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Nucleic Acids Research Mar 2015DNA bis-intercalators are widely used in molecular biology with applications ranging from DNA imaging to anticancer pharmacology. Two fundamental aspects of these...
DNA bis-intercalators are widely used in molecular biology with applications ranging from DNA imaging to anticancer pharmacology. Two fundamental aspects of these ligands are the lifetime of the bis-intercalated complexes and their sequence selectivity. Here, we perform single-molecule optical tweezers experiments with the peptide Thiocoraline showing, for the first time, that bis-intercalation is driven by a very slow off-rate that steeply decreases with applied force. This feature reveals the existence of a long-lived (minutes) mono-intercalated intermediate that contributes to the extremely long lifetime of the complex (hours). We further exploit this particularly slow kinetics to determine the thermodynamics of binding and persistence length of bis-intercalated DNA for a given fraction of bound ligand, a measurement inaccessible in previous studies of faster intercalating agents. We also develop a novel single-molecule footprinting technique based on DNA unzipping and determine the preferred binding sites of Thiocoraline with one base-pair resolution. This fast and radiolabelling-free footprinting technique provides direct access to the binding sites of small ligands to nucleic acids without the need of cleavage agents. Overall, our results provide new insights into the binding pathway of bis-intercalators and the reported selectivity might be of relevance for this and other anticancer drugs interfering with DNA replication and transcription in carcinogenic cell lines.
Topics: Algorithms; DNA; DNA Footprinting; Depsipeptides; Elasticity; Intercalating Agents; Kinetics; Ligands; Models, Molecular; Nucleic Acid Conformation; Optical Tweezers; Protein Binding; Thermodynamics; Time Factors
PubMed: 25690887
DOI: 10.1093/nar/gkv087 -
Methods in Molecular Biology (Clifton,... 2009The association of proteins with the DNA double helix can interfere with the accessibility of the latter to nucleases. This is particularly true when using bulky...
The association of proteins with the DNA double helix can interfere with the accessibility of the latter to nucleases. This is particularly true when using bulky nucleases such as DNAse I. The DNAse I footprinting method was developed to make use of this phenomenon in the study of DNA-protein interactions; it consists in comparing the pattern of fragments produced by the partial digestion of DNA in the absence of a protein to that produced by partial digestion of DNA in the presence of a protein. Normally, when the two sets of fragments are separated side by side on a gel, the fragments produced in the presence of the protein will feature blank regions (indicating protection) and/or enhanced cleavage sites (indicating increased availability). This technique can furthermore reveal if multiple sites for a DNA-binding protein are present on a same fragment, and allow the comparison of their respective affinities.
Topics: DNA Footprinting; Deoxyribonuclease I
PubMed: 19378157
DOI: 10.1007/978-1-60327-015-1_3 -
Methods in Molecular Biology (Clifton,... 2017The transition of bacteria from a planktonic lifestyle to a collaborative, sessile biofilm lifestyle is a regulated process orchestrated by the intracellular...
The transition of bacteria from a planktonic lifestyle to a collaborative, sessile biofilm lifestyle is a regulated process orchestrated by the intracellular second-messenger c-di-GMP (bis-(3'-5')-cyclic dimeric guanosine monophosphate). To modulate this transition, c-di-GMP acts at the transcriptional, posttranscriptional, and posttranslational levels. In this chapter, we describe a method to study of how a transcriptional regulator modulates gene expression in response to c-di-GMP binding. DNase I footprinting is a valuable tool for use in analyzing how regulatory proteins bind to DNA, the location of their binding sites or how c-di-GMP affects their binding to DNA. This chapter describes a protocol for nonradiochemical DNase I footprinting experiments using a capillary electrophoresis method based on the interaction of the Pseudomonas aeruginosa FleQ protein with the promoter regions of biofilm-related genes.
Topics: Binding Sites; Cyclic GMP; DNA Footprinting; DNA, Bacterial; DNA-Binding Proteins; Deoxyribonuclease I; Electrophoresis, Capillary; Fluorescent Dyes; Promoter Regions, Genetic; Staining and Labeling; Transcription Factors
PubMed: 28889304
DOI: 10.1007/978-1-4939-7240-1_24 -
Nucleic Acids Research Jul 2004Oligodeoxyribonucleotides (5'-phosphorylated) of varying lengths were capped using a polyamide linker to form thermodynamically stable, endcapped DNA duplexes containing...
Oligodeoxyribonucleotides (5'-phosphorylated) of varying lengths were capped using a polyamide linker to form thermodynamically stable, endcapped DNA duplexes containing 8-14 bp. We have employed these endcapped DNA duplexes as tools to determine the DNA footprint of T4 DNA ligase. By high-performance liquid chromatography and PAGE analysis of the ligation mixtures of the endcapped DNA duplexes, we have found that by varying the lengths and the position of the nick, we can determine the minimal DNA-binding site as well as the mode of binding (symmetrical or asymmetrical binding) by the enzyme. The results of the study revealed that a 11 bp endcapped duplex was the shortest duplex effectively ligated. Dependence of ligation efficiency on nick position demonstrates that T4 DNA ligase bound asymmetrically to its DNA substrate. The use of a set of thermodynamically stable endcapped deoxyribonucleoside duplexes as a tool to elucidate the DNA footprint provides an efficient strategy for footprinting, which avoids ambiguities associated with chemical and biochemical footprinting methods.
Topics: Base Sequence; Binding Sites; Chromatography, High Pressure Liquid; DNA; DNA Footprinting; DNA Ligases; DNA-Binding Proteins; Oligodeoxyribonucleotides; Polyethylene Glycols; Protein Binding
PubMed: 15263063
DOI: 10.1093/nar/gnh103 -
Methods in Molecular Biology (Clifton,... 2009The in cellulo analysis of DNA protein interactions and chromatin structure is very important to better understand the mechanisms involved in the regulation of gene...
The in cellulo analysis of DNA protein interactions and chromatin structure is very important to better understand the mechanisms involved in the regulation of gene expression. The nuclease-hypersensitive sites and sequences bound by transcription factors often correspond to genetic regulatory elements. Using the Ligation-mediated polymerase chain reaction (LMPCR) technology, it is possible to precisely analyze these DNA sequences to demonstrate the existence of DNA-protein interactions or unusual DNA structures directly in living cells. Indeed, the ideal chromatin substrate is, of course, found inside intact cells. LMPCR, a genomic-sequencing, technique that map DNA single-strand breaks at the sequence level of resolution, is the method of choice for in cellulo footprinting and DNA structure studies because it can be used to investigate any complex genomes, including human. The detailed conventional and automated LMPCR protocols are presented in this chapter.
Topics: Base Sequence; Cell Survival; Chromatin; DNA; DNA Breaks, Single-Stranded; DNA Footprinting; DNA Methylation; Deoxyribonuclease I; Molecular Sequence Data; Polymerase Chain Reaction; Protein Binding; Pyrimidine Dimers; Sequence Analysis, DNA; Sulfuric Acid Esters; Templates, Genetic; Ultraviolet Rays
PubMed: 19378174
DOI: 10.1007/978-1-60327-015-1_20 -
Nature Biotechnology Feb 2002Gene transcription is regulated by proteins that bind specific DNA sequences and control the initiation of RNA synthesis. A major challenge is to map all of the...
Gene transcription is regulated by proteins that bind specific DNA sequences and control the initiation of RNA synthesis. A major challenge is to map all of the regulatory sites in the genome and to identify the proteins that bind them. Because members of transcription factor families often exhibit similar sequence preferences, methods for determining intermolecular contacts in protein-DNA interfaces must be sensitive to even subtle structural differences. The most detailed structural views of protein-DNA interfaces have been obtained through X-ray crystallography and NMR spectroscopy, and these methods have revolutionized the understanding of the structural determinants of sequence-specific recognition. Neither crystallography nor NMR, however, is particularly well-suited to high-throughput applications such as pan-genomic elucidation of regulatory sequences; in addition, these methods yield no information on the energetic contribution of particular contacts. Here we report a straightforward, high-resolution biochemical method for mapping, at single-nucleotide resolution, DNA bases that are subject to sequence-specific contacts by regulatory proteins.
Topics: Base Sequence; Binding Sites; Crystallography, X-Ray; DNA; DNA Footprinting; Humans; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Models, Genetic; Molecular Sequence Data; Promoter Regions, Genetic; Protein Binding; Sequence Analysis, DNA; Time Factors
PubMed: 11821865
DOI: 10.1038/nbt0202-183 -
Cold Spring Harbor Protocols Jan 2012This article describes approaches for identifying proteins that bind conserved or functional DNA motifs. It discusses the use of consensus sequence databases to identify... (Review)
Review
This article describes approaches for identifying proteins that bind conserved or functional DNA motifs. It discusses the use of consensus sequence databases to identify candidate proteins capable of binding a DNA motif of interest and then explains the potential uses of sophisticated mass spectrometry technology. DNA-binding proteins are most commonly identified by electrophoretic mobility-shift assay (EMSA) or DNase I footprinting. Each of these methods is described, and their advantages and limitations are outlined. It is important to stress that each of the strategies discussed in this article may identify one or more proteins that bind a DNA element of interest. However, none of the strategies will necessarily lead to the protein that is the functionally relevant regulator of the control element in the context of the endogenous locus. Regardless of how a DNA-binding protein is isolated and identified, it merely becomes a candidate regulator of the gene of interest.
Topics: Consensus Sequence; DNA; DNA Footprinting; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Mass Spectrometry; Protein Binding
PubMed: 22194258
DOI: 10.1101/pdb.top067470 -
Methods in Molecular Biology (Clifton,... 2009Structural studies of DNA-protein complexes reveal networks of contacts between proteins and the phosphates, sugars and bases of DNA. A range of biochemical methods,...
Structural studies of DNA-protein complexes reveal networks of contacts between proteins and the phosphates, sugars and bases of DNA. A range of biochemical methods, termed chemical footprinting, aim to determine the functional groups on DNA which are protected in solution by bound protein against modification or where chemical pre-modification interferes with subsequent protein binding. One of these approaches, termed ethylation interference footprinting, reveals which backbone phosphate groups are contacted by protein and the positions where the DNA-protein interface is so tight that the modification cannot be accommodated. This chapter describes the steps necessary to perform an ethylation interference experiment, including modification of DNA using ethylnitrosourea, fractionation of the products based on their affinities for a DNA-binding protein and analysis of the "bound" and "free" fractions to reveal sites critical for complex formation. This is illustrated using results from our experiments with the Escherichia coli methionine repressor, MetJ.
Topics: Bacterial Proteins; Base Sequence; Chemical Fractionation; DNA Footprinting; DNA, Bacterial; Electrophoretic Mobility Shift Assay; Escherichia coli; Ethylnitrosourea; Isotope Labeling; Models, Molecular; Molecular Sequence Data; Radioisotopes; Repressor Proteins; Sequence Analysis, DNA
PubMed: 19378163
DOI: 10.1007/978-1-60327-015-1_9 -
Nucleic Acids Research May 2002A variety of methods are available to analyze protein-DNA interactions in vivo. Two of the most prominent of these methods are chromatin immunoprecipitation (ChIP) and...
A variety of methods are available to analyze protein-DNA interactions in vivo. Two of the most prominent of these methods are chromatin immunoprecipitation (ChIP) and in vivo footprinting. Both of these procedures have specific limitations. For example, the ChIP assay fails to document where exactly a protein binds in vivo. The precipitation of a specific segment of DNA with antibodies directed against DNA-binding proteins does not necessarily indicate that the protein directly interacts with a sequence in the precipitate but could rather reflect protein-protein interactions. Furthermore, the results of in vivo footprinting studies are inconclusive if a DNA sequence is analyzed that is bound by a specific protein in only a certain fraction of cells. Finally, in vivo footprinting does not indicate which protein is bound at a specific site. We have developed a new procedure that combines the ChIP assay and DMS footprinting techniques. Using this method we show here that antibodies specific for USF1 and NF-E2 precipitate the murine beta-globin promoter in MEL cells. DMS footprinting analysis of the DNA precipitated with NF-E2 antibodies revealed a protection over a partial NF-E2-binding site in the beta-globin downstream promoter region. We believe that this novel method will generally benefit investigators interested in analyzing protein-DNA interactions in vivo.
Topics: Animals; Base Sequence; Chromatin; DNA; DNA Footprinting; DNA-Binding Proteins; Erythroid-Specific DNA-Binding Factors; Globins; Humans; Molecular Sequence Data; NF-E2 Transcription Factor; NF-E2 Transcription Factor, p45 Subunit; Precipitin Tests; Promoter Regions, Genetic; Protein Binding; Transcription Factors; Tumor Cells, Cultured; Upstream Stimulatory Factors
PubMed: 12000849
DOI: 10.1093/nar/30.10.e44 -
Methods in Molecular Biology (Clifton,... 2001
Topics: Autoradiography; Base Sequence; DNA; DNA Footprinting; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Indicators and Reagents; Lasers; Oligodeoxyribonucleotides; Phosphorus Radioisotopes; Sequence Analysis, DNA; Ultraviolet Rays
PubMed: 11357584
DOI: 10.1385/1-59259-208-2:161