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Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity

Werther, Rachel; Hallinan, Jazmine P.; Lambert, Abigail R.; Havens, Kyle; Pogson, Mark; Jarjour, Jordan; Galizi, Roberto; Windbichler, Nikolai; Crisanti, Andrea; Nolan, Tony; Stoddard, Barry L.

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Authors

Rachel Werther

Jazmine P. Hallinan

Abigail R. Lambert

Kyle Havens

Mark Pogson

Jordan Jarjour

Nikolai Windbichler

Andrea Crisanti

Tony Nolan

Barry L. Stoddard



Abstract

The retargeting of protein-DNA specificity, outside of extremely modular DNA binding proteins such as TAL effectors, has generally proved to be quite challenging. Here, we describe structural analyses of five different extensively retargeted variants of a single homing endonuclease, that have been shown to function efficiently in ex vivo and in vivo applications. The redesigned proteins harbor mutations at up to 53 residues (18%) of their amino acid sequence, primarily distributed across the DNA binding surface, making them among the most significantly reengineered ligand-binding proteins to date. Specificity is derived from the combined contributions of DNA-contacting residues and of neighboring residues that influence local structural organization. Changes in specificity are facilitated by the ability of all those residues to readily exchange both form and function. The fidelity of recognition is not precisely correlated with the fraction or total number of residues in the protein-DNA interface that are actually involved in DNA contacts, including directional hydrogen bonds. The plasticity of the DNA-recognition surface of this protein, which allows substantial retargeting of recognition specificity without requiring significant alteration of the surrounding protein architecture, reflects the ability of the corresponding genetic elements to maintain mobility and persistence in the face of genetic drift within potential host target sites.

Journal Article Type Article
Acceptance Date Jun 12, 2017
Publication Date Jun 20, 2017
Publicly Available Date Mar 29, 2024
Journal Nucleic Acids Research
Print ISSN 1362-4962
Publisher Oxford University Press
Pages 8621 - 8634
DOI https://doi.org/10.1093/nar/gkx544
Keywords crystallographic, plasticity, meganuclease
Publisher URL https://academic.oup.com/nar/article/45/14/8621/3871306
PMID 28637173

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