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Nucleic Acids Research Sep 2017Nucleosomes are the most abundant protein-DNA complexes in eukaryotes that provide compaction of genomic DNA and are implicated in regulation of transcription, DNA...
Nucleosomes are the most abundant protein-DNA complexes in eukaryotes that provide compaction of genomic DNA and are implicated in regulation of transcription, DNA replication and repair. The details of DNA positioning on the nucleosome and the DNA conformation can provide key regulatory signals. Hydroxyl-radical footprinting (HRF) of protein-DNA complexes is a chemical technique that probes nucleosome organization in solution with a high precision unattainable by other methods. In this work we propose an integrative modeling method for constructing high-resolution atomistic models of nucleosomes based on HRF experiments. Our method precisely identifies DNA positioning on nucleosome by combining HRF data for both DNA strands with the pseudo-symmetry constraints. We performed high-resolution HRF for Saccharomyces cerevisiae centromeric nucleosome of unknown structure and characterized it using our integrative modeling approach. Our model provides the basis for further understanding the cooperative engagement and interplay between Cse4p protein and the A-tracts important for centromere function.
Topics: Algorithms; Centromere; Chromosomal Proteins, Non-Histone; DNA; DNA Cleavage; DNA Footprinting; DNA-Binding Proteins; Hydroxyl Radical; Models, Molecular; Nucleic Acid Conformation; Nucleosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 28934480
DOI: 10.1093/nar/gkx616 -
Protoplasma Oct 2012Phylogenetic footprinting is a method for the discovery of regulatory elements in a set of homologous regulatory regions, usually collected from multiple species. It... (Review)
Review
Phylogenetic footprinting is a method for the discovery of regulatory elements in a set of homologous regulatory regions, usually collected from multiple species. It does so by identifying the best conserved motifs in those homologous regions. There are two popular sets of methods-alignment-based and motif-based, which are generally employed for phylogenetic methods. However, serious efforts have lacked to develop a tool exclusively for phylogenetic footprinting, based on either of these methods. Nevertheless, a number of software and tools exist that can be applied for prediction of phylogenetic footprinting with variable degree of success. The output from these tools may get affected by a number of factors associated with current state of knowledge, techniques and other resources available. We here present a critical apprehension of various phylogenetic approaches with reference to prokaryotes outlining the available resources and also discussing various factors affecting footprinting in order to make a clear idea about the proper use of this approach on prokaryotes.
Topics: Bacteria; Binding Sites; DNA Footprinting; Genome, Bacterial; Genomics; Phylogeny
PubMed: 22113593
DOI: 10.1007/s00709-011-0351-9 -
Methods in Molecular Biology (Clifton,... 2011In the following chapter we describe methods and protocols to analyze the interaction of proteins with DNA using footprinting and related techniques based on the...
In the following chapter we describe methods and protocols to analyze the interaction of proteins with DNA using footprinting and related techniques based on the modification of DNA with either hydroxyl radicals or methylating agents. Footprinting, based on the protection from chemical modification of DNA through the specific binding of a protein, gives information about the nucleotides that are in close contact with the protein upon binding. The derived missing nucleoside and interference techniques identify nucleotides that are energetically important for protein binding. These methods are highly valuable to study in detail the interaction of a transcription factor with nucleotides on both strands of its target DNA sequence.
Topics: Base Sequence; Binding Sites; DNA Footprinting; DNA, Plant; DNA-Binding Proteins; Hydroxyl Radical; Methylation; Models, Molecular; Sequence Analysis, DNA; Staining and Labeling; Transcription Factors
PubMed: 21720958
DOI: 10.1007/978-1-61779-154-3_15 -
Methods in Molecular Biology (Clifton,... 2009DNA footprinting is a widely used method to locate the binding sites of protein on the DNA. It is based on the observation that a protein bound to DNA protects it from...
DNA footprinting is a widely used method to locate the binding sites of protein on the DNA. It is based on the observation that a protein bound to DNA protects it from degradation by an enzyme or chemical reagent.Exonuclease III is a suitable probe to analyze the boundaries of a protein when it is necessary to eliminate any excess unbound DNA from the reaction to avoid background problems. In combination with biotin-labeled DNA that is bound to streptavidin-coated magnetic particles, information on the precise position of a DNA bound protein is available within a few hours. The position of the archaeal RNA polymerase at different stages of transcription in the Pyrococcus furiosus in vitro transcription system was analyzed by this method.
Topics: DNA; DNA Footprinting; Exodeoxyribonucleases; Isotope Labeling; Pyrococcus furiosus; Sequence Analysis, DNA; Templates, Genetic; Transcription, Genetic
PubMed: 19378158
DOI: 10.1007/978-1-60327-015-1_4 -
Methods in Molecular Biology (Clifton,... 2009This unit details the use of hydroxyl radicals to characterize protein-DNA interactions. This method may be used to assess the exact location of contacts between a...
This unit details the use of hydroxyl radicals to characterize protein-DNA interactions. This method may be used to assess the exact location of contacts between a protein and its cognate DNA and details of the complex structure. We describe several methods to prepare DNA templates for footprinting and ways to avoid many of the pitfalls associated with the use of hydroxyl radical footprinting. In addition, we describe in detail one example of the application of this technique.
Topics: Base Sequence; DNA; DNA Footprinting; Humans; Hydroxyl Radical; Isotope Labeling; Molecular Sequence Data; MutS Homolog 2 Protein
PubMed: 19378159
DOI: 10.1007/978-1-60327-015-1_5 -
Nucleic Acids Research 2004We have cloned and expressed the ahdIC gene of the AhdI restriction-modification system and have purified the resulting controller (C) protein to homogeneity. The...
We have cloned and expressed the ahdIC gene of the AhdI restriction-modification system and have purified the resulting controller (C) protein to homogeneity. The protein sequence shows a HTH motif typical of that found in many transcriptional regulators. C.AhdI is found to form a homodimer of 16.7 kDa; sedimentation equilibrium experiments show that the dimer dissociates into monomers at low concentration, with a dissociation constant of 2.5 microM. DNase I and Exo III footprinting were used to determine the C.AhdI DNA-binding site, which is found approximately 30 bp upstream of the ahdIC operon. The intact homodimer binds cooperatively to a 35 bp fragment of DNA containing the C-protein binding site with a dissociation constant of 5-6 nM, as judged both by gel retardation analysis and by surface plasmon resonance, although in practice the affinity for DNA is dominated by protein dimerization as DNA binding by the monomer is negligible. The location of the C-operator upstream of both ahdIC and ahdIR suggests that C.AhdI may act as a positive regulator of the expression of both genes, and could act as a molecular switch that is critically dependent on the K(d) for the monomer-dimer equilibrium. Moreover, the structure and location of the C.AhdI binding site with respect to the putative -35 box preceding the C-gene suggests a possible mechanism for autoregulation of C.AhdI expression.
Topics: Amino Acid Sequence; Base Sequence; Binding Sites; DNA; DNA Footprinting; DNA Restriction-Modification Enzymes; DNA-Binding Proteins; Dimerization; Gene Expression Regulation; Gene Order; Molecular Sequence Data; Operon; Surface Plasmon Resonance; Transcription Factors
PubMed: 15590905
DOI: 10.1093/nar/gkh975 -
Methods in Molecular Biology (Clifton,... 2021Chromatin accessibility is directly linked with transcription in eukaryotes. Accessible regions associated with regulatory proteins are highly sensitive to DNase I...
Chromatin accessibility is directly linked with transcription in eukaryotes. Accessible regions associated with regulatory proteins are highly sensitive to DNase I digestion and are termed DNase I hypersensitive sites (DHSs). DHSs can be identified by DNase I digestion, followed by high-throughput DNA sequencing (DNase-seq). The single-base-pair resolution digestion patterns from DNase-seq allows identifying transcription factor (TF) footprints of local DNA protection that predict TF-DNA binding. The identification of differential footprinting between two conditions allows mapping relevant TF regulatory interactions. Here, we provide step-by-step instructions to build gene regulatory networks from DNase-seq data. Our pipeline includes steps for DHSs calling, identification of differential TF footprints between treatment and control conditions, and construction of gene regulatory networks. Even though the data we used in this example was obtained from Arabidopsis thaliana, the workflow developed in this guide can be adapted to work with DNase-seq data from any organism with a sequenced genome.
Topics: Arabidopsis; Chromatin; Chromosome Mapping; DNA Footprinting; Deoxyribonuclease I; Gene Expression Regulation; Gene Regulatory Networks; Genomics; High-Throughput Nucleotide Sequencing; Protein Binding; Software; Transcription Factors
PubMed: 34251618
DOI: 10.1007/978-1-0716-1534-8_3 -
Biochimica Et Biophysica Acta Oct 2013G-quadruplexes are polymorphic non-canonical nucleic acid conformations involved both in physiological and pathological processes. Given the high degree of folding...
BACKGROUND
G-quadruplexes are polymorphic non-canonical nucleic acid conformations involved both in physiological and pathological processes. Given the high degree of folding heterogeneity and comparable conformational stabilities, different G-quadruplex forms can occur simultaneously, hence rendering the use of basic instrumental methods for structure determination, like X-ray diffraction or NMR, hardly useful. Footprinting techniques represent valuable and relatively rapid alternative to characterize DNA folding. The natural diterpenoid clerocidin is an alkylating agent that specifically reacts at single-stranded DNA regions, with different mechanisms depending on the exposed nucleotide.
METHODS
Clerocidin was used to footprint G-quadruplex structures formed by telomeric and oncogene promoter sequences (c-myc, bcl-2, c-kit2), and by the thrombin binding aptamer.
RESULTS
The easy modulability of CL reactivity towards DNA bases permitted to discriminate fully and partially protected sites, highlights stretched portions of the G-quadruplex conformation, and discriminate among topologies adopted by one sequence in different environmental conditions. Importantly, CL displayed the unique property to allow detection of G-quadruplex folding within a duplex context.
CONCLUSIONS
CL is a finely performing new tool to unveil G-quadruplex arrangements in DNA sequences under genomically relevant conditions.
GENERAL SIGNIFICANCE
Nucleic acid G-quadruplex structures are an emerging research field because of the recent indication of their involvement in a series of key biological functions, in particular in regulation of proliferation-associated gene expression. The use of clerocidin as footprinting agent to identify G-quadruplex structures under genomically relevant conditions may allow detection of new G-quadruplex-based regulatory regions.
Topics: Base Sequence; Circular Dichroism; DNA Footprinting; DNA Primers; Diterpenes; G-Quadruplexes; Nucleic Acid Conformation
PubMed: 23747297
DOI: 10.1016/j.bbagen.2013.05.039 -
Methods in Molecular Biology (Clifton,... 2010Polypurine/polypyrimidine (pPu/pPy) tracts, which exist in the promoter regions of many growth-related genes, have been proposed to be very dynamic in their...
Polypurine/polypyrimidine (pPu/pPy) tracts, which exist in the promoter regions of many growth-related genes, have been proposed to be very dynamic in their conformation. In this chapter, we describe a detailed protocol for DNase I and S1 nuclease footprinting experiments with supercoiled plasmid DNA containing the promoter regions to probe whether there are conformational transitions to B-type DNA, melted DNA, and G-quadruplex structures within this tract. This is demonstrated with the proximal promoter region of the human vascular endothelial growth factor (VEGF) gene, which also contains multiple binding sites for Sp1 and Egr-1 transcription factors.
Topics: Base Sequence; DNA Footprinting; DNA Primers; DNA, Superhelical; Deoxyribonucleases; Electrophoresis; Humans; Nucleic Acid Denaturation; Plasmids; Polymerase Chain Reaction; Promoter Regions, Genetic; Vascular Endothelial Growth Factor A
PubMed: 19997887
DOI: 10.1007/978-1-60327-418-0_14 -
Nucleic Acids Research 2007The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By...
The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the time, risk of sample loss and complexity of footprinting, whilst dramatically increasing the yield of data (30-fold). A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive 'one compound-one line' scheme. Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA. The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents.
Topics: Antigens, Neoplasm; Benzodiazepines; DNA Footprinting; DNA Topoisomerases, Type II; DNA-Binding Proteins; Deoxyribonuclease I; Drug Evaluation, Preclinical; Humans; Infrared Rays; Promoter Regions, Genetic; Pyrroles
PubMed: 17586817
DOI: 10.1093/nar/gkm467