Fluorescent microplate-based analysis of protein-DNA interactions. I: Immobilized protein

A simple protein-DNA interaction analysis has been developed using a high-affinity/high-specificity zinc finger protein. In essence, purified protein samples are immobilized directly onto the surface of microplate wells, and fluorescently labeled DNA is added in solution. After incubation and washing, bound DNA is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.2 nM DNA. Since the detection of bound DNA is noninvasive and the protein-DNA interaction is not disrupted during detection, iterative readings may be taken from the same well, after successive alterations in interaction conditions, if required. In this respect, the assay may therefore be considered real time and permits appropriate interaction conditions to be determined quantitatively. The assay format is ideally suited to investigate the interactions of purified unlabeled DNA binding proteins in a high-throughput format.     

Proteolytic DNA for mapping protein-DNA interactions

We describe a technique to determine sites on proteins involved in protein-DNA interactions. DNA was synthesized via polymerase chain reaction (PCR) to produce four polynucleotide products with phosphorothioate nucleotides at the A, T, G, or C residues. Limited conjugation with the chemical protease FeBABE results in the surface of DNA being randomly labeled at the phosphorothioate sites with this protein-cleaving reagent. After formation of a protein-DNA complex, the proteolytic DNA can be activated to cleave the protein backbone at sites near the DNA. This technique was used to study the bacterial RNA polymerase/lacUV5 DNA open promoter complex, about which significant structural information is available. Cleavage sites on the two largest subunits of RNA polymerase, beta and beta', agree well with a recent model based on the crystal structure of the core enzyme alpha(2)betabeta' [Naryshkin, N., Revyakin, A., Kim, Y., Mekler, V., and Ebright, R. H. (2000) Cell 101, 601-611]. The cleavage site present on alpha supports previous studies regarding DNA binding regions of the alpha subunit. Cleavage sites identified throughout the sigma(70) subunit help to orient it with respect to the open promoter complex.     

An analysis of the relationship between hydration and protein-DNA interactions.

Eleven protein-DNA crystal structures were analyzed to test the hypothesis that hydration sites predicted in the first hydration shell of DNA mark the positions where protein residues hydrogen-bond to DNA. For nine of those structures, protein atoms, which form hydrogen bonds to DNA bases, were found within 1.5 A of the predicted hydration positions in 86% of the interactions. The correspondence of the predicted hydration sites with the hydrogen-bonded protein side chains was significantly higher for bases inside the conserved DNA recognition sequences than outside those regions. In two CAP-DNA complexes, predicted base hydration sites correctly marked 71% (within 1.5 A) of protein atoms, which form hydrogen bonds to DNA bases. Phosphate hydration was compared to actual protein binding sites in one CAP-DNA complex with 78% marked contacts within 2.0 A. These data suggest that hydration sites mark the binding sites at protein-DNA interfaces.     

Insights into protein-DNA interactions through structure network analysis

Protein-DNA interactions are crucial for many cellular processes. Now with the increased availability of structures of protein-DNA complexes, gaining deeper insights into the nature of protein-DNA interactions has become possible. Earlier, investigations have characterized the interface properties by considering pairwise interactions. However, the information communicated along the interfaces is rarely a pairwise phenomenon, and we feel that a global picture can be obtained by considering a protein-DNA complex as a network of noncovalently interacting systems. Furthermore, most of the earlier investigations have been carried out from the protein point of view (protein-centric), and the present network approach aims to combine both the protein-centric and the DNA-centric points of view. Part of the study involves the development of methodology to investigate protein-DNA graphs/networks with the development of key parameters. A network representation provides a holistic view of the interacting surface and has been reported here for the first time. The second part of the study involves the analyses of these graphs in terms of clusters of interacting residues and the identification of highly connected residues (hubs) along the protein-DNA interface. A predominance of deoxyribose-amino acid clusters in beta-sheet proteins, distinction of the interface clusters in helix-turn-helix, and the zipper-type proteins would not have been possible by conventional pairwise interaction analysis. Additionally, we propose a potential classification scheme for a set of protein-DNA complexes on the basis of the protein-DNA interface clusters. This provides a general idea of how the proteins interact with the different components of DNA in different complexes. Thus, we believe that the present graph-based method provides a deeper insight into the analysis of the protein-DNA recognition mechanisms by throwing more light on the nature and the specificity of these interactions.     

Improved band shift assay for the simultaneous analysis of protein-DNA interactions and enzymatic functions of DNA polymerases.

A simple method to assay the major properties of DNA polymerases such as template binding, polymerase and exonuclease activities in one step is exemplified with the DNA polymerases of E. coli, bacteriophage T4 and herpes simplex virus. Combining the advantages of the band-shift assay with the resolving power of polyacrylamide gradient gel electrophoresis, the procedure is particularly useful for a rapid functional analysis of mutant polymerases as well as inhibitors of DNA replication.     

Immobilized enzyme cellulose hollow fibers: II. Kinetic analysis

Immobilized enzyme hollow fibers may be useful in the purification or treatment of whole blood under clinical conditions. In this study, catalytically pure heparinase was immobilized to cellulose to analyze the feasibility for the removal of heparin's anticoagulant activity from whole blood. The kinetics of catalytically pure heparinase immobilized to regenerated cellulose hollow fibers were quantified with respect to mass transfer coefficient and enzyme loading. The kinetic analysis showed that increases in the mass transfer coefficient of heparin in the fiber lumen decreased the apparent Michaelis constant while increases in enzyme activity immobilized to the fiber lumen increased the apparent Michaelis constant. The apparent Michaelis constant was an order of magnitude greater than the intrinsic K(m) value for the system. The intrinsic K(m) value for heparinase-cellulose is 0.4 +/- 0.3 mg/mL (N = 6) and it is the same order of magnitude as the K(m) value for soluble heparinase.     

Tramtrack protein-DNA interactions. A cross-linking study

Interaction of the Tramtrack protein from Drosophila melanogaster with DNA was analyzed by a cross-linking method. Tramtrack residues cross-linkable to the partially depurinated DNA were identified by direct sequencing. The N-terminal alpha-amino group of the protein DNA-binding domain was found to be the major product of cross-linking. The location of the N terminus on the DNA was determined by identification of the DNA bases that were cross-linked to the protein alpha-amino group. We conclude that accessory N-terminal peptide preceding the first zinc finger of Tramtrack directly interacts with DNA, both in specific and nonspecific DNA-protein complexes. Our finding explains the role in the protein binding of the DNA bases outside of the direct interaction with the zinc fingers.     

Probing protein-DNA interactions by unzipping a single DNA double helix

We present unzipping force analysis of protein association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex. The capacity of UFAPA to spatially locate protein-DNA interactions is demonstrated by noncatalytic restriction mapping on a 4-kb DNA with three restriction enzymes (BsoBI, XhoI, and EcoRI). A restriction map for a given restriction enzyme was generated with an accuracy of approximately 25 bp. UFAPA also allows direct determination of the site-specific equilibrium association constant (K(A)) for a DNA-binding protein. This capability is demonstrated by measuring the cation concentration dependence of K(A) for EcoRI binding. The measured values are in good agreement with previous measurements of K(A) over an intermediate range of cation concentration. These results demonstrate the potential utility of UFAPA for future studies of site-specific protein-DNA interactions.     

Analytical ultracentrifugation studies of translin: analysis of protein-DNA interactions using a single-stranded fluorogenic oligonucleotide.

Translin is a recently identified nucleic acid binding protein that appears to be involved in the recognition of conserved sequences found at many chromosomal breakpoints. Previous reports indicate that, based on gel filtration analysis and electron microscopy of protein-DNA complexes, translin forms an octameric structure that binds the DNA. In this study, we further examine the possibility of self-association of translin and its interactions with DNA by analytical ultracentrifugation. Sedimentation velocity analysis of translin indicates that the predominant species sediments with a sedimentation coefficient of 8.5 S and has a frictional ratio, f/f(omicron), of 1.35; these data are consistent with the presence of an octamer with an ellipsoidal configuration; a small amount of a component with significantly higher mass is also present. Equilibrium sedimentation studies of translin at three different protein concentrations also indicate that the predominant species present is an octamer with a minor fraction of aggregated species. Neither monomer nor dimer was detected. Sedimentation equilibrium studies of translin with an FITC-labeled single-stranded oligonucleotide were performed to examine the interaction. A novel analysis method has been developed to analyze protein-nucleic acid interactions based on global fitting of scans of 280 and 490 nm to appropriate mathematical models. Utilizing this method, it was determined that the DNA binding species of translin is an octamer binding a single-stranded oligonucleotide with a DeltaG degrees value of -9.49 +/- 0.12 kcal/mol, corresponding to a dissociation constant, K(d), of 84 +/- 17 nM. On the basis of this evidence and electron microscopy, it is envisioned that translin forms an annular structure of eight subunits, hydrodynamically an oblate ellipsoid, which binds DNA at chromosomal breakpoints.     

Thermodynamics of sequence-specific protein-DNA interactions

The molecular recognition processes in sequence-specific protein-DNA interactions are complex. The only feature common to all sequence-specific protein-DNA structures is a large interaction interface, which displays a high degree of complementarity in terms of shape, polarity and electrostatics. Many molecular mechanisms act in concert to form the specific interface. These include conformational changes in DNA and protein, dehydration of surfaces, reorganization of ion atmospheres, and changes in dynamics. Here we review the current understanding of how different mechanisms contribute to the thermodynamics of the binding equilibrium and the stabilizing effect of the different types of noncovalent interactions found in protein-DNA complexes. The relation to the thermodynamics of small molecule-DNA binding and protein folding is also briefly discussed.     

DNA deformation energy as an indirect recognition mechanism in protein-DNA interactions

Proteins that bind to specific locations in genomic DNA control many basic cellular functions. Proteins detect their binding sites using both direct and indirect recognition mechanisms. Deformation energy, which models the energy required to bend DNA from its native shape to its shape when bound to a protein, has been shown to be an indirect recognition mechanism for one particular protein, integration host factor (IHF). This work extends the analysis of deformation to two other DNA-binding proteins, CRP and SRF, and two endonucleases, I-Crel and I-Ppol. Known binding sites for all five proteins showed statistically significant differences in mean deformation energy as compared to random sequences. Binding sites for the three DNA-binding proteins and one of the endonucleases had mean deformation energies lower than random sequences. Binding sites for I-Ppol had mean deformation energy higher than random sequences. Classifiers that were trained using the deformation energy at each base pair step showed good cross-validated accuracy when classifying unseen sequences as binders or nonbinders. These results support DNA deformation energy as an indirect recognition mechanism across a wider range of DNA-binding proteins. Deformation energy may also have a predictive capacity for the underlying catalytic mechanism of DNA-binding enzymes     

A new analytical scale DNA affinity binding assay for analyses of specific protein-DNA interactions.

We describe a rapid analytical assay for identification of proteins binding to specific DNA sequences. The DAPSTER assay (DNA affinity preincubation specificity test of recognition assay) is a DNA affinity chromatography-based microassay that can discriminate between specific and nonspecific protein-DNA interactions. The assay is sensitive and can detect protein-DNA interactions and larger multicomponent complexes that can be missed by other analytical methods. Here we describe in detail the optimization and utilization of the DAPSTER assay to isolate AP-1 complexes and associated proteins in multimeric complexes bound to the AP-1 DNA element.     

Water-mediated protein-DNA interactions: the relationship of thermodynamics to structural detail.

The elucidation of a relationship between the thermodynamic parameters and the structural changes accompanying biomolecular interactions could lead to predictive algorithms. For example, based on some knowledge of the structure of a target molecule the affinities of ligands could be determined with obvious implications for the pharmaceutical industry. In attempting to relate the thermodynamic and structural changes on formation of a protein-DNA complex, the correlation between change in heat capacity and burial of surface area has proved successful. However, this correlation appears to break down when water molecules are included in the binding interface. Here we present data that support the hypothesis that bound water molecules have to be considered as contributing to the change in heat capacity and could, thus, be used in ligand design.     

Gene 5 protein-DNA complex: modeling binding interactions

A helical (not toroidal) complex consisting of eight gene 5 protein dimers per turn is proposed for the extension of DNA from dimer to dimer using known bond length constraints, postulated protein-nucleic acid interactions (determined from NMR and chemical modification studies), other physical properties of the complex, and data from electron micrographs. The binding channel has been dictated by these known parameters and the relative ease of geometrically fitting these constituents. This channel is different from that previously reported by other modelers. The channel lies underneath the long arm "claw-like" extension of the monomer, so that it rests inside the outer surface of the protein complex. An explanation is proposed for the two binding modes, n = 4 (the predominate mode) and n = 3, based on the weak binding interaction of Tyrosine 34. Also, the site of the less mobile nucleic acid base as reported from ESR studies (S.-C. Kao, E.V. Bobst, G.T. Pauly and A.M. Bobst, J. Biom. Struc. Dyn. 3,261 (1985)) is postulated as involving the fourth nucleotide, and this particular base is stacked between Tyrosine 34 and Phenylalanine 73'.     

Binding site selection analysis of protein-DNA interactions via solid phase sequencing of oligonucleotide mixtures.

By combining the concept of degenerate oligonucleotide mutagenesis (1,2,3,4) and the convenience of solid phase chemical DNA sequencing (5), we have developed a rapid procedure for determining the specificity of DNA-binding proteins in vitro. Starting with a degenerate oligonucleotide mixture, the technique assays for alternative nucleotides in fractions that are bound or non-bound to the protein of interest. In contrast to previous approaches using degenerate oligonucleotides, it does not involve cloning but rather employs direct sequencing of the oligonucleotide mixtures after attachment to a solid support. Solid state processing obviates the need for both DNA extractions from polyacrylamide gels and time-consuming ethanol precipitations. Because of its convenience and sensitivity, this binding site selection analysis is well suited to determining rapidly the sequence preference of DNA-binding proteins that are available in small amounts, and complements well established approaches like methylation interference or missing contact assays. The solid phase reaction protocol we propose can also improve these latter approaches.     

Application of fluorescamine to the study of protein-DNA interactions.

The reactivity of alpha-amino groups of basic proteins towards fluorescamine is essentially abolished if salt linkages with DNA phosphate groups are formed. This observation prompted the elaboration of a very general assay which allows the determination of binding parameters for the interaction of proteins with DNA and chromatin. Protamines, labeled with fluorescamine prior to their binding by DNA appear to be useful probes to monitor the formation and nature of DNA-protein complexes.