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DNA footprinting

    DNA footprinting is a method of investigating the sequence specificity of DNA-binding proteins This technique can be used to study protein-DNA interactions within a cell
    The regulation of transcription has been studied extensively and yet there is still so much that is not known about upstream and downstream enhancers and silencers and their control over transcription at specific genes within the genome Elucidating which transcription factors bind to these regions of DNA will help unravel the complexities of transcriptional controlMore…

    Method

    The wet lab methodology is summarized below with appropriate selection of reagents discussed later in the text
    1. Label one strand of a homogeneous solution of double-stranded DNA molecules of region of interest ideally between 50 to 200 bps in length
    2. Cut the strand using a chemical or enzyme that will cleave at random (sequence-independent)
      • One sample in the absence of a DNA-binding protein of interest
      • The other in the presence of that DNA-binding protein A ligand that specifically binds a region within the DNA fragment will protect the DNA it is bound to from the cleavage agent
    3. Run both samples side by side on a polyacrylamide gel electrophoresis
      • The DNA absent of protein will be cut at random locations and thus when it is run on a gel will produce a ladder-like distribution
      • The DNA plus protein will result in ladder distribution with a break in it, where the DNA is protected from the cleavage agent
    4. Run a Maxam-Gilbert chemical DNA sequencing along side the samples on the polyacrylamide gel which allows the prediction of the exact location of ligand binding site

    Labeling

    • Radioactivity has been traditionally used to label DNA fragments for footprinting analysis as the method was originally developed from the Maxam-Gilbert chemical sequencing technique Radioactive labeling is very sensitive and is optimal for visualizing small amounts of DNA
    • Fluorescence is a desirable advancement due to the hazards of using radio-chemicals However it has been more difficult to optimize because it is not always sensitive enough to detect the low concentrations of the target DNA strands used in DNA footprinting experiments Electrophoretic sequencing gels or capillary electrophoresis have been successful in analyzing footprinting of fluorescently tagged fragments

    Cleavage agent

    A variety of cleavage agents can be chosen Ideally a desireable agent is one that is sequence neutral easy to use and is easy to control Unfortunately none available meet all these all of these standards so an appropriate agent can be chosen depending on your DNA sequence and ligand of interest The following cleavage agents are described in detail:
    • “DNase I:” a large protein that functions as a double-strand endonuclease It binds the minor groove of DNA and cleaves the phosphodiester backbone It is a good cleavage agent for footprinting because its size makes it easily physically hindered Thus is more likely to have its action blocked by a bound protein on a DNA sequence In addition the DNase I enzyme is easily controlled by adding EDTA to stop the reaction There are however some limitations in using DNase I. The enzyme does not cut DNA randomly; its activity is affected by local DNA structure and sequence and therefore results in an uneven ladder This can limit the precision of predicting a protein’s binding site on the DNA molecule
    • “Hydroxyl radicals:” are created from the Fenton reaction which involves reducing Fe2+ with H2O2 to form free hydroxyl molecules These hydroxyl molecules react with the DNA backbone resulting in a break Due to their small size the resulting DNA footprint has high resolution Unlike DNase I they have no sequence dependence and result in a much more evenly distributed ladder The negative aspect of using hydroxyl radicals is that they are more time consuming to use due to a slower reaction and digestion time
    • “Ultraviolet irradiation:” can be used to excite nucleic acids and create photoreactions which results in damaged bases in the DNA strand Photoreactions can include intra- or inter-strand reactions reactions with a solvent or protein crosslinking
    The workflow for this method has an additional step once both your protected and unprotected DNA has been treated there is subsequent primer extension of the cleaved products The extension will terminate upon reaching a damaged base and thus when the PCR products are run side-by-side on a gel; the protected sample will show an additional band where the DNA was crosslinked with a bound proteinAdvantages of using UV are that it reacts very quickly and can therefore capture interactions that are only momentary Additionally it can be applied to in vivo experiments because UV can penetrate cell membranes A disadvantage is that the gel can be difficult to interpret as the bound protein does not protect the DNA it merely alters the photoreactions in the vicinity

    Advanced Applications

    Quantitative footprintingIn vivo footprintingRefer to genome-wide applications

    History

    In 1978 David Galas and Albert Schmitz developed the DNA footprinting technique to study the binding specificity of the lac repressor protein [1] It was originally a modification of the Maxam-Gilbert chemical sequencing technique

    References

    Galas D and Schmitz A. (1978) DNAse footprinting: a simple method for the detection of protein-DNA binding specificity Nucleic Acids Research 5(9):3157-70Geiselmann J and Boccard F. (2001) Ultraviolet-laser footprinting Methods in Molecular Biology 148:161-73Hampshire A, Rusling D, Broughton-Head V, and Fox K. (2007) Footprinting: A method for determining the sequence selectivity affinity and kinetics of DNA-binding ligands Methods 42:128-140LeBlanc B and Moss T. (2001) DNase I Footprinting Methods in Molecular Biology 148: 31-8Zaychikov E, Schickor P, Denissova L, and Heumann H. (2001) Hydroxyl radical footprinting Methods in Molecular Biology 148: 49-61