Abstract
Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity—offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.Electronic supplementary materialThe online version of this article (doi:10.1007/s10822-014-9765-8) contains supplementary material, which is available to authorized users.
Highlights
Zinc finger nucleases (ZFN) hydrolyze DNA at a specific sequence
Three or four zinc finger (ZF) units are responsible for the site-specific DNA-binding, and in most ZFN-s a FokI nuclease domain linked to the ZF-s cleaves DNA [1, 2]
NColE7 was divided into two parts using its special features: the HNHmotif at the C-terminus of the protein is only functional in the presence of the positively charged N-terminal sequence
Summary
Zinc finger nucleases (ZFN) hydrolyze DNA at a specific sequence. Three or four zinc finger (ZF) units are responsible for the site-specific DNA-binding, and in most ZFN-s a FokI nuclease domain linked to the ZF-s cleaves DNA [1, 2]. The pair of two ZFN-s recognize 18–24 base pairs This allows for specific targeting of sequences in plant or mammalian genomes, including the human genome [3]. The double-strand break in DNA induces homology-directed repair in the presence of a suitable template [4] This could offer a promising opportunity to cure monogenetic diseases [5]. Even though the FokI nuclease is a nonspecific nuclease, in the natural enzyme it is under negative allosteric control of the specific DNA-binding domain [6, 7]. The emerging new nucleases for genome editing are transcription activator-like effector nucleases (TALENs) and RNA-guided engineered
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