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

Rui Xiao1, David D. Moore1

1 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.21
DOI:  10.1002/0471142727.mb2121s94
Online Posting Date:  April, 2011
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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 N6‐adenine methylation in nearby sites in the genomic DNA. Methylated DNA fragments are enriched by immunopreciptation with an antibody that recognizes N6‐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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Basic Protocol 1: DamIP for Studying DNA‐Protein Interactions In Vivo

  • 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

  • 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

  • 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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Literature Cited

Literature Cited
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