ABSTRACT
Telomeres, complexes of DNA and proteins, protect ends of linear chromosomes. In humans, the two shelterin proteins TRF1 and TIN2, along with cohesin subunit SA1, were proposed to mediate telomere cohesion. Although the ability of the TRF1-TIN2 and TRF1-SA1 systems to compact telomeric DNA by DNA-DNA bridging has been reported, the function of the full ternary TRF1-TIN2-SA1 system has not been explored in detail. Here, we quantify the compaction of nanochannel-stretched DNA by the ternary system, as well as its constituents, and obtain estimates of the relative impact of its constituents and their interactions. We find that TRF1, TIN2, and SA1 work synergistically to cause a compaction of the DNA substrate, and that maximal compaction occurs if all three proteins are present. By altering the sequence with which DNA substrates are exposed to proteins, we establish that compaction by TRF1 and TIN2 can proceed through binding of TRF1 to DNA, followed by compaction as TIN2 recognizes the previously bound TRF1. We further establish that SA1 alone can also lead to a compaction, and that compaction in a combined system of all three proteins can be understood as an additive effect of TRF1-TIN2 and SA1-based compaction. Atomic force microscopy of intermolecular aggregation confirms that a combination of TRF1, TIN2, and SA1 together drive strong intermolecular aggregation as it would be required during chromosome cohesion.
Subject(s)
Telomere , Telomeric Repeat Binding Protein 1 , Humans , Telomeric Repeat Binding Protein 1/chemistry , Telomeric Repeat Binding Protein 1/genetics , Telomeric Repeat Binding Protein 1/metabolism , Telomere/metabolism , Shelterin Complex , DNAABSTRACT
The transmission of light through sub-wavelength apertures (zero-mode waveguides, ZMW) in metal films is well-explored. It introduces both an amplitude modulation as well as a phase shift to the oscillating electromagnetic field. We propose a nanophotonic interferometer by bringing two ZMW (â¼100 nm diameter) in proximity and monitoring the distribution of transmitted light in the back-focal plane of collecting microscope objective (1.3 N.A.). We demonstrate that both an asymmetry induced by the binding of a quantum dot in one of the two ZMW, as well as an asymmetry in ZMW diameter yield qualitatively similar transmission patterns. We find that the complex pattern can be quantified through a scalar measure of asymmetry along the symmetry axis of the aperture pair. In a combined experimental and computational exploration of detectors with differing ZMW diameters, we find that the scalar asymmetry is a monotonous function of the diameter difference of the two apertures, and that the scalar asymmetry measure is higher if the sample is slightly displaced from the focal plane of the collecting microscope objective. An optimization of the detector geometry determined that the maximum response is achieved at an aperture separation that is comparable to the wavelength on the exit side of the sensor. For small separations of apertures, on the order of a quarter of the wavelength and less, the signal is strongly polarization dependent, while for larger separations, on the order of the wavelength or larger, the signal becomes essentially polarization-independent.
ABSTRACT
The methyl-CpG-binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status.
Subject(s)
DNA Methylation , DNA-Binding Proteins , CpG Islands , DNA-Binding Proteins/metabolism , Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Nucleosomes , Protein Binding , Transcription Factors/metabolism , Humans , Single Molecule ImagingABSTRACT
The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2-TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein-DNA structures, and monitoring of DNA-DNA and DNA-RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA-dsDNA, dsDNA-ssDNA and dsDNA-ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.
Subject(s)
DNA/metabolism , Telomere-Binding Proteins/metabolism , Telomere/metabolism , Telomeric Repeat Binding Protein 2/metabolism , Animals , DNA/chemistry , DNA/genetics , HeLa Cells , Humans , Mice, Inbred C57BL , Microscopy, Atomic Force , Nucleic Acid Conformation , Protein Binding , Shelterin Complex/genetics , Shelterin Complex/metabolism , Telomere/genetics , Telomere-Binding Proteins/genetics , Telomeric Repeat Binding Protein 2/geneticsABSTRACT
TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.
Subject(s)
Cell Adhesion Molecules/metabolism , Telomere-Binding Proteins/metabolism , Telomeric Repeat Binding Protein 2/metabolism , Cell Adhesion Molecules/physiology , DNA/metabolism , DNA-Binding Proteins/metabolism , Humans , Microscopy, Atomic Force/methods , Models, Molecular , Multiprotein Complexes/metabolism , Protein Binding , Protein Isoforms/metabolism , Shelterin Complex/metabolism , Shelterin Complex/physiology , Telomere/metabolism , Telomere-Binding Proteins/physiology , Telomeric Repeat Binding Protein 1/metabolism , Telomeric Repeat Binding Protein 1/physiology , Telomeric Repeat Binding Protein 2/physiologyABSTRACT
Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , R-Loop Structures , RNA-Binding Proteins/metabolism , Binding Sites , DNA/metabolism , RNA/metabolism , CohesinsABSTRACT
BACKGROUND: MeCP2 and MBD2 are members of a family of proteins that possess a domain that selectively binds 5-methylcytosine in a CpG context. Members of the family interact with other proteins to modulate DNA packing. Stretching of DNA-protein complexes in nanofluidic channels with a cross-section of a few persistence lengths allows us to probe the degree of compaction by proteins. RESULTS: We demonstrate DNA compaction by MeCP2 while MBD2 does not affect DNA configuration. By using atomic force microscopy (AFM), we determined that the mechanism for compaction by MeCP2 is the formation of bridges between distant DNA stretches and the formation of loops. CONCLUSIONS: Despite sharing a similar specific DNA-binding domain, the impact of full-length 5-methylcytosine-binding proteins can vary drastically between strong compaction of DNA and no discernable large-scale impact of protein binding. We demonstrate that ATTO 565-labeled MBD2 is a good candidate as a staining agent for epigenetic mapping.
Subject(s)
5-Methylcytosine/metabolism , DNA Methylation , DNA-Binding Proteins/metabolism , DNA/chemistry , Methyl-CpG-Binding Protein 2/metabolism , Microfluidics/methods , 5-Methylcytosine/chemistry , Binding Sites , DNA/metabolism , DNA-Binding Proteins/chemistry , Epigenomics/methods , Humans , Methyl-CpG-Binding Protein 2/chemistry , Microfluidics/instrumentation , Microscopy, Atomic Force/methods , Protein BindingABSTRACT
Nanofluidic devices have channel dimensions which come to within one order of magnitude of the Debye length of common aqueous solutions. Conventionally, external driving is used to create concentration polarization of ions and biomolecules in nanofluidic devices. Here we show that long-range ionic strength gradients intrinsic to all nanofluidic devices, even at equilibrium, also drive a drift of macromolecules. To demonstrate the effect, we confine long DNA to straight nanochannels of constant, rectangular cross-section (100 × 100 nm2) which are connected to large microfluidic reservoirs. The motion of DNA is observed in absence of any driving. We find that at low ionic strengths, molecules in nanochannels migrate toward the nano-micro interface, while they are undergoing purely diffusive motion in high salt. Using numerical models, we demonstrate that the motion is consistent with the ionic strength gradient at the micro-nano interface even at equilibrium, and that the dominant cause of the drift is diffusophoresis.
Subject(s)
Nanotechnology/methods , DNA/chemistry , Diffusion , Ions/chemistry , Macromolecular Substances/chemistry , Microfluidics/methods , Osmolar ConcentrationABSTRACT
Complex manipulations of DNA in a nanofluidic device require channels with branches and junctions. However, the dynamic response of DNA in such nanofluidic networks is relatively unexplored. Here, the transport of DNA in a 2D metamaterial made by arrays of nanochannel junctions is investigated. The mechanism of transport is explained as Brownian motion through an energy landscape formed by the combination of the confinement free energy of DNA and the effective potential of hydrodynamic flow, which both can be tuned independently within the device. For the quantitative understanding of DNA transport, a dynamic mean-field model of DNA at a nanochannel junction is proposed. It is shown that the dynamics of DNA in a nanofluidic device with branched channels and junctions is well described by the model.
Subject(s)
DNA/chemistry , Nanotechnology/methods , HydrodynamicsABSTRACT
We report that long double-stranded DNA confined to quasi-1D nanochannels undergoes superdiffusive motion under the action of the enzyme T4 DNA ligase in the presence of necessary co-factors. Inside the confined environment of the nanochannel, double-stranded DNA molecules stretch out due to self-avoiding interactions. In absence of a catalytically active enzyme, we see classical diffusion of the center of mass. However, cooperative interactions of proteins with the DNA can lead to directed motion of DNA molecules inside the nanochannel. Here we show directed motion in this configuration for three different proteins (T4 DNA ligase, MutS, E. coli DNA ligase) in the presence of their energetic co-factors (ATP, NAD+).
Subject(s)
DNA Ligases/metabolism , DNA/metabolism , Escherichia coli Proteins/metabolism , MutS DNA Mismatch-Binding Protein/metabolism , Adenosine Triphosphate/metabolism , DNA Ligases/physiology , DNA-Binding Proteins/genetics , Diffusion , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/physiology , Motion , MutS DNA Mismatch-Binding Protein/physiology , NAD/metabolismABSTRACT
We show that DNA carrying 5-methylcytosine modifications or methylated DNA (m-DNA) can be distinguished from DNA with unmodified cytosine by Raman spectroscopy enhanced by both a bowtie nanoantenna and excitation resonance. In particular, m-DNA can be identified by a peak near 1000 cm-1 and changes in the Raman peaks in the 1200-1700 cm-1 band that are enhanced by the ring-absorption resonance. The identification is robust to the use of resonance Raman and nanoantenna excitation used to obtain significant signal improvement. The primary differences are three additional Raman peaks with methylation at 1014, 1239, and 1639 cm-1 and spectral intensity inversion at 1324 (C5=C6) and 1473 cm-1 (C4=N3) in m-DNA compared to that of DNA with unmodified cytosine. We attribute this to the proximity of the methyl group to the antenna, which brings the (C5=C6) mode closer to experiencing a stronger near-field enhancement. We also show distinct Raman spectral features attributed to the transition of DNA from a hydrated state, when dissolved, to a dried/denatured state. We observe a general broadening of the larger lines and a transfer of spectral weight from the â¼1470 cm-1 vibration to the two higher-energy lines of the dried m-DNA solution. We attribute the new spectral characteristics to DNA softening under high salt conditions and find that the m-DNA is still distinguishable via the â¼1000 cm-1 peak and distribution of the signal in the 1200-1700 cm-1 band. The nanoantenna gain exceeds 20,000, whereas the real signal ratio is much less because of a low average enhanced region occupancy even with these relatively high DNA concentrations. It is improved when fixed DNA in a salt crystal lies near the nanoantenna. The Raman resonance gain profile is consistent with A-term expectations, and the resonance is found at â¼259 nm excitation wavelength.
Subject(s)
DNA Methylation , Nanotechnology , Spectrum Analysis, Raman , 5-Methylcytosine/metabolismABSTRACT
Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids, mediated by the cohesin protein complex, which also plays crucial roles in diverse genome maintenance pathways. Current models attribute DNA binding by cohesin to entrapment of dsDNA by the cohesin ring subunits (SMC1, SMC3, and RAD21 in humans). However, the biophysical properties and activities of the fourth core cohesin subunit SA2 (STAG2) are largely unknown. Here, using single-molecule atomic force and fluorescence microscopy imaging as well as fluorescence anisotropy measurements, we established that SA2 binds to both dsDNA and ssDNA, albeit with a higher binding affinity for ssDNA. We observed that SA2 can switch between the 1D diffusing (search) mode on dsDNA and stable binding (recognition) mode at ssDNA gaps. Although SA2 does not specifically bind to centromeric or telomeric sequences, it does recognize DNA structures often associated with DNA replication and double-strand break repair, such as a double-stranded end, single-stranded overhang, flap, fork, and ssDNA gap. SA2 loss leads to a defect in homologous recombination-mediated DNA double-strand break repair. These results suggest that SA2 functions at intermediate DNA structures during DNA transactions in genome maintenance pathways. These findings have important implications for understanding the function of cohesin in these pathways.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Cell Cycle Proteins/chemistry , Chromosomal Proteins, Non-Histone/chemistry , DNA Repair/genetics , DNA Repair/physiology , DNA Replication/physiology , Fluorescence Polarization , Genomic Instability/genetics , Genomic Instability/physiology , Microscopy, Atomic Force , Microscopy, Fluorescence , Protein Binding/genetics , Protein Binding/physiology , CohesinsABSTRACT
The methylcytosine-binding domain 2 (MBD2) protein recruits the nucleosome remodeling and deacetylase complex (NuRD) to methylated DNA to modify chromatin and regulate transcription. Importantly, MBD2 functions within CpG islands that contain 100s to 1000s of potential binding sites. Since NuRD physically rearranges nucleosomes, the dynamic mobility of this complex is directly related to function. In these studies, we use NMR and single-molecule atomic force microscopy and fluorescence imaging to study DNA binding dynamics of MBD2. Single-molecule fluorescence tracking on DNA tightropes containing regions with CpG-rich and CpG-free regions reveals that MBD2 carries out unbiased 1D diffusion on CpG-rich DNA but subdiffusion on CpG-free DNA. In contrast, the protein stably and statically binds to methylated CpG (mCpG) regions. The intrinsically disordered region (IDR) on MBD2 both reduces exchange between mCpG sites along the DNA as well as the dissociation from DNA, acting like an anchor that restricts the dynamic mobility of the MBD domain. Unexpectedly, MBD2 binding to methylated CpGs induces DNA bending that is augmented by the IDR region of the protein. These results suggest that MBD2 targets NuRD to unmethylated or methylated CpG islands where its distinct dynamic binding modes help maintain open or closed chromatin, respectively.
Subject(s)
5-Methylcytosine/chemistry , CpG Islands , DNA-Binding Proteins/chemistry , DNA/chemistry , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Nucleosomes/metabolism , 5-Methylcytosine/metabolism , Animals , Binding Sites , Chickens , Cloning, Molecular , DNA/metabolism , DNA Methylation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Fluorescence Polarization , Gene Expression , Humans , Magnetic Resonance Spectroscopy , Mi-2 Nucleosome Remodeling and Deacetylase Complex/ultrastructure , Nucleic Acid Conformation , Nucleosomes/ultrastructure , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Single Molecule ImagingABSTRACT
Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA-DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA-DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding.
Subject(s)
Chromosome Segregation/genetics , Nuclear Proteins/genetics , Telomere-Binding Proteins/genetics , Telomere/genetics , Telomeric Repeat Binding Protein 1/genetics , AT-Hook Motifs/genetics , Chromatids/genetics , Chromatids/ultrastructure , DNA-Binding Proteins/genetics , Humans , Microscopy, Atomic Force , Mitosis/genetics , Nuclear Proteins/metabolism , Telomere/ultrastructure , Telomere-Binding Proteins/metabolism , Telomeric Repeat Binding Protein 1/metabolismABSTRACT
Shelterin protein TRF2 modulates telomere structures by promoting dsDNA compaction and T-loop formation. Advancement of our understanding of the mechanism underlying TRF2-mediated DNA compaction requires additional information regarding DNA paths in TRF2-DNA complexes. To uncover the location of DNA inside protein-DNA complexes, we recently developed the Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM) imaging technique. DREEM imaging shows that in contrast to chromatin with DNA wrapping around histones, large TRF2-DNA complexes (with volumes larger than TRF2 tetramers) compact DNA inside TRF2 with portions of folded DNA appearing at the edge of these complexes. Supporting coarse-grained molecular dynamics simulations uncover the structural requirement and sequential steps during TRF2-mediated DNA compaction and result in folded DNA structures with protruding DNA loops as seen in DREEM imaging. Revealing DNA paths in TRF2 complexes provides new mechanistic insights into structure-function relationships underlying telomere maintenance pathways.
Subject(s)
DNA/chemistry , Multiprotein Complexes/chemistry , Nucleic Acid Conformation , Telomeric Repeat Binding Protein 2/chemistry , DNA/metabolism , HeLa Cells , Humans , Microscopy , Multiprotein Complexes/metabolism , Static Electricity , Telomeric Repeat Binding Protein 2/metabolismABSTRACT
Recent studies have shown that double-stranded DNA can collapse in the presence of a strong electric field. Here we provide an in-depth study of the collapse of DNA under weak confinement in microchannels as a function of buffer strength, driving frequency, applied electric-field strength, and molecule size. We find that the critical electric field at which DNA molecules collapse (tens of kV/m) is strongly dependent on driving frequency (100-800 Hz) and molecular size (20-160 kbp), and weakly dependent on the ionic strength (8-60 mM). We argue that an apparent stretching at very high electric fields is an artifact of the finite frame time of video microscopy.
Subject(s)
DNA Contamination , DNA/chemistry , Electromagnetic Phenomena , Ions/chemistry , Microscopy, Fluorescence , Video RecordingABSTRACT
We present a nanofluidic device for targeted manipulations in the quarternary structure of single DNA molecules. We demonstrate the folding and unfolding of hairpin-shaped regions, similar to chromatin loops. These loops are stable for minutes at nanochannel junctions. We demonstrate continuous scanning of two DNA segments that occupy a common nanovolume. We present a model governing the stability of loop folds and discuss how the system achieves specific DNA configurations without operator intervention.
Subject(s)
DNA/chemistry , Equipment Design , Lab-On-A-Chip Devices , Microscopy, Fluorescence , Nanotechnology/instrumentation , Nucleic Acid ConformationABSTRACT
Intercalating fluorescent probes are widely used to visualize DNA in studies on DNA-protein interactions. Some require the presence of adenosine triphosphate (ATP). We have investigated the mechanical properties of DNA stained with the fluorescent intercalating dyes YOYO-1 and YOYO-3 as a function of ATP concentrations (up to 2 mM) by stretching single molecules in nanofluidic channels with a channel cross-section as small as roughly 100×100 nm2. The presence of ATP reduces the length of the DNA by up to 11 %. On the other hand, negligible effects are found if DNA is visualized with the minor groove-binding probe 4',6-diamidino-2-phenylindole. The apparent drop in extension under nanoconfinement is attributed to an interaction of the dye and ATP, and the resulting expulsion of YOYO-1 from the double helix.
ABSTRACT
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.
ABSTRACT
Thermal fluctuations, geometric exclusion, and external driving all govern the mechanical response of dense particulate suspensions. Here, we measure the stress-strain response of quasi-two-dimensional flow-stabilized microsphere heaps in a regime in which all three effects are present using a microfluidic device. We observe that the elastic modulus and the mean interparticle separation of the heaps are tunable via the confining stress provided by the fluid flow. Furthermore, the measured stress-strain curves exhibit a universal nonlinear shape, which can be predicted from a thermal van der Waals equation of state with excluded volume. This analysis indicates that many-body interactions contribute a significant fraction of the stress supported by the heap.