DamID

DamID (DNA adenine methyltransferase identification) is a molecular biology protocol used to map the binding sites of DNA- and chromatin-binding proteins in eukaryotes. DamID identifies binding sites by expressing the proposed DNA-binding protein as a fusion protein with DNA methyltransferase. Binding of the protein of interest to DNA localizes the methyltransferase in the region of the binding site. Adenosine methylation does not occur naturally in eukaryotes and therefore adenine methylation in any region can be concluded to have been caused by the fusion protein, implying the region is located near a binding site. DamID is an alternate method to ChIP-on-chip.

N6-methyladenine (m6A) is the product of the addition of a methyl group (CH₃) at position 6 of the adenine. This modified nucleotide is absent from the vast majority of eukaryotes, but is widespread in bacterial genomes, as part of the restriction modification or DNA repair systems. In Escherichia coli, adenine methylation is catalyzed by the adenine methyltransferase Dam (DNA adenine methyltransferase), which catalyses adenine methylation exclusively in the palindromic sequence GATC. Ectopic expression of Dam in eukaryotic cells leads to methylation of adenine in GATC sequences without any other noticeable side effect.

Based on this, DamID consists in fusing Dam to a protein of interest (usually a protein that interacts with DNA such as transcription factors) or a chromatin component. The protein of interest thus targets Dam to its cognate in vivo binding site, resulting in the methylation of neighboring GATCs. The presence of m6A, coinciding with the binding sites of the proteins of interest, is revealed by methyl PCR.

In this assay the genome is digested by DpnI, which cuts only methylated GATCs. Double-stranded adapters with a known sequence are then ligated to the ends generated by DpnI. A PCR with primers matching the adaptors is then carried out, leading to the specific amplification of genomic fragments flanked by methylated GATCs. In practice, ligation products are digested by DpnII prior PCR amplification. This enzyme cuts non-methylated GATCs, ensuring that only fragments flanked by consecutive methylated GATCs are amplified.

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