Proteins that shape DNA

During the last five years, considerable accumulation of data on nucleic acids metabolism leads to the discovery of a number of proteins designed to change the conformation of DNA and to "shape" it. Experimental results emphasize the importance of the conformation and the flexibility of DNA itself in such interactions. The mutual recognition of nucleic acids by proteins may be or not dependent on the nnucleotide sequence and in most cases is accompanied by conformational changes in the proteins involved. Among these are proteins that bind in stoichiometric amounts to DNA, proteins that promote the separation of the two strands in a duplex, and finally proteins that change the topology of DNA.     

Purification of plasmid and high molecular mass DNA using PEG-salt two-phase extraction

A method for the rapid preparation of DNA is described. The method utilizes a polymer (polyethylene glycol) and salt solution to form a two-phase system. A crude source of DNA is added to a phase-forming mixture, it is mixed and phase separation occurs. Under the appropriate conditions, the nucleic acids remain in the lower (salt-rich) phase, while the proteins, cellular debris and other constituents are in the upper phase (polymer-rich) or are precipitated at the interphase region. Incorporation of protein denaturants (detergents and chaotropes) stop the action of liberated nucleases in the sample. The nucleic acids are obtained in an intact state and in a form suitable for further manipulation, as shown by gel electrophoresis and DNA restriction digestion. This method describes the conditions of the two-phase systems that are important for the separation of nucleic acids and proteins. The important phase-forming conditions shown in this paper are pH, polymer molecular weight and concentration, salt type and concentration and the addition of detergents and chaotropic agents. With the use of these extraction conditions, proteins can be moved selectively from the lower to the upper phase. The paper describes a method for DNA isolation that is rapid, simple and economical.     

Do nucleic acids moonlight as molecular chaperones?

Organisms use molecular chaperones to combat the unfolding and aggregation of proteins. While protein chaperones have been widely studied, here we demonstrate that DNA and RNA exhibit potent chaperone activity in vitro. Nucleic acids suppress the aggregation of classic chaperone substrates up to 300-fold more effectively than the protein chaperone GroEL. Additionally, RNA cooperates with the DnaK chaperone system to refold purified luciferase. Our findings reveal a possible new role for nucleic acids within the cell: that nucleic acids directly participate in maintaining proteostasis by preventing protein aggregation.     

The HMG domain of the ROX1 protein mediates repression of HEM13 through overlapping DNA binding and oligomerization functions.

The ROX1 gene of Saccharomyces cerevisiae encodes a protein required for the repression of genes expressed under anaerobic conditions. ROX1 belongs to a family of DNA binding proteins which contain the high mobility group motif (HMG domain). To ascertain whether the HMG domain of ROX1 is required for specific DNA binding we synthesized a series of ROX1 protein derivatives, either in vitro or in Escherichia coli as fusions to glutathione S-transferase (GST) protein, and tested them for their ability to bind to DNA. Both ROX1 proteins that were synthesized in vitro and GST-ROX1 fusion proteins containing the intact HMG domain were able to bind to specific target DNA sequences. In contrast, ROX1 proteins which contained deletions within the HMG domain were no longer capable of binding to DNA. The oligomerization of ROX1 in vitro was demonstrated using affinity-purified GST-ROXI protein and ROX1 labelled with [35S]methionine. Using various ROX1 protein derivatives we were able to demonstrate that the domain required for ROX1-ROX1 interaction resides within the N-terminal 100 amino acids which constitute the HMG domain. Therefore, the HMG domain is required for both DNA binding activity and oligomerization of ROX1.     

Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system

Single-pulse (approximately 8 ns) ultraviolet laser excitation of protein-nucleic acid complexes can result in efficient and rapid covalent cross-linking of proteins to nucleic acids. The reaction produces no nucleic acid-nucleic acid or protein-protein cross-links, and no nucleic acid degradation. The efficiency of cross-linking is dependent on the wavelength of the exciting radiation, on the nucleotide composition of the nucleic acid, and on the total photon flux. The yield of cross-links/laser pulse is largest between 245 and 280 nm; cross-links are obtained with far UV photons (200-240 nm) as well, but in this range appreciable protein degradation is also observed. The method has been calibrated using the phage T4-coded gene 32 (single-stranded DNA-binding) protein interaction with oligonucleotides, for which binding constants have been measured previously by standard physical chemical methods (Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104). Photoactivation occurs primarily through the nucleotide residues of DNA and RNA at excitation wavelengths greater than 245 nm, with reaction through thymidine being greatly favored. The nucleotide residues may be ranked in order of decreasing photoreactivity as: dT much greater than dC greater than rU greater than rC, dA, dG. Cross-linking appears to be a single-photon process and occurs through single nucleotide (dT) residues; pyrimidine dimer formation is not involved. Preliminary studies of the individual proteins of the five-protein T4 DNA replication complex show that gene 43 protein (polymerase), gene 32 protein, and gene 44 and 45 (polymerase accessory) proteins all make contact with DNA, and can be cross-linked to it, whereas gene 62 (polymerase accessory) protein cannot. A survey of other nucleic acid-binding proteins has shown that E. coli RNA polymerase, DNA polymerase I, and rho protein can all be cross-linked to various nucleic acids by the laser technique. The potential uses of this procedure in probing protein-nucleic acid interactions are discussed.     

Denatured Mammalian Protein Mixtures Exhibit Unusually High Solubility in Nucleic Acid-Free Pure Water

Preventing protein aggregation is a major goal of biotechnology. Since protein aggregates are mainly comprised of unfolded proteins, protecting against denaturation is likely to assist solubility in an aqueous medium. Contrary to this concept, we found denatured total cellular protein mixture from mammalian cell kept high solubility in pure water when the mixture was nucleic acids free. The lysates were prepared from total cellular protein pellet extracted by using guanidinium thiocyanate-phenol-chloroform mixture of TRIzol, denatured and reduced total protein mixtures remained soluble after extensive dialysis against pure water. The total cell protein lysates contained fully disordered proteins that readily formed large aggregates upon contact with nucleic acids or salts. These findings suggested that the highly flexible mixtures of disordered proteins, which have fully ionized side chains, are protected against aggregation. Interestingly, this unusual solubility is characteristic of protein mixtures from higher eukaryotes, whereas most prokaryotic protein mixtures were aggregated under identical conditions. This unusual solubility of unfolded protein mixtures could have implications for the study of intrinsically disordered proteins in a variety of cells.     

DNA is a template for accelerating the aggregation of copper, zinc superoxide dismutase

The proteinaceous aggregates rich in copper, zinc superoxide dismutase (SOD1) have been shown to be involved in pathogenesis of amyotrophic lateral sclerosis (ALS). Since negatively charged species such as nucleic acids have frequently been found associated with the proteinaceous deposits in the tissues of patients with amyloid diseases, we examined here the aggregation behavior of SOD1 in the presence of DNA under acidic conditions that facilitate protein aggregation. Several forms of double-stranded DNA were tested to trigger SOD1 aggregation by light scattering, single- and double-fluorescence imaging with dyes, atomic force microscopy, and direct observations under visible light. The results reveal that DNA acts as a template for accelerating the formation of SOD1 aggregates and is incorporated into SOD1 aggregates. The spherical and ellipsoidal SOD1 aggregates were characterized in both hydrated and dried states and have morphology similar to those identified in the diseased neurons. Light scattering experiments indicate that the aggregation first undergoes a rapid phase where the aggregates with average diameters of 40-80 nm rapidly form in <2 min, and then passes through a slow phase where the average diameters of aggregates were increased to at least 200-260 nm in 2 h. All forms of DNAs tested can lead to the aggregation of SOD1 at nanomolar levels. The association of SOD1 with DNA, driven by electrostatic interactions between both, can restrict the orientation of SOD1 molecules and increase a SOD1 population along DNA strands. This facilitates the hydrophobic interactions between SOD1 molecules, as indicated by hydrophobic probe binding and chemical denaturant treatment experiments. Demonstration of the DNA-accelerated aggregation of SOD1 might establish a possible role of DNA in the pathogenesis of some diseases because of the ubiquitous expression of SOD1 and the coexistence of SOD1 and DNA in the crowded molecular environment of a cell.     

Electron microscopic mapping of proteins bound to herpes simplex virus DNA.

Exonuclease digestion experiments have suggested that there is a protein(s) bound close to one or both ends of herpes simplex virus-1 (HSV) DNA. The existence of such bound proteins has been positively demonstrated and their positions on the HSV genome determined by application of a newly developed method for electron microscopic mapping of proteins bound to nucleic acids. Purified HSV DNA was treated with dinitrofluorobenzene under conditions that covalently attach the dinitrophenyl (DNP) group to the proteins in a protein-nucleic acid complex. The HSV DNA-protein-(DNP)n complex was treated with rabbit anti-DNP IgG, and, in some cases, additionally treated with monovalent Fab fragments of goat anti-rabbit IgG, and mounted for examination in the electron microscope. Electron opaque dots representing the protein-(DNP)n-(IgG)m complex were seen on the HSV DNA. Direct measurements of the positions of the protein, as well as partial denaturation mapping, indicate that there are four positions for protein bound to HSV DNA: two near but not at the two ends and two at sites corresponding to the internal inverted repeats of the ends. These results suggest that there is a specific protein binding sequence within the direct terminal repeat of HSV DNA. The previous observation that HSV DNA is more sensitive to digestion by a 3' than by a 5' exonuclease then indicates that the bound protein(s) is more intimately associated with one strand of the specific sequence than with the complementary strand.     

An electron microscopic method for the mapping of proteins attached to nucleic acids.

An electron microscopic method for demonstrating the presence of and mapping the positions of proteins specifically bound to nucleic acids is described. The nucleic acid-protein complex is treated with dinitrofluorobenzene under conditions such that dinitrophenyl (DNP) groups are attached to nucleophilic groups on the protein, with only a low level of random attachment to the nuclei acid. This product is treated with rabbit anti-DNP IgG. The position of the protein-(DNP)n(IgG)m complex on the nucleic acid strand can be observed by electron microscopy by protein free spreading methods and, in many cases, by cytochrome-c spreading. If necessary for visualization by the latter method, the size of the labeled region can be increased by treatment with goat anti-rabbit IgG. High efficiency of electron microscopic labeling is achieved. Examples studied are: the adenovirus-2 DNA terminal protein, a protein covalently bound to SV40 DNA, DNA polymerase I bound to DNA, E. coli RNA polymerase bound to T7 DNA, and proteins UV crosslinked to avian sarcoma virus RNA.     

Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins

The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experiments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be ~10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are consistent with the primary function of Gag as a nucleic acid binding and packaging protein and the primary function of the NC proteins as nucleic acid chaperones. Also, our results suggest that NCp7's capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nucleic acid strand annealing by inducing electrostatic attraction between strands, likely optimize the fully processed NC protein to facilitate complex nucleic acid secondary structure rearrangements. In contrast, Gag's stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization.     

NMR studies of dynamics in RNA and DNA by 13C relaxation

RNA and DNA molecules experience motions on a wide range of time scales, ranging from rapid localized motions to much slower collective motions of entire helical domains. The many functions of RNA in biology very often require this molecule to change its conformation in response to biological signals in the form of small molecules, proteins or other nucleic acids, whereas local motions in DNA may facilitate protein recognition and allow enzymes acting on DNA to access functional groups on the bases that would otherwise be buried in Watson-Crick base pairs. Although these statements make a compelling case to study the sequence dependent dynamics in nucleic acids, there are few residue-specific studies of nucleic acid dynamics. Fortunately, NMR studies of dynamics of nucleic acids and nucleic acids-protein complexes are gaining increased attention. The aim of this review is to provide an update of the recent progress in studies of nucleic acid dynamics by NMR based on the application of solution relaxation techniques.     

Purification of the gene 0.3 protein of bacteriophage T7, an inhibitor of the DNA restriction system of Escherichia coli

The gene 0.3 protein of bacteriophage T7 prevents the DNA restriction system of EScherichia coli from interfering with T7 infection. A mutant strain of T7 that greatly overproduces the 0.3 protein has been constructed and used for purification of this protein. The 0.3 protein ws found to be extremely acidic and can be separated from virtually all other proteins of the infected cell by chromatography on DEAE-cellulose. Residual contaminating proteins and nucleic acids can be removed by gel filtration, but an even simpler final purification is possible, because under appropriate conditions the 0.3 protein is soluble in high concentrations of ethanol. Thus, a simple, essentially two-step purification can produce about 50 mg of pure 0.3 protein from 30 liters of culture. The purified protein appears to be a dimer of identical subunits. AS expected from its known function during infection, the purified 0.3 protein inhibits the nuclease and ATPase activities of partially purified Eco B, the DNA restriction enzyme of E. coli B, but it does not interfere with several different type II endonucleases tested. The inhibition of Eco B appears to require stoichiometric rather than catalytic amounts of 0.3 protein.     

Evaluation of weak interactions of proteins and organic cations with DNA duplex structures

Molecular interactions and reactions in living cells occur with high background concentrations of organic compounds including proteins. Uncharged water-soluble polymers are commonly used cosolutes in studies on molecular crowding, and most studies argue about the effects of intracellular crowding based on results obtained using polymer cosolutes. Further investigations using protein crowders and organic cations are important in understanding the effects of cellular environments on nucleic acids with negatively charged surfaces. We assessed the effects of using model globular proteins, serum proteins, histone proteins, structurally flexible polypeptides, di- and polyamines, and uncharged polymers. Thermal stability analysis of DNA oligonucleotide structures revealed that unlike conventional polymer cosolutes, basic globular proteins (lysozyme and cytochrome c) at high concentrations stabilized long internal and bulge loop structures but not fully matched duplexes. The selective stabilization of long loop structures suggests preferential binding to unpaired nucleotides in loops through weak electrostatic interactions. Furthermore, the ability of the proteins to stabilize the loop structures was enhanced under macromolecular crowding conditions. Remarkably, the effects of basic proteins on the stability of fully matched duplexes were dissimilar to those of basic amino-acid-rich polypeptides and polyamines. This study provides new insights into the interaction of nucleic acid structures with organic cations.     

Studies of the domain structure of mammalian DNA polymerase beta. Identification of a discrete template binding domain

Characterization of the domain structure of DNA polymerase beta is reported. Large scale overproduction of the rat protein in Escherichia coli was achieved, and the purified recombinant protein was verified by sequencing tryptic peptides. This protein is both a single-stranded DNA binding protein and a DNA polymerase consisting of one polypeptide chain of 334 amino acids. As revealed by controlled proteolysis experiments, the protein is organized in two relatively protease-resistant segments linked by a short protease-sensitive region. One of these protease-resistant segments represents the NH2-terminal 20% of the protein. This NH2-terminal domain (of about 75 residues) has strong affinity for single-stranded nucleic acids. The other protease-resistant segment, representing the COOH-terminal domain of approximately 250 residues, does not bind to nucleic acids. Neither domain, tested as purified proteins, has substantial DNA polymerase activity. The results suggest that the NH2-terminal domain is principally responsible for the template binding activity of the intact protein.     

Thermodynamic modulation of folding and aggregation energy landscape by DNA binding of functional domains of TDP-43

TDP-43 is a vital nucleic acid binding protein which forms stress-induced aberrant aggregates in around 97% cases of ALS, a fatal neurodegenerative disease. The functional tandem RRM domain of the protein (TDP-43^tRRM) has been shown to undergo amyloid-like aggregation under stress in a pH-dependent fashion. However, the underlying thermodynamic and molecular basis of aggregation and how the energy landscape of folding, stability, and aggregation are coupled and modulated by nucleic acid binding is poorly understood. Here, we show that the pH stress thermodynamically destabilizes the native protein and systematically populates the unfolded-like aggregation-prone molecules which leads to amyloid-like aggregation. We observed that specific DNA binding inhibits aggregation and populates native-like compact monomeric state even under low-pH stress as measured by circular dichroism, ANS binding, size exclusion chromatography, and transmission electron microscopy. We show that DNA-binding thermodynamically stabilizes and populates the native state even under stress and reduces the population of unfolded-like aggregation-prone molecules which leads to systematic aggregation inhibition. Our results suggest that thermodynamic modulation of the folding and aggregation energy landscape by nucleic-acid-like molecules could be a promising approach for effective therapeutic intervention in TDP-43-associated proteinopathies.     

Rapid assembly and disassembly of complementary DNA strands through an equilibrium intermediate state mediated by A1 hnRNP protein.

A1 hnRNP protein, which rapidly renatures complementary strands of nucleic acids in vitro, affects both the equilibrium and kinetic properties of the reaction (single-stranded DNA in equilibrium with double-stranded DNA). A1 lowers the melting transition of duplex DNA. However, at temperatures above this new Tm, both single- and double-stranded DNAs are present at equilibrium and are rapidly interconverting. Although the ratio of single and double strands under these conditions is a function of both the A1 protein and complementary DNA strand concentrations, it is not strongly affected by further increases in temperature. These surprising results demonstrate that A1 does not act as a simple catalyst in promoting renaturation and indicate how A1 and other proteins could act to speed the turnover of intermediate complexes in important biological processes.     

Effects of a protecting osmolyte on the ion atmosphere surrounding DNA duplexes

Osmolytes are small, chemically diverse, organic solutes that function as an essential component of cellular stress response. Protecting osmolytes enhance protein stability via preferential exclusion, and nonprotecting osmolytes, such as urea, destabilize protein structures. Although much is known about osmolyte effects on proteins, less is understood about osmolyte effects on nucleic acids and their counterion atmospheres. Nonprotecting osmolytes destabilize nucleic acid structures, but effects of protecting osmolytes depend on numerous factors including the type of nucleic acid and the complexity of the functional fold. To begin quantifying protecting osmolyte effects on nucleic acid interactions, we used small-angle X-ray scattering (SAXS) techniques to monitor DNA duplexes in the presence of sucrose. This protecting osmolyte is a commonly used contrast matching agent in SAXS studies of protein-nucleic acid complexes; thus, it is important to characterize interaction changes induced by sucrose. Measurements of interactions between duplexes showed no dependence on the presence of up to 30% sucrose, except under high Mg(2+) conditions where stacking interactions were disfavored. The number of excess ions associated with DNA duplexes, reported by anomalous small-angle X-ray scattering (ASAXS) experiments, was sucrose independent. Although protecting osmolytes can destabilize secondary structures, our results suggest that ion atmospheres of individual duplexes remain unperturbed by sucrose.     

Biophysical effect of amino acids on the prevention of protein aggregation

Each protein folds into a unique and native structure spontaneously. However, during the unfolding or refolding process, a protein often tends to form aggregates. To establish a method to prevent undesirable protein aggregation and to increase the stability of native protein structures under deterioration conditions, two types of aggregation conditions, thermal unfolding-induced aggregation and dilution-induced aggregation from denatured state, were studied in the presence of additional amino acids and ions using lysozyme as a model protein. Among 15 amino acids tested, arginine exhibited the best results in preventing the formation of aggregates in both cases. Further biophysical studies revealed that arginine did not change the thermal denaturation temperature (T(m)) of the lysozyme. The preventive effect of arginine on aggregation was not dependent on the size or isoelectric point of eight kinds of proteins tested.     

DNA framework-engineered electrochemical biosensors

Self-assembled DNA nanostructures have shown remarkable potential in the engineering of biosensing interfaces, which can improve the performance of various biosensors. In particular, by exploiting the structural rigidity and programmability of the framework nucleic acids with high precision, molecular recognition on the electrochemical biosensing interface has been significantly enhanced, leading to the development of highly sensitive and specific biosensors for nucleic acids, small molecules,proteins, and cells. In this review, we summarize recent advances in DNA framework-engineered biosensing interfaces and the application of corresponding electrochemical biosensors.