Unraveling DNA Essay

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Unraveling the molecular mechanism of DNA binding by Transcription-activator like effectors

Sequence-specific DNA targeting of nucleases, recombinases and transcriptional activators is a powerful tool to manipulate the sequence or regulate the expression of the gene of interest. While Zinc fingers specific to DNA trinucleotides, coupled to different effector domains have been employed for targeted manipulation of the genome with considerable success, we are limited by the off-target toxicity caused by trinucleotide specific zinc fingers. Recently, it has been shown that Xanthomonas secreted virulence factors called transcription-activator like effectors (TALEs), which contain 1.5 to 33.5 tandem repeats bind sequence
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In the crystal structure presented by Deng et al., the central region of an engineered TALE dHax3 shows a super helical assembly with an external diameter of 60A and a pitch also of 60A with 11 repeat domains per helical turn. This structure strongly suggests that dHax3 wraps around the DNA helix. Each repeat of dHax3 consists of 34 amino acids, 3 to 11 of which form a short helix ‘a’ and 15 to 33 form helix ‘b’. These two helices are connected by the RVD loop that contains residue 12, usually a His or Asn, that stabilizes the local conformation of RVD loop and residue 13 that recognizes a DNA base. In dHax3, residue 14 is shown to be a conserved Glycine. Helices ‘a’ and ‘b’ stack together using Van der Waals interactions. Interestingly, the pitch of dHax3 in DNA-free form is 60A but goes down to 35A when bound to the DNA but maintains 11 repeat domains per helical turn. This compression of TALEs is attributed to the conformational plasticity of residues 23 to 34 of the repeat domains. The inner region of the repeat domains is electrostatically positively charged with residues like Lys 16 and Gln 17 on helix ‘b’, which can hydrogen bond with the negatively charged phosphate groups of the DNA. The hypervariable di-residues HD recognize base ‘C’, NS recognize base ‘A’ and NG recognize base ‘T’. In the case of NGà T recognition, glycine at position 13 helps accommodate the 5’ methyl
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