA becomes entangled? Is it possible that the mechanical properties of DNA can influence interactions of the genome even in the absence of proteins? Recent experiments have suggested that the handedness of DNA may affect the stability of DNA-DNA pairs.Using a single-molecule approach, we employ a unique 4-way optical trap to wrap two separate DNA molecules around one another. By displacing optically trapped beads, it is possible to slide one DNA molecule along the other. Using triangulation, we are then able to track the location at which the DNA molecules are entwined. This analysis reveals that a small but clear friction is present only when sliding the DNA molecule through a right-handed wrap. Moreover, when sliding the DNA further, tension is built up and then released, giving rise to an abrupt stick-release force pattern. Strikingly, the latter is once again found predominantly in the case of a right-handed wrap. By staining the DNA molecules with the force-sensitive fluorescent dye Sytox, we are able to both visualize and measure this build-up and release of force. The stick-release behavior occurs only after a force of ∼65 pN has been applied. This suggests that the DNA molecules interact at their point of contact via melting of base-pairs, possibly creating a 3- or 4-stranded complex.The existence of chiral and force dependent interactions between bare DNA molecules has strong implications for the study of DNA in confined environments and raises interesting questions as to their potential importance in vivo.

Time-dependent bending rigidity and helical twist of DNA by rearrangement of bound HU protein

HU is a protein that plays a role in various bacterial processes including compaction, transcription and replication of the genome. Here, we use atomic force microscopy to study the effect of HU on the stiffness and supercoiling of double-stranded DNA. First, we measured the persistence length, height profile, contour length and bending angle distribution of the DNA–HU complex after different incubation times of HU with linear DNA. We found that the persistence and contour length depend on the incubation time. At high concentrations of HU, DNA molecules first become stiff with a larger value of the persistence length. The persistence length then decreases over time and the molecules regain the flexibility of bare DNA after ∼2 h. Concurrently, the contour length shows a slight increase. Second, we measured the change in topology of closed circular relaxed DNA following binding of HU. Here, we observed that HU induces supercoiling over a similar time span as the measured change in persistence length. Our observations can be rationalized in terms of the formation of a nucleoprotein filament followed by a structural rearrangement of the bound HU on DNA. The rearrangement results in a change in topology, an increase in bending flexibility and an increase in contour length through a decrease in helical pitch of the duplex.

Kinesin KIFC1 actively transports bare double-stranded DNA

During the past years, exogenous DNA molecules have been used in gene and molecular therapy. At present, it is not known how these DNA molecules reach the cell nucleus. We used an in cell single-molecule approach to observe the motion of exogenous short DNA molecules in the cytoplasm of eukaryotic cells. Our observations suggest an active transport of the DNA along the cytoskeleton filaments. We used an in vitro motility assay, in which the motion of single-DNA molecules along cytoskeleton filaments in cell extracts is monitored; we demonstrate that microtubule-associated motors are involved in this transport. Precipitation of DNA-bound proteins and mass spectrometry analyses reveal the preferential binding of the kinesin KIFC1 on DNA. Cell extract depletion of kinesin KIFC1 significantly decreases DNA motion, confirming the active implication of this molecular motor in the intracellular DNA transport.

A Quantitative Kinetic Model of Eukaryotic Transcription Elongation from Single-Molecule Experiments

Transcription by RNA polymerase II (Pol II) is an important point of control for eukaryotic gene expression, and has been extensively studied by structural, biochemical, and biophysical methods. During transcription elongation, Pol II moves processively along the DNA template and synthesizes RNA, one nucleotide at a time. A comprehensive kinetic characterization of this process, the transcription elongation cycle, incorporating both the on-pathway nucleotide addition phase and the off-pathway pausing phase, is still lacking. We used an optical trapping assay to follow transcription by individual yeast Pol II on bare or nucleosomal DNA. The single-molecule technique employed here allowed us to separate analyze the nucleotide addition phase and pausing phase separately, and arrived at a kinetic model that quantitatively describes both phases of transcription elongation. This model was used to measure the effects of a point mutation in the trigger loop motif on the on- and off-pathway dynamics of transcription, and can serve as a general framework to study the roles of various transcription and chromatin remodeling factors in transcription.

Natural phage nanoparticle-mediated real-time immuno-PCR for ultrasensitive detection of protein marker

Current immuno-PCR methods either use bare or nanostructured DNA as a reporter or combine phage display for antigen binding and reporting, however, they are often complex to carry out and lack universality. We present a novel and universal design of immuno-PCR termed natural phage nanoparticle-mediated real-time immuno-PCR.