DamID

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Principle of the DamID. Dam (green) is bound to the DNA-binding protein (orange), which methylates neighboring GATC sequences.

DamID ( English DNA adenine methyltransferase identification ) is a biochemical method for determining protein-DNA interactions in eukaryotes .

principle

The DNA sequences to which proteins bind are detected by a fusion protein of the DNA-binding protein and a DNA methyltransferase (Dam). After binding of the DNA-binding protein is carried out a methylation of adenosine residues in adjacent GATC sequences by the attached DNA methyltransferase to N6-Methyladeninresten which do not occur in eukaryotes. The DNA sequences of the binding are determined by identifying the methylated adenosines. The partially methylated DNA is treated with the restriction endonuclease Dpn I , which cuts DNA at methylated GATC sequences. The ends of the DNA fragments are ligated with oligonucleotides . This labeled DNA is replicated in a polymerase chain reaction with primers , the sequences of which are complementary to the attached oligonucleotides. This means that only sequences that border on DNA methylation are copied.

Alternative methods are e.g. B. EMSA , ChIP , ChIP-on-Chip or ChIP-Seq . Compared to the ChIP methods, no antibody is required, but the involvement of post-translational modifications in the DNA binding by the protein cannot be checked. Since only DNA sequences with which the Dam was detected are detected and no protein-DNA interaction is detected directly, incorrect results arise with proteins containing DNA clips, such as RNA polymerase , which do not have a fixed position on the DNA but slide along the DNA. Due to the PCR, only DNA sequences between two GATC sequences are amplified. The mean distance between two consecutive GATC sequences is about 205 bases in Drosophila (FlyBase release 5), 260 in mice (Mm9) and 264 in humans (HG19). Transfected plasmids should be produced in Dam-negative E. coli strains, since bacterial Dam-methylated sequences lead to false positive results.

Web links

Individual evidence

  1. ↑ van Steensel B, Henikoff S: Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase . In: Nat. Biotechnol. . 18, No. 4, April 2000, pp. 424-8. doi : 10.1038 / 74487 . PMID 10748524 .
  2. ↑ Germann S, Juul-Jensen T, Letarnec B, Gaudin V: DamID, a new tool for studying plant chromatin profiling in vivo, and its use to identify putative LHP1 target loci . In: Plant J . 48, No. 1, October 2006, pp. 153-63. doi : 10.1111 / j.1365-313X.2006.02859.x . PMID 16972870 .
  3. ↑ Vogel MJ, Peric-Hupkes D, van Steensel B: Detection of in vivo protein-DNA interactions using DamID in mammalian cells . In: Nat Protoc . 2, No. 6, 2007, pp. 1467-78. doi : 10.1038 / nprot.2007.148 . PMID 17545983 .
  4. ↑ Greil F, Moorman C, van Steensel B: DamID: mapping of in vivo protein-genome interactions using tethered DNA adenine methyltransferase . In: Meth. Enzymol. . 410, 2006, pp. 342-59. doi : 10.1016 / S0076-6879 (06) 10016-6 . PMID 16938559 .
  5. ^ Brooks JE, Roberts RJ: Modification profiles of bacterial genomes . In: Nucleic Acids Res . 10, No. 3, 1982, pp. 913-34. doi : 10.1093 / nar / 10.3.913 . PMID 6278441 . PMC 326211 (free full text).