DamIP: Using Mutant DNA Adenine Methyltransferase to Study DNA‐Protein Interactions In Vivo
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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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PDF or HTML at Wiley Online LibraryTable 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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PDF or HTML at Wiley Online LibraryMaterials
Basic Protocol 1: DamIP for Studying DNA‐Protein Interactions In Vivo
Materials
Support Protocol 1: Preparation of Sonicated Methylated Genomic DNA for DamIP
Materials
Support Protocol 2: Antibody Pretreatment for DamIP
Materials
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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
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Literature Cited
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