Quantifying DNA-protein interactions by double-stranded DNA arrays.

We have created double-stranded oligonucleotide arrays to perform highly parallel investigations of DNA-protein interactions. Arrays of single-stranded DNA oligonucleotides, synthesized by a combination of photolithography and solid-state chemistry, have been used for a variety of applications, including large-scale mRNA expression monitoring, genotyping, and sequence-variation analysis. We converted a single-stranded to a double-stranded array by synthesizing a constant sequence at every position on an array and then annealing and enzymatically extending a complementary primer. The efficiency of second-strand synthesis was demonstrated by incorporation of fluorescently labeled dNTPs (2'-deoxyribonucleoside 5'-triphosphates) and by terminal transferase addition of a fluorescently labeled ddNTP. The accuracy of second-strand synthesis was demonstrated by digestion of the arrayed double-stranded DNA (dsDNA) on the array with sequence-specific restriction enzymes. We showed dam methylation of dsDNA arrays by digestion with DpnI, which cleaves when its recognition site is methylated. This digestion demonstrated that the dsDNA arrays can be further biochemically modified and that the DNA is accessible for interaction with DNA-binding proteins. This dsDNA array approach could be extended to explore the spectrum of sequence-specific protein binding sites in genomes.     

Probing DNA-protein interactions in vitro with the CpG DNA methyltransferase.

A sensitive method was devised to monitor the in vitro binding of nuclear proteins from HeLa cells presumably to the major groove of DNA. Upon the incubation of DNA with nuclear extracts, the complexed DNA was incubated with the CpG DNA methyltransferase from Spiroplasma species. Subsequently, the DNA was repurified, and the location of the methylated cytidine residues was determined by the hydrazine reaction of the DNA sequencing method. By using as DNA substrate the VAI (virus associated) region of human adenovirus type 2 (Ad2) DNA or specific Alu sequences associated with a number of human genes, it was documented that those segments of DNA that were protected by bound proteins against the reaction with DNasel also escaped in vitro methylation by the CpG DNA methyltransferase. This new footprinting method provides a sensitive indicator for in vitro DNA--protein interactions which are specific for the major groove of DNA.     

The effect of HhaI methylation on DNA local structure.

The effect of methylating the central cytosine in the sequence GCGC on the sensitivity to digestion by deoxyribonuclease I was investigated. Examination of five such sites shows that the extra methyl group renders the bond on its 5' side more susceptible to cleavage, and this is interpreted by suggesting that it causes small changes in the local DNA phosphate orientation.     

DNA annealing and DNA-protein interactions by capillary electrophoresis

This work deals with annealing of single-stranded DNA and the binding of a serum respond factor to a DNA probe containing specific binding site. Capillary electrophoresis (CE) method is explored and compared with the mobility-shift gel electrophoresis (GE) procedure. The results indicate the CE method offers direct and rapid annealing of the DNA strands. It requires no prior incubation with additives (polynucleotides, proteins) to reduce nonspecific DNA-protein interactions. Unwanted nonspecific interactions are not observed in the CE method. The presence of a fluorescein tag to the DNA probe yields identical results to those with the radioactive label. A fluorescein tag in the CE work can be used without any adverse effects. The dissociation constant (Kd) of this protein-DNA complex by the CE method was similar to those determined by the GE method (approximately 10(-6) M). The proposed method is extremely powerful, highly sensitive, quantitative, and fast. It can determine even very small conformational differences of the DNA probe.     

Attenuation of DNA-protein interactions associated with intrinsic, sequence-dependent DNA curvature.

Inherently curved DNA segments, associated with short runs of adenines, have been identified in many gene regulatory regions, yet their physiological significance remains unknown. The observations reported in this study indicate that intrinsically bent nucleic acid fragments are characterized by substantially attenuated affinities toward DNA-binding proteins involved in structural functions, such as H1 histone and protamine, as well as toward various DNA-modifying enzymes including ligases and exo- and endonucleases. Two mechanisms might be responsible for the altered binding properties. According to the first mechanism, the attenuated binding affinities and the bending represent two independent consequences of the unique structural parameters exhibited by A-tracts. Indeed, analysis of the degradation products obtained upon exposure of the curved sequences to various chemical nucleases points toward the narrowing of the DNA minor groove, a conformational modulation known to characterize A-tracts and to run along the axially-bent motifs, as a potential determinant of the observed binding attenuation. Alternatively, the conformational constraints which result from the stable bending might act to modulate the strength of DNA-protein interactions. Although the factor directly responsible for the altered binding affinities revealed by the bent sequences cannot as yet be conclusively resolved, it is proposed that a reiteration of this specific factor, being either an A-tract or a bend, in phase with the DNA helical repeat acts to amplify the modulation of the binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studying DNA-protein interactions using NMR.

NMR spectroscopy is emerging as a powerful tool in molecular biology and biotechnology; one aspect of which is the use of one- and two-dimensional NMR methodologies to investigate the interactions of proteins with DNA. The dynamic and structural information which NMR can provide, on the changes in conformation and molecular flexibility, complements X-ray crystallography data and enables mechanistic models of DNA-protein interactions to be formulated.     

The effect of gamma radiation on DNA methylation

The effect of 60Co gamma radiation on DNA methylation was studied in four cultured cell lines. In all cases a dose-dependent decrease in 5-methylcytosine was observed at 24, 48, and 72 h postexposure to 0.5-10 Gy. Nuclear DNA methyltransferase activity decreased while cytoplasmic activity increased in irradiated (10 Gy) V79A03 cells as compared to controls. No DNA demethylase activity was detected in the nuclei of control or irradiated V79A03 cells. Additionally, gamma radiation resulted in the differentiation of C-1300 N1E-115 cells, a mouse neuroblastoma line, in a dose- and time-dependent manner. These results are consistent with the hypothesis that (1) genes may be turned on following radiation via a mechanism involving hypomethylation of cytosine and (2) radiation-induced hypomethylation results from decreased intranuclear levels of DNA methyltransferase.     

Polyamines alter sequence-specific DNA-protein interactions.

The polyamines are abundant biogenic cations implicated in many biological processes. Despite a plethora of evidence on polyamine-induced DNA conformational changes, no thorough study of their effects on the activities of sequence-specific DNA binding proteins has been performed. We describe the in vitro effects of polyamines on the activities of purified, representative DNA-binding proteins, and on complex protein mixtures. Polyamines at physiological concentrations enhance the binding of several proteins to DNA (e.g. USF, TFE3, Ig/EBP, NF-IL6, YY1 and ICP-4, a herpes simplex virus gene regulator), but inhibit others (e.g. Oct-1). The degree of enhancement correlates with cationic charge; divalent putrescine is ineffective whereas tetravalent spermine is more potent than trivalent spermidine. Polyamine effects on USF and ICP-4 result from increased rate of complex formation rather than a decreased rate of dissociation. DNAse I footprint analysis indicated that polyamines do not alter DNA-protein contacts. Polyamines also facilitate formation of complexes involving binding of more than one protein on a DNA fragment.     

Thymine methyls and DNA-protein interactions.

Evidence is summarized showing that thymine methyls are as important in the recognition of specific sequences by proteins as are the more widely recognized hydrogen bonding sites of bases in the major groove (1). Strongest evidence has come from experiments using functional group mutagenesis (2) in which thymines in a specific recognition sequence (e.g., promoters, operators and restriction sites) are replaced by oligonucleotide synthesis with methyl-free uracil or cytosine and 5-methylcytosine. Such experiments have shown that thymine methyls can provide contact points via van der Waals interactions with amino acid side chains of specific DNA binding proteins. Actual contact between a thymine methyl and carbons of a glutamine side chain has been observed in a cocrystal of the phage 434 repressor and its operator by X-ray analysis. The issue of why thymine occurs in DNA is discussed in light of these findings.     

The effect of long-chain bases on polysialic acid-mediated membrane interactions

Negatively-charged polysialic acid (polySia) chains are usually membrane-bound and are often expressed on the surface of neuroinvasive bacterial cells, neural cells, and tumor cells. PolySia can mediate both repulsive and attractive cis interactions between membrane components, and trans interactions between membranes. Positively-charged long-chain bases are widely present in cells, are often localized in membranes and can function as bioactive lipids. Here we use Langmuir monolayer technique, fluorescence spectroscopy and electron microscopy of lipid vesicles to study the role of a simple long-chain base, octadecylamine (ODA), in both cis and trans interactions mediated by polySia in model membranes composed of ODA and dioleoylphospatidycholine (DOPC). When added free to an aqueous solution, polySia increases the collapse pressure of ODA/DOPC monolayers, reduces the effect of ODA on the limiting molecular area, inverses the values of excess area per molecule and of excess free energy of mixing from positive to negative, and induces fusion of ODA/DOPC vesicles. These results suggest that a polySia chain can act as a multi-bridge that mediates cis interactions between different components of a lipid membrane, disrupts membrane aggregates, and mediates trans interactions between lipids in apposing membranes. These observations imply that polySia in cellular systems can act in a similar way.     

The effect of sequence specific DNA methylation on restriction endonuclease cleavage.

Sequence specific DNA methylation sometimes results in the protection of some or all of a restriction endonucleases' cleavage sites. This is usually, but not always, the result of methylation of one or both strands of DNA at the site characteristic of the corresponding "cognate" modification methylase. The known effects of sequence specific methylation on restriction endonucleases are compiled.     

Free radicals-mediated induction of oxidized DNA bases and DNA-protein cross-links by nickel chloride

Using the comet assay, we showed that nickel chloride at 250-1000 microM induced DNA damage in human lymphocytes, measured as the change in comet tail moment, which increased with nickel concentration up to 500 microM and then decreased. Observed increase might follow from the induction of strand breaks or/and alkali-labile sites (ALS) by nickel, whereas decrease from its induction of DNA-DNA and/or DNA-protein cross-links. Proteinase K caused an increase in the tail moment, suggesting that nickel chloride at 1000 microM might cross-link DNA with nuclear proteins. Lymphocytes exposed to NiCl(2) and treated with enzymes recognizing oxidized and alkylated bases: endonuclease III (Endo III), formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), displayed greater extent of DNA damage than those not treated with these enzymes, indicating the induction of oxidized and alkylated bases by nickel. The incubation of lymphocytes with spin traps, 5,5-dimethyl-pyrroline N-oxide (DMPO) and PBN decreased the extent of DNA damage, which might follow from the production of free radicals by nickel. The pre-treatment with Vitamin C at 10 microM and Vitamin E at 25 microM decreased the tail moment of the cells exposed to NiCl(2) at the concentrations of the metal causing strand breaks or/and ALS. The results obtained suggest that free radicals may be involved in the formation of strand breaks or/and ALS in DNA as well as DNA-protein cross-links induced by NiCl(2). Nickel chloride can also alkylate DNA bases. Our results support thesis on multiple, free radicals-based genotoxicity pathways of nickel.     

Base substitution specificity of DNA polymerase beta depends on interactions in the DNA minor groove.

To examine the hypothesis that interactions between a DNA polymerase and the DNA minor groove are critical for accurate DNA synthesis, we studied the fidelity of DNA polymerase beta mutants at residue Arg(283), where arginine, which interacts with the minor groove at the active site, is replaced by alanine or lysine. Alanine substitution, removing minor groove interactions, strongly reduces polymerase selectivity for all single-base mispairs examined. In contrast, the lysine substitution, which retains significant interactions with the minor groove, has wild-type-like selectivity for T.dGMP and A.dGMP mispairs but reduced selectivity for T.dCMP and A.dCMP mispairs. Examination of DNA crystal structures of these four mispairs indicates that the two mispairs excluded by the lysine mutant have an atom (N2) in an unfavorable position in the minor groove, while the two mispairs permitted by the lysine mutant do not. These results suggest that unfavorable interactions between an active site amino acid side chain and mispair-specific atoms in the minor groove contribute to DNA polymerase specificity.     

Bleomycin-dependent damage to the bases in DNA is a minor side reaction.

The antitumor antibiotic bleomycin degrades DNA in the presence of ferric ions and H2O2 or in the presence of ferric ions, oxygen, and ascorbic acid. When DNA degradation is measured as formation of base propenals by the thiobarbituric acid assay, it is not inhibited by superoxide dismutase and scavengers of the hydroxyl radical or by catalase (except that catalase inhibits in the bleomycin/ferric ion/H2O2 system by removing H2O2). Using the technique of gas chromatography/mass spectrometry with selected-ion monitoring, we show that DNA degradation is accompanied by formation of small amounts of modified DNA bases. The products formed are identical with those generated when hydroxyl radicals react with DNA bases. Base modification is significantly inhibited by catalase and partially inhibited by scavengers of the hydroxyl radical and by superoxide dismutase. We suggest that the bleomycin-oxo-iron ion complex that cleaves the DNA to form base propenals can decompose in a minor side reaction to generate hydroxyl radical, which accounts for the base modification in DNA. However, hydroxyl radical makes no detectable contribution to the base propenal formation.     

Studying DNA modifications and DNA-protein interactions in vivo. A window onto the native genome

The study of native genomes has been greatly facilitated by the use of direct genomic sequencing and footprinting strategies. This review provides an overview of the techniques involved and presents some highlights of the biological results obtained with these methods.