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        DamIP: Using Mutant DNA Adenine Methyltransferase to Study DNA‐Protein Interactions In Vivo

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        • Abstract
        • Table of Contents
        • Materials
        • Figures
        • Literature Cited

        Abstract

         

        DamIP is a new method for studying DNA?protein interaction in vivo. A mutant form of DNA adenine methyltransferase (DamK9A) from E. coli is fused to the protein of interest and expressed. The fusion protein will bind to target binding sites and introduce N 6 ?adenine methylation in nearby sites in the genomic DNA. Methylated DNA fragments are enriched by immunopreciptation with an antibody that recognizes N 6 ?methyladenine, and can then be used for further analysis, e.g., real?time PCR, microarray, or high?throughput sequencing. This method is simple and does not require protein?DNA crosslinking or a specific antibody to the protein of interest. This unit describes the application of this method for identification of DNA binding sites in vivo. Curr. Protoc. Mol. Biol. 94:21.21.1?21.21.10. © 2011 by John Wiley & Sons, Inc.

        Keywords: DNA adenine methyltransferase; transcription factor binding sites; DamIP; immunoprecipitation; chromatin immunoprecipitation

             
         
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        Table of Contents

        • Introduction
        • Strategic Planning
        • Basic Protocol 1: DamIP for Studying DNA‐Protein Interactions In Vivo
        • Support Protocol 1: Preparation of Sonicated Methylated Genomic DNA for DamIP
        • Support Protocol 2: Antibody Pretreatment for DamIP
        • Reagents and Solutions
        • Commentary
        • Literature Cited
        • Figures
             
         
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        Materials

        Basic Protocol 1: DamIP for Studying DNA‐Protein Interactions In Vivo

          Materials
        • Sonicated methylated genomic DNA (see protocol 2 )
        • TE buffer ( appendix 22 )
        • 10× DamIP buffer (see recipe )
        • Pretreated antibodies for m6A and control (see protocol 3 )
        • A/G Plus agarose beads (Santa Cruz Biotechnologies, sc‐2003)
        • Digestion buffer (see recipe )
        • Proteinase K, DNase‐free
        • QIAquick PCR purification kit (Qiagen; optional)
        • TE buffer ( appendix 22 )
        • 1.7‐ml microcentrifuge tubes
        • Boiling water bath
        • Benchtop centrifuge
        • Additional reagents and equipment for DNA purification (unit 2.1 ; optional)

        Support Protocol 1: Preparation of Sonicated Methylated Genomic DNA for DamIP

          Materials
        • Cells expressing DamK9A fusion protein or control DamK9A only
        • Phosphate‐buffered saline (PBS; appendix 22 )
        • Lysis buffer (see recipe )
        • RNase A, DNase‐free
        • Proteinase K, DNase‐free
        • TE buffer ( appendix 22 )
        • Water baths, 37° and 55°C
        • Microtip sonicator (e.g., Branson Sonifier 250)
        • Additional reagents and equipment for harvesting and counting cells ( appendix 3F ), DNA purification (unit 2.1 ), DNA quantitation ( appendix 3D ), and agarose gel electrophoresis (unit 2.5 )

        Support Protocol 2: Antibody Pretreatment for DamIP

          Materials
        • Eukaryotic genomic DNA: salmon sperm DNA or DNA purified from cells
        • Buffer A: 10 mM potassium phosphate buffer, pH 8.0 ( appendix 22 )
        • CNBr‐activated agarose (Sigma, C9210)
        • 1 mM HCl, 4°C
        • 0.2 M glycine, adjusted to pH 8.0 with NaOH
        • Buffer B: buffer A containing 1 M KCl
        • TE buffer ( appendix 22 ), with and without 0.05% (w/v) NaN 3
        • 0.2 M NaOH
        • 1× DamIP buffer (see recipe )
        • Affinity‐purified antibody against m6A (Megabase Research or Synaptic Systems)
        • 15‐ml Falcon tube
        • Boiling water bath
        • Benchtop centrifuge, refrigerated
        • Additional reagents and equipment for DNA purification (unit 2.1 ) and quantitation ( appendix 3D )
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        Figures

        •   Figure 21.21.1 Illustration of the DamIP procedure. DamK9A or DamK9A fusion protein is expressed in cells. Genomic DNA is purified, sonicated, and denatured before being mixed with anti‐m6A antibodies. Methylated DNA recognized by the antibody is enriched and then analyzed by qPCR, microarray analysis, or next‐generation sequencing. (Figure adapted from Xiao et al., .)
          View Image
        •   Figure 21.21.2 Denaturation is important for recognition of m6A by antibodies. Unmethylated or methylated plasmids were mixed with affinity‐purified m6A antibody (AP‐m6A) and enriched fragments were quantified by real‐time PCR. Denaturation of DNA by heating greatly increased the affinity of m6A antibody for methylated DNA.
          View Image
        •   Figure 21.21.3 Representative DamIP result. Primers were designed to amplify genomic regions with (FSIP) or without (SCA7) known hER binding. DNA enriched by DamIP from MCF‐7 cells transfected with DamK9A‐hER (Dam‐ER) or Dam alone was analyzed by qPCR. Data are presented as mean ± SEM.
          View Image

        Videos

        Literature Cited

        Literature Cited
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