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Methods in Molecular Biology (Clifton,... 1997
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Nature Protocols 2008Hydroxyl radical footprinting has been widely used for studying the structure of DNA and DNA-protein complexes. The high reactivity and lack of base specificity of the...
Hydroxyl radical footprinting has been widely used for studying the structure of DNA and DNA-protein complexes. The high reactivity and lack of base specificity of the hydroxyl radical makes it an excellent probe for high-resolution footprinting of DNA-protein complexes; this technique can provide structural detail that is not achievable using DNase I footprinting. Hydroxyl radical footprinting experiments can be carried out using readily available and inexpensive reagents and lab equipment. This method involves using the hydroxyl radical to cleave a nucleic acid molecule that is bound to a protein, followed by separating the cleavage products on a denaturing electrophoresis gel to identify the protein-binding sites on the nucleic acid molecule. We describe a protocol for hydroxyl radical footprinting of DNA-protein complexes, along with a troubleshooting guide, that allows researchers to obtain efficient cleavage of DNA in the presence and absence of proteins. This protocol can be completed in 2 d.
Topics: Binding Sites; DNA; DNA Footprinting; Hydroxyl Radical; Macromolecular Substances; Protein Binding; Protein Footprinting; Proteins
PubMed: 18546600
DOI: 10.1038/nprot.2008.72 -
Carcinogenesis Aug 2006Many genotoxic carcinogens are known to leave unique signatures on cancer-related genes. The signature of carcinogens is manifested by the induction of characteristic... (Review)
Review
Many genotoxic carcinogens are known to leave unique signatures on cancer-related genes. The signature of carcinogens is manifested by the induction of characteristic mutations at distinctive nucleotide positions along oncogenes and/or tumor suppressor genes. Often, the nucleotide positions, wherein mutations occur, co-localize with the sites of initial DNA damage induced by the respective carcinogens. Thus, DNA damage-targeted mutation can be a predictor of carcinogenicity of genotoxins. Today, genomic sequencing technologies for investigating human cancer etiology are based on DNA-lesion footprinting in conjunction with mutagenicity analysis of genotoxic carcinogens. In this review article, we discuss the ligation-mediated PCR and terminal transferase-dependent-PCR, two versatile DNA-lesion footprinting techniques. We highlight the in vitro shuttle vector-based mutation systems for investigating site-specific mutagenicity of carcinogens and the in vivo transgenic rodent mutation systems for exploring DNA damaging and mutagenic properties of carcinogens. We present examples of application of each of these methodologies to human cancer etiology, and provide prospective views on investigations using these technologies for carcinogenicity testing.
Topics: DNA Damage; DNA Footprinting; DNA, Neoplasm; Humans; Mutagenicity Tests; Neoplasms
PubMed: 16344267
DOI: 10.1093/carcin/bgi311 -
Methods in Molecular Biology (Clifton,... 2015The in cellulo analysis of protein-DNA interactions and chromatin structure is very important to better understand the mechanisms involved in the regulation of gene...
The in cellulo analysis of protein-DNA 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 complex animal genomes, including human. The detailed conventional and automated LMPCR protocols are presented in this chapter.
Topics: Chromatin; DNA; DNA Breaks, Single-Stranded; DNA Footprinting; DNA-Binding Proteins; Gene Expression; High-Throughput Nucleotide Sequencing; Humans; Polymerase Chain Reaction; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid
PubMed: 26404143
DOI: 10.1007/978-1-4939-2877-4_4 -
Methods in Molecular Biology (Clifton,... 1999
Review
Topics: Animals; Base Sequence; Cross-Linking Reagents; DNA; DNA Footprinting; Lasers; Molecular Sequence Data; Proteins; Ultraviolet Rays
PubMed: 10804534
DOI: 10.1385/1-59259-681-9:481 -
Nature Protocols Nov 2018Hydroxyl-radical footprinting (HRF) is a powerful method for probing structures of nucleic acid-protein complexes with single-nucleotide resolution in solution. To tap...
Hydroxyl-radical footprinting (HRF) is a powerful method for probing structures of nucleic acid-protein complexes with single-nucleotide resolution in solution. To tap the full quantitative potential of HRF, we describe a protocol, hydroxyl-radical footprinting interpretation for DNA (HYDROID), to quantify HRF data and integrate them with atomistic structural models. The stages of the HYDROID protocol are extraction of the lane profiles from gel images, quantification of the DNA cleavage frequency at each nucleotide and theoretical estimation of the DNA cleavage frequency from atomistic structural models, followed by comparison of experimental and theoretical results. Example scripts for each step of HRF data analysis and interpretation are provided for several nucleosome systems; they can be easily adapted to analyze user data. As input, HYDROID requires polyacrylamide gel electrophoresis (PAGE) images of HRF products and optionally can use a molecular model of the DNA-protein complex. The HYDROID protocol can be used to quantify HRF over DNA regions of up to 100 nucleotides per gel image. In addition, it can be applied to the analysis of RNA-protein complexes and free RNA or DNA molecules in solution. Compared with other methods reported to date, HYDROID is unique in its ability to simultaneously integrate HRF data with the analysis of atomistic structural models. HYDROID is freely available. The complete protocol takes ~3 h. Users should be familiar with the command-line interface, the Python scripting language and Protein Data Bank (PDB) file formats. A graphical user interface (GUI) with basic functionality (HYDROID_GUI) is also available.
Topics: DNA; DNA Cleavage; DNA Footprinting; Electrophoresis, Polyacrylamide Gel; Humans; Hydroxyl Radical; Models, Molecular; Nucleosomes; Protein Footprinting; Proteins; Software; Solutions
PubMed: 30341436
DOI: 10.1038/s41596-018-0048-z -
Genome Research Jul 2020Transcription is tightly regulated by -regulatory DNA elements where transcription factors (TFs) can bind. Thus, identification of TF binding sites (TFBSs) is key to...
Transcription is tightly regulated by -regulatory DNA elements where transcription factors (TFs) can bind. Thus, identification of TF binding sites (TFBSs) is key to understanding gene expression and whole regulatory networks within a cell. The standard approaches used for TFBS prediction, such as position weight matrices (PWMs) and chromatin immunoprecipitation followed by sequencing (ChIP-seq), are widely used but have their drawbacks, including high false-positive rates and limited antibody availability, respectively. Several computational footprinting algorithms have been developed to detect TFBSs by investigating chromatin accessibility patterns; however, these also have limitations. We have developed a footprinting method to predict TF footprints in active chromatin elements (TRACE) to improve the prediction of TFBS footprints. TRACE incorporates DNase-seq data and PWMs within a multivariate hidden Markov model (HMM) to detect footprint-like regions with matching motifs. TRACE is an unsupervised method that accurately annotates binding sites for specific TFs automatically with no requirement for pregenerated candidate binding sites or ChIP-seq training data. Compared with published footprinting algorithms, TRACE has the best overall performance with the distinct advantage of targeting multiple motifs in a single model.
Topics: Binding Sites; Cell Line; Chromatin; DNA Footprinting; Deoxyribonucleases; Humans; K562 Cells; Markov Chains; Nucleotide Motifs; Sequence Analysis, DNA; Transcription Factors
PubMed: 32660981
DOI: 10.1101/gr.258228.119 -
Methods in Molecular Biology (Clifton,... 1999
Review
Topics: Animals; DNA Footprinting; DNA-Binding Proteins; Xenopus laevis
PubMed: 10503236
DOI: 10.1385/1-59259-678-9:199 -
Methods in Molecular Biology (Clifton,... 2009Defining the precise promoter DNA sequence motifs where nuclear receptors and other transcription factors bind is an essential prerequisite for understanding how these...
Defining the precise promoter DNA sequence motifs where nuclear receptors and other transcription factors bind is an essential prerequisite for understanding how these proteins modulate the expression of their specific target genes. The purpose of this chapter is to provide the reader with a detailed guide with respect to the materials and the key methods required to perform this type of DNA-binding analysis. Irrespective of whether starting with purified DNA-binding proteins or somewhat crude cellular extracts, the tried-and-true procedures described here will enable one to accurately access the capacity of specific proteins to bind to DNA as well as to determine the exact sequences and DNA contact nucleotides involved. For illustrative purposes, we primarily have used the interaction of the androgen receptor with the rat probasin proximal promoter as our model system.
Topics: Androgen-Binding Protein; Animals; Base Sequence; DNA Footprinting; Electrophoretic Mobility Shift Assay; Methylation; Molecular Sequence Data; Promoter Regions, Genetic; Protein Binding; Rats; Receptors, Androgen
PubMed: 19117141
DOI: 10.1007/978-1-60327-575-0_6 -
Cell Systems Mar 2017DNA in cells is predominantly B-form double helix. Though certain DNA sequences in vitro may fold into other structures, such as triplex, left-handed Z form, or...
DNA in cells is predominantly B-form double helix. Though certain DNA sequences in vitro may fold into other structures, such as triplex, left-handed Z form, or quadruplex DNA, the stability and prevalence of these structures in vivo are not known. Here, using computational analysis of sequence motifs, RNA polymerase II binding data, and genome-wide potassium permanganate-dependent nuclease footprinting data, we map thousands of putative non-B DNA sites at high resolution in mouse B cells. Computational analysis associates these non-B DNAs with particular structures and indicates that they form at locations compatible with an involvement in gene regulation. Further analyses support the notion that non-B DNA structure formation influences the occupancy and positioning of nucleosomes in chromatin. These results suggest that non-B DNAs contribute to the control of a variety of critical cellular and organismal processes.
Topics: Animals; Chromatin; Computational Biology; DNA; DNA Footprinting; DNA, Single-Stranded; Fungal Proteins; G-Quadruplexes; Gene Expression Regulation; Genome; Mammals; Manganese Compounds; Mice; Nucleic Acid Conformation; Nucleosomes; Oxides; Protein Binding; Single-Strand Specific DNA and RNA Endonucleases
PubMed: 28237796
DOI: 10.1016/j.cels.2017.01.013