Visualization of DNA and RNA molecules, and protein-DNA complexes for electron microscopy

This article provides step-by step instructions for the preparation of double- and single-stranded DNA and RNA molecules and protein-DNA complexes for electron microscopy (EM). Absorption, spreading, staining, dark-field imaging, and metal shadowing techniques are described in detail. A number of examples are illustrated on analysis of DNA replication, DNA repair and DNA recombination to demonstrate the usefulness of the technique for EM visualisation. Application of immunogold labeling of specific protein in DNA-protein complexes is also covered.     

Measurement of DNA-protein crosslinks in mammalian cells without X-irradiation

To study the mechanisms of formation and repair of DNA-protein crosslinks in mammalian cells, the best general method to assay these lesions is the Kohn membrane alkaline elution procedure. Use of this sensitive technique requires the introduction of random strand breaks in the DNA by X-irradiation to reduce the very high molecular weight so that it elutes off the filter at an appropriate rate. This report describes an alternative method for fragmenting the DNA in the absence of X-irradiation equipment. Convenient reproducible elution rates of DNA from various mouse and human cells in culture without X-irradiation result from elution through polyvinyl chloride filters with 75 mM sodium hydroxide (0.33 ml/min) instead of the standard 20 mM EDTA-tetrapropylammonium hydroxide, pH 12.2 (0.03 to 0.04 ml/min). Dose-dependent retardation of the DNA elution was observed over the range 0 to 30 microM trans-platinum(II)diamminedichloride, and proteinase K treatment during cell lysis restored the elution rate to that of the untreated control cell DNA. In the absence of X-irradiation, this elution method measures DNA-protein crosslinks with higher sensitivity and equivalent reproducibility as the air-burst procedure.     

A new electron microscopic method for studying protein-nucleic acid interactions.

A new method for preparation of nucleic acid specimens for electron microscopy has been adapted to study the interaction of proteins with DNA. Both a detergent and a basic protein are added to the DNA-protein solution before spreading on a hypophase containing 0.2 m ammonium acetate. This method has been tested using T7 DNA and Escherichia coli RNA polymerase. Specifically bound enzyme molecules were clearly visible on the well extended DNA molecules; the binding sites were located at 0.59, 1.24, 1.57, and 1.86% of the total length of T7 DNA. Under carefully controlled conditions, 40–85% of the DNA molecules specifically bound at least one enzyme molecule.     

Quantitative aspects of the cytochemical Feulgen-DNA procedure studied on model systems and cell nuclei

Quantitative aspects of DNA losses during fixation and pararosaniline(SO2)-Feulgen staining of microscopic preparations were studied. The preparation of a new cytochemical model, consisting of DNA-protein layers (with thicknesses between 0.1 and 5.0 micrometer) on microscopic glass slides is described and potentialities and limitations of this model are discussed. Polyacrylamide films into which high molecular weight calf thymus DNA or chicken erythrocyte nuclei had been constrained served as another model. As biological objects chicken erythrocyte nuclei and rat liver nuclei either in suspension or on microscopical glass slides were used. The experimental results indicate a loss of about 5% of the DNA due to the fixation procedure applied. Hydrolysis in 5 N HCl at room temperature, staining with the pararosaniline-Schiff medium and rinsing with sulfurous acid induced losses of DNA too, varying in amount depending on the type of preparation used. About 10% of the original DNA content is lost in total from chicken erythrocyte nuclei and rat liver nuclei dried on microscopical glass slides, from chicken erythrocyte nuclei constrained in polyacrylamide films, and from DNA-protein layers on microscopic glass slides. For nuclei fixed and stained in suspension the total losses amount to about 40%. The differences in losses between various types of preparations are discussed. Biochemically, the content of DNA originally present per chicken erythrocyte nucleus was determined to be 2.52 pg, a value, which is in good accordance with reliable biochemical data published already. It is shown that calibration of cytochemical staining intensities into biochemical units or absolute amounts of material by use of a model system, is only reliable when it is known or to be expected that both the loss of material due to fixation and staining, and the stoichiometric relation between material present and dye molecules is identical. The same holds for the application of internal biological reference systems.     

Fast gel electrophoresis to analyze DNA-protein interactions

A rapid method for electrophoresis of DNA-protein complexes is described. Popular "gel-shift" assays are performed using Pharmacia PhastSystem with its convenience of pre-cast gels and buffer strips and microprocessor-controlled high-resolution separation. Using this system, the products of a DNA binding reaction (DNA-protein complexes) can be separated from "free" DNA in less than one hour. DNA fragments as well as oligonucleotides have been used as targets with partially purified extracts containing sequence-specific DNA binding proteins. We present here a comparison of results of gel-shift assays obtained by conventional techniques and by our rapid "PhastShift" method which reduces the time, effort and technical expertise necessary to obtain reproducible results.     

A convenient method of aligning large DNA molecules on bare mica surfaces for atomic force microscopy.

Large DNA molecules remain difficult to be imaged by atomic force microscopy (AFM) because of the tendency of aggregation. A method is described to align long DNA fibers in a single direction on unmodified mica to facilitate AFM studies. The clear background, minimal overstretching, high reproducibility and convenience of this aligning procedure make it useful for physical mapping of genome regions and the studies of DNA-protein complexes.     

Receptors for neurotensin in canine small intestine.

We have previously characterized the neurotensin receptors on the circular smooth muscle (CM) of the canine small intestine (1). In the present studies, using radioligand binding technique, neurotensin receptors were localized on the membranes from deep muscular (DMP) and the submucous plexus while no binding was observed on either the longitudinal smooth muscle or myenteric plexus membranes. The high affinity binding sites (Kd 0.1-0.2 nM) on DMP membranes were similar to those on CM; the low affinity component was of much lower affinity (Kd approximately 40 nM). DMP had 4-6 times higher density of binding sites than the CM. The recognition properties of DMP receptors were similar to those on the CM and reduced sulfhydryl groups were required for the binding activity. The action of neurotensin on the contractility of the canine small intestine, therefore, appears to be through a direct action on the circular smooth muscle and through the prejunctional action on the DMP neurons through distinct receptors. Thiol groups in the neurotensin receptors may be important for the receptor function.     

The major herpes simplex virus DNA-binding protein holds single-stranded DNA in an extended configuration

Properties of the major DNA-binding protein found in herpes simplex virus-infected cells were investigated by using a filter binding assay and electron microscopy. Filter binding indicated that the stoichiometry of binding of the protein with single-stranded DNA is approximately 40 nucleotides per protein molecule at saturation. Strong clustering of the protein in DNA-protein complexes, indicative of cooperative binding, was seen with the electron microscope. Measurements of single-stranded fd DNA molecules saturated with protein and spread for electron microscopy by using both the aqueous and formamide spreading techniques indicated that the DNA is held in an extended configuration with a base spacing of approximately 0.13 nm per base.     

DARTs: A DNA-based in vitro polypeptide display technology

Display technologies link proteins with the genes that encode them, providing a means of selecting proteins with desired properties through the process of directed evolution. Here, we describe DNA/protein attachment and recovery tools (DARTs), a novel polypeptide display technology that utilizes the Agrobacterium tumefaciens protein VirD2 to generate DNA-protein hybrid molecules. The resulting DNA-protein hybrids are small, robust, and are not expected to be subject to the synthesis and selection biases associated with viral- and cell-based display systems. We demonstrated that these DNA-protein hybrids could be used to display a variety of peptides that bind to appropriate antibodies for immunodetection and immunopurification. Further, the DNA components of the hybrid molecules can hybridize to complementary DNA molecules in solution or on a solid substrate. Because full-length VirD2 self-associated, we constructed a truncation that did not self-associate but still exhibited DNA linking activity and efficiently displayed peptides. Finally, we purified DNA-protein hybrids using their displayed peptide epitopes and amplified their DNA components by polymerase chain reaction. We suggest that the DART polypeptide display system will be valuable for performing directed evolution and generating protein arrays.     

Quantitative aspects of the cytochemical Feulgen-DNA procedure studied on model systems and cell nuclei

Summary Quantitative aspects of DNA losses during fixation and pararosaniline(SO₂)-Feulgen staining of microscopic preparations were studied. The preparation of a new cytochemical model, consisting of DNA-protein layers (with thicknesses between 0.1 and 5.0 m) on microscopic glass slides is described and potentialities and limitations of this model are discussed.Polyacrylamide films into which high molecular weight calf thymus DNA or chicken erythrocyte nuclei had been constrained served as another model. As biological objects chicken erythrocyte nuclei and rat liver nuclei either in suspension or on microscopical glass slides were used.The experimental results indicate a loss of about 5% of the DNA due to the fixation procedure applied. Hydrolysis in 5 N HCl at room temperature, staining with the pararosaniline-Schiff medium and rinsing with sulfurous acid induced losses of DNA too, varying in amount depending on the type of preparation used. About 10% of the original DNA content is lost in total from chicken erythrocyte nuclei and rat liver nuclei dried on microscopical glass slides, from chicken erythrocyte nuclei constrained in polyacrylamide films, and from DNA-protein layers on microscopic glass slides. For nuclei fixed and stained in suspension the total losses amount to about 40%. The differences in losses between various types of preparations are discussed. Biochemically, the content of DNA originally present per chicken eythrocyte nucleus was determined to be 2.52 pg, a value, which is in good accordance with reliable biochemical data published already. It is shown that calibration of cytochemical staining intensities into biochemical units or absolute amounts of material by use of a model system, is only reliable when it is known or to be expected that both the loss of material due to fixation and staining, and the stoichiometric relation between material present and dye molecules is identical. The same holds for the application of internal biological reference systems.Supported by grant no. 28-394 of the Praeventiefonds, The HagueIn receipt of a grant from Het Ministerie van Onderwijs en Wetenschappen, Afdeling Buitenlandse Betrekkingen, The Netherlands     

Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging

Single-molecule techniques such as optical tweezers and fluorescence imaging are powerful tools for probing the biophysics of DNA and DNA-protein interactions. The application of these methods requires efficient approaches for creating designed DNA structures with labels for binding to a surface or microscopic beads. In this paper, we develop a simple and fast technique for making a diverse range of such DNA constructs by combining PCR amplicons and synthetic oligonucleotides using golden gate assembly rules. We demonstrate high yield fabrication of torsionally-constrained duplex DNA up to 10 kbp in length and a variety of DNA hairpin structures. We also show how tethering to a cross-linked antibody substrate significantly enhances measurement lifetime under high force. This rapid and adaptable fabrication method streamlines the assembly of DNA constructs for single molecule biophysics.     

Fractionation of type V collagen from carcinomatous and dysplasic breast in the presence of alkaline potassium chloride

A simple, one-step fractionation technique suitable for the finest quantitation of type V collagen from acidic mixtures of interstitial collagens is reported. The technique is based on the higher solubility of this collagen type in alkaline KCl-phosphate solution, and the purity of the sample has been monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopic analyses. The latter reveal that the majority of the molecules are endowed with a thread portion and a terminal knob, and show a low tendency to originate ordered and reproducible polymers at low temperature.     

Protein-DNA interactions in genetic recombination

The formation of DNA-protein complexes that are capable of interaction with other DNA molecules is necessary for efficient genetic recombination. How do such complexes form, and how are homologous DNA sequences brought into alignment? Physical and biochemical studies of recombination enzymes from bacteria indicate that these proteins provide the structural framework within which the genetic exchanges occur.     

Plasmid partitioning and the spreading of P1 partition protein ParB

Bacterial plasmids of low copy number, P1 prophage among them, are actively partitioned to nascent daughter cells. The process is typically mediated by a pair of plasmid-encoded proteins and a cis-acting DNA site or cluster of sites, referred to as the plasmid centromere. P1 ParB protein, which binds to the P1 centromere (parS), can spread for several kilobases along flanking DNA. We argue that studies of mutant ParB that demonstrated a strong correlation between spreading capacity and the ability to engage in partitioning may be misleading, and describe here a critical test of the dependence of partitioning on the spreading of the wild-type protein. Physical constraints imposed on the spreading of P1 ParB were found to have only a minor, but reproducible, effect on partitioning. We conclude that, whereas extensive ParB spreading is not required for partitioning, spreading may have an auxiliary role in the process.     

The use of ultraviolet light in the fractionation of chromatin containing unsubstituted and bromodeoxyuridine-substituted DNA.

Two procedures are described for the fractionation of chromatin containing unsubstituted (LL) DNA and DNA unifilarly substituted with bromodeoxyuridine (HL). The two procedures rely upon the sensitivity of bromodeoxyuridine-containing DNA to UV light to induce either strand breakage or protein crosslinking. When a mixture of LL and HL chromatin is irradiated with UV light, the HL DNA fragments into molecules of smaller molecular weight than the LL DNA and crosslinks more chromosomal protein than the LL DNA. LL and HL chromatin can be fractionated on the basis of size by centrifuging through a neutral sucrose gradient. The HL DNA-protein adducts that are generated by the UV light have a unique buoyant density and may be isolated by isopycnic centrifugation in CS2SO4. The ability to fractionate LL and HL chromatin permits certain studies on the structure of replicating chromatin.     

Measuring intermolecular rupture forces with a combined TIRF-optical trap microscope and DNA curtains

We report a new approach to probing DNA-protein interactions by combining optical tweezers with a high-throughput DNA curtains technique. Here we determine the forces required to remove the individual lipid-anchored DNA molecules from the bilayer. We demonstrate that DNA anchored to the bilayer through a single biotin-streptavidin linkage withstands ∼20 pN before being pulled free from the bilayer, whereas molecules anchored to the bilayer through multiple attachment points can withstand ?65 pN; access to this higher force regime is sufficient to probe the responses of protein-DNA interactions to force changes. As a proof-of-principle, we concurrently visualized DNA-bound fluorescently-tagged RNA polymerase while simultaneously stretching the DNA molecules. This work presents a step towards a powerful experimental platform that will enable concurrent visualization of DNA curtains while applying defined forces through optical tweezers.     

High-throughput single-molecule analysis of DNA-protein interactions by tethered particle motion

Tethered particle motion (TPM) monitors the variations in the effective length of a single DNA molecule by tracking the Brownian motion of a bead tethered to a support by the DNA molecule. Providing information about DNA conformations in real time, this technique enables a refined characterization of DNA-protein interactions. To increase the output of this powerful but time-consuming single-molecule assay, we have developed a biochip for the simultaneous acquisition of data from more than 500 single DNA molecules. The controlled positioning of individual DNA molecules is achieved by self-assembly on nanoscale arrays fabricated through a standard microcontact printing method. We demonstrate the capacity of our biochip to study biological processes by applying our method to explore the enzymatic activity of the T7 bacteriophage exonuclease. Our single molecule observations shed new light on its behaviour that had only been examined in bulk assays previously and, more specifically, on its processivity.