Optimization of a radiolabel DNA-binding assay in cultured mammalian cells

An improved protocol for the radiolabel DNA-binding assay, which gives a high yield of highly pure DNA has been developed by use of mouse lymphoma cells. The critical difference from previously published methods is the use of enzymatic degradation of proteins in the later DNA purification steps rather than during the homogenisation procedure. Different DNA-purification methodologies were first compared and the protocol of choice was optimized later on; both steps were performed with [(35)S]-labelled amino acids for labelling of cellular protein, which enabled both the quantification of cellular protein contaminating the DNA sample and the distinction between cellular and enzyme-derived protein. The assay was later evaluated and shown to give reproducible results based on the data obtained with benzo[a]pyrene (B[a]P) and doxorubicin in two different laboratories. In addition, two further reference compounds, dopamine and diazepam and one proprietary AstraZeneca compound were also tested in mouse lymphoma cells in one laboratory. The two compounds B[a]P and doxorubicin were identified as suitable positive controls for routine testing in the presence and absence of S9, respectively. Exposing 90-100×10(6) cells to (14)C-labelled compound with a molar radioactivity of 2MBq/μmol, yields approximately 500μg DNA with <3% total protein contamination, of which approximately 7% is of cellular origin (<0.2%). The detection level is approximately 2adducts/10(8) dNTP.     

A solid-phase assay to screen monoclonal antibodies against DNA-binding protein.

A method is described for selecting monoclonal antibodies (mAb) against DNA-binding protein. The protocol involves a non-radioactive solid-phase DNA binding assay using a 96-well plate. Because the solid-phase assay is highly specific and sensitive, partially purified antigen is sufficient for the immunization, and mAb screening can be performed with crude cell extract as the antigen. MAbs obtained by this method could supershift the DNA-protein complex in the electromobility shift assay, and were sufficient for immunoscreening of a cDNA expression library.     

A rapid solid-phase fluorimetric assay for measuring bacterial adherence,using DNA-binding stains

In this report, we describe the validation of a rapid, single-step, microtiter plate method for quantifying bacterial adherence, based on fluorescent labeling of microorganisms with cell-permeable fluorescent DNA-binding probes. We have tested the binding to saliva-coated microtiter plates of bacteria, including Helicobacter pylori and viridans streptococci (S. mitis, S. gordonii, S. sanguis), known to interact with salivary components. Furthermore, we tested the short-term and longer-term temporal stability of a saliva-mediated adherence of these bacteria in a healthy population (N=30). The assay exhibited excellent reliability statistics, yielding within-assay variability coefficients ranging from 4.9% to 11%. A range of approximately 5 x 10(4)-1 x 10(7) cells could be detected. This method may be generally applicable to study surface binding of virtually any microbial species, while obviating the need of radioactive materials or specific antibodies for quantification, thus providing a procedure that is useful to both basic and clinical research.     

Fluorescence resonance energy transfer-based assay for DNA-binding protein tagged by green fluorescent protein

Specific interaction between green fluorescent protein (GFP)-tagged human alpha- or gamma-enolase(97-242) (alpha or gammaENO(97-242)) and the rhodamine-labeled DNA fragment containing the c-myc P2 promoter was detected by a fluorescence resonance energy transfer (FRET)-based assay, designated as a "real-time FRET assay." The approach of donor (GFP) and acceptor (rhodamine) was caused by the association between ENO(97-242) and the c-myc P2 promoter, and the time-dependent increase in fluorescence intensity of the reaction mixture was observed at ex=400 nm and em=590 nm. The relative affinity (R(as)) of ENO(97-242) mutants to the wild type was investigated with a real-time FRET assay, and it was clarified that the amino acids that participated in the interaction existed comparatively broadly. Although it was difficult to measure the absolute value of the affinity for the binding protein by using this method, it was possible to investigate the relative affinity of mutants for the wild type. A real-time FRET assay using the GFP-tagged protein could be used as not only a qualitative, but also as a quantitative analysis, this being the best for investigating the key amino acids in binding proteins.     

Binding mode and affinity studies of DNA-binding agents using topoisomerase I DNA unwinding assay

A topoisomerase I DNA unwinding assay has been used to determine the relative DNA-binding affinities of a model pair of homologous naphthalene diimides. Binding affinity data were corroborated using calorimetric (ITC) and spectrophotometric (titration and T(m)) studies, with substituent size playing a significant role in binding. The assay was also used to investigate the mode of binding adopted by several known DNA-binding agents, including SYBR Green and PicoGreen. Some of the compounds exhibited unexpected binding modes.     

Using electrostatic potentials to predict DNA-binding sites on DNA-binding proteins

A method to detect DNA-binding sites on the surface of a protein structure is important for functional annotation. This work describes the analysis of residue patches on the surface of DNA-binding proteins and the development of a method of predicting DNA-binding sites using a single feature of these surface patches. Surface patches and the DNA-binding sites were initially analysed for accessibility, electrostatic potential, residue propensity, hydrophobicity and residue conservation. From this, it was observed that the DNA-binding sites were, in general, amongst the top 10% of patches with the largest positive electrostatic scores. This knowledge led to the development of a prediction method in which patches of surface residues were selected such that they excluded residues with negative electrostatic scores. This method was used to make predictions for a data set of 56 non-homologous DNA-binding proteins. Correct predictions made for 68% of the data set.     

Cytoplasmic DNA-binding proteins

Cytoplasmic DNA-binding proteins were isolated from Chinese hamster liver, kidney and tissue culture cells by DNA-polyacrylamide chromatography. With homologous Chinese hamster DNA, and with calf thymus DNA, 1.4% of the proteins were bound to the column. With single-stranded DNA and with heterologous Micrococcus lysodeikticus DNA there was only 0.3% binding, suggesting the proteins preferentially bind to double-stranded DNA and show some sequence specificity. By a nitrocellulose filter assay the bound proteins had at least a 4- to 7-fold greater affinity for DNA than bulk cytoplasmic protein. SDS gel electrophoresis showed that specific proteins were being markedly concentrated by the column and it was primarily the high molecular weight proteins of 65 000 D and over which showed sequence specificity. Some proteins appeared in common with different organs, others were unique. These studies thus define a group of high molecular weight, cytoplasmic proteins which bind to native DNA with a degree of sequence specificity. Their possible relationship to gene regulation is discussed.     

DNA-binding studies with 6BT and 5I: implications for DNA-binding/carcinogenicity and DNA-binding/mutagenicity correlations

The divergent activities of a reported carcinogen/noncarcinogen pair of monoazo dyes related to the hepatocarcinogen Butter Yellow (DAB) are currently under investigation in our laboratories. As part of these studies we have determined (a) target organ distribution after oral dosing to rats and (b) covalent binding of 14C-labelled compound to DNA. In DNA-binding studies, 3 rat liver-metabolising systems were employed: in vivo (whole liver), isolated intact hepatocytes, and liver subcellular fractions. Distribution studies revealed that comparable levels of both compounds were detected in the liver at similar times after dosing, and these in vivo tissue concentrations were used for in vitro DNA-binding studies. At this 'in vivo equivalent dose', the carcinogen was consistently bound to DNA more effectively, and the difference (ratio of DNA binding) between the 2 compounds was far greater in vivo. In subsequent studies, covalent DNA binding to bacterial (Salmonella) DNA was assessed at the in vivo equivalent dose. In contrast to the afore-mentioned findings in mammalian systems, the carcinogen was bound less effectively to DNA, and gave fewer revertant counts/plate when the 2 compounds were bound to an equivalent extent. These data are discussed in view of their implications for DNA-binding/carcinogenicity correlations, and with respect to the relationship between DNA binding and mutagenicity in the Salmonella assay.     

Residue-level prediction of DNA-binding sites and its application on DNA-binding protein predictions

Protein-DNA interactions are crucial to many cellular activities such as expression-control and DNA-repair. These interactions between amino acids and nucleotides are highly specific and any aberrance at the binding site can render the interaction completely incompetent. In this study, we have three aims focusing on DNA-binding residues on the protein surface: to develop an automated approach for fast and reliable recognition of DNA-binding sites; to improve the prediction by distance-dependent refinement; use these predictions to identify DNA-binding proteins. We use a support vector machines (SVM)-based approach to harness the features of the DNA-binding residues to distinguish them from non-binding residues. Features used for distinction include the residue's identity, charge, solvent accessibility, average potential, the secondary structure it is embedded in, neighboring residues, and location in a cationic patch. These features collected from 50 proteins are used to train SVM. Testing is then performed on another set of 37 proteins, much larger than any testing set used in previous studies. The testing set has no more than 20% sequence identity not only among its pairs, but also with the proteins in the training set, thus removing any undesired redundancy due to homology. This set also has proteins with an unseen DNA-binding structural class not present in the training set. With the above features, an accuracy of 66% with balanced sensitivity and specificity is achieved without relying on homology or evolutionary information. We then develop a post-processing scheme to improve the prediction using the relative location of the predicted residues. Balanced success is then achieved with average sensitivity, specificity and accuracy pegged at 71.3%, 69.3% and 70.5%, respectively. Average net prediction is also around 70%. Finally, we show that the number of predicted DNA-binding residues can be used to differentiate DNA-binding proteins from non-DNA-binding proteins with an accuracy of 78%. Results presented here demonstrate that machine-learning can be applied to automated identification of DNA-binding residues and that the success rate can be ameliorated as more features are added. Such functional site prediction protocols can be useful in guiding consequent works such as site-directed mutagenesis and macromolecular docking.     

Nonintercalative DNA-binding antitumour compounds

Summary A family of compounds which appear to bind reversibly to double stranded DNA without intercalation between DNA base pairs has been defined. Methods are described by which this non-intercalative binding can be characterised using ultraviolet spectrometry, fluorimetry with ethidium as a probe, viscometry and other hydrodynamic techniques, circular dichroism and nuclear magnetic resonance spectrometry. Antibiotics which fall into this family include the antibiotics distamycin A, netropsin, mithramycin, chromomycin and olivomycin. Synthetic antitumour agents include diarylamidines such as berenil, phthalanilides, aromatic bisguanylhydrazones and bisquaternary ammonium heterocycles. A survey has been made of the general requirements of this family of compounds for DNA binding and biological activity. Binding of drugs to the minor groove of the DNA double helix appears to be the most likely mechanism for the antitumour action of these compounds.