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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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) NaN₃
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., .)

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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.

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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.

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Literature Cited

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