Unraveling DNA

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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 specifically to the sense strand of the DNA helix. The specificity of this binding is directed by the repeat variable di-residues (RVDs) present on a short loop of every repeat of the TALE. In the Science January 2013 issue, Deng et al. present the structural basis for sequence specific binding of TALEs to the sense strand of the DNA using a crystal structure. They solved the crystal structures of an engineered TALE named dHax3, crystallized both, in its free form and in complex with the DNA at 2.4A resolution. This structure elucidates the molecular mechanism of TALE binding to DNA and provides a foundation for the rational design of DNA-specific TALEs with different effector domains for site-specific manipulations of the genome. Bacterial TALEs modulate the target gene expression in host cells by acting as transcriptional activators. TALEs are made up of 33 to 35 tandem repeat domains. Each domain consists of 33 to 35 amino acids forming two alpha helices connected by a short loop and recognizes ... ... middle of paper ... ...ses, TALEs are shown to bind degenerate DNA sequences, albeit with much lower toxicity than zinc fingers (Carlson, Fahrenkrug et al. 2012). Thus along with a high potential of TALEs, the scientific community today is faced with a number of challenges which need to be answered before TALEs can be used as robust effectors of targeted gene manipulation. In this regard, the structural information about TALE interaction with the DNA which Deng et al. present in this paper is an important first step towards rational design of target DNA specific TALEs that can carry out a variety of effector functions. References: Carlson, D. F., et al. (2012). "Targeting DNA With Fingers and TALENs." Mol Ther Nucleic Acids 1: e3. Gaj, T., et al. (2013). "ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering." Trends Biotechnol 31(7): 397-405.
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