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

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 driftmore » within potential host target sites.« less

Authors:
 [1];  [1];  [1];  [2];  [2];  [2];  [3];  [3];  [3];  [3]; ORCiD logo [1]
  1. Basic Sciences Division, Fred Hutchinson Cancer Research Center,Seattle, WA (United States)
  2. Bluebird Bio Inc., Seattle, WA (United States)
  3. Imperial College of London, Department of Life Sciences, South Kensington Campus, London (United Kingdom)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS); Bill and Melinda Gates Foundation; Fred Hutchinson Cancer Center; Bluebird Bio, Inc.
OSTI Identifier:
1625567
Grant/Contract Number:  
AC02-05CH11231; R01 GM105691
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 45; Journal Issue: 14; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English
Subject:
Biochemistry & Molecular Biology

Citation Formats

Werther, Rachel, Hallinan, Jazmine P., Lambert, Abigail R., Havens, Kyle, Pogson, Mark, Jarjour, Jordan, Galizi, Roberto, Windbichler, Nikolai, Crisanti, Andrea, Nolan, Tony, and Stoddard, Barry L. Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity. United States: N. p., 2017. Web. doi:10.1093/nar/gkx544.
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. Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity. United States. https://doi.org/10.1093/nar/gkx544
Werther, Rachel, Hallinan, Jazmine P., Lambert, Abigail R., Havens, Kyle, Pogson, Mark, Jarjour, Jordan, Galizi, Roberto, Windbichler, Nikolai, Crisanti, Andrea, Nolan, Tony, and Stoddard, Barry L. Tue . "Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity". United States. https://doi.org/10.1093/nar/gkx544. https://www.osti.gov/servlets/purl/1625567.
@article{osti_1625567,
title = {Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity},
author = {Werther, Rachel and Hallinan, Jazmine P. and Lambert, Abigail R. and Havens, Kyle and Pogson, Mark and Jarjour, Jordan and Galizi, Roberto and Windbichler, Nikolai and Crisanti, Andrea and Nolan, Tony and Stoddard, Barry L.},
abstractNote = {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.},
doi = {10.1093/nar/gkx544},
url = {https://www.osti.gov/biblio/1625567}, journal = {Nucleic Acids Research},
issn = {0305-1048},
number = 14,
volume = 45,
place = {United States},
year = {2017},
month = {6}
}

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