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Title: Modifying a covarying protein–DNA interaction changes substrate preference of a site-specific endonuclease

Abstract

Abstract Identifying and validating intermolecular covariation between proteins and their DNA-binding sites can provide insights into mechanisms that regulate selectivity and starting points for engineering new specificity. LAGLIDADG homing endonucleases (meganucleases) can be engineered to bind non-native target sites for gene-editing applications, but not all redesigns successfully reprogram specificity. To gain a global overview of residues that influence meganuclease specificity, we used information theory to identify protein–DNA covariation. Directed evolution experiments of one predicted pair, 227/+3, revealed variants with surprising shifts in I-OnuI substrate preference at the central 4 bases where cleavage occurs. Structural studies showed significant remodeling distant from the covarying position, including restructuring of an inter-hairpin loop, DNA distortions near the scissile phosphates, and new base-specific contacts. Our findings are consistent with a model whereby the functional impacts of covariation can be indirectly propagated to neighboring residues outside of direct contact range, allowing meganucleases to adapt to target site variation and indirectly expand the sequence space accessible for cleavage. We suggest that some engineered meganucleases may have unexpected cleavage profiles that were not rationally incorporated during the design process.

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1]
  1. Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON N6A 5C1, Canada
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1570046
Grant/Contract Number:  
DE AC02 06CH 11357
Resource Type:
Published Article
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Name: Nucleic Acids Research Journal Volume: 47 Journal Issue: 20; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United Kingdom
Language:
English

Citation Formats

Laforet, Marc, McMurrough, Thomas A., Vu, Michael, Brown, Christopher M., Zhang, Kun, Junop, Murray S., Gloor, Gregory B., and Edgell, David R. Modifying a covarying protein–DNA interaction changes substrate preference of a site-specific endonuclease. United Kingdom: N. p., 2019. Web. doi:10.1093/nar/gkz866.
Laforet, Marc, McMurrough, Thomas A., Vu, Michael, Brown, Christopher M., Zhang, Kun, Junop, Murray S., Gloor, Gregory B., & Edgell, David R. Modifying a covarying protein–DNA interaction changes substrate preference of a site-specific endonuclease. United Kingdom. doi:10.1093/nar/gkz866.
Laforet, Marc, McMurrough, Thomas A., Vu, Michael, Brown, Christopher M., Zhang, Kun, Junop, Murray S., Gloor, Gregory B., and Edgell, David R. Fri . "Modifying a covarying protein–DNA interaction changes substrate preference of a site-specific endonuclease". United Kingdom. doi:10.1093/nar/gkz866.
@article{osti_1570046,
title = {Modifying a covarying protein–DNA interaction changes substrate preference of a site-specific endonuclease},
author = {Laforet, Marc and McMurrough, Thomas A. and Vu, Michael and Brown, Christopher M. and Zhang, Kun and Junop, Murray S. and Gloor, Gregory B. and Edgell, David R.},
abstractNote = {Abstract Identifying and validating intermolecular covariation between proteins and their DNA-binding sites can provide insights into mechanisms that regulate selectivity and starting points for engineering new specificity. LAGLIDADG homing endonucleases (meganucleases) can be engineered to bind non-native target sites for gene-editing applications, but not all redesigns successfully reprogram specificity. To gain a global overview of residues that influence meganuclease specificity, we used information theory to identify protein–DNA covariation. Directed evolution experiments of one predicted pair, 227/+3, revealed variants with surprising shifts in I-OnuI substrate preference at the central 4 bases where cleavage occurs. Structural studies showed significant remodeling distant from the covarying position, including restructuring of an inter-hairpin loop, DNA distortions near the scissile phosphates, and new base-specific contacts. Our findings are consistent with a model whereby the functional impacts of covariation can be indirectly propagated to neighboring residues outside of direct contact range, allowing meganucleases to adapt to target site variation and indirectly expand the sequence space accessible for cleavage. We suggest that some engineered meganucleases may have unexpected cleavage profiles that were not rationally incorporated during the design process.},
doi = {10.1093/nar/gkz866},
journal = {Nucleic Acids Research},
number = 20,
volume = 47,
place = {United Kingdom},
year = {2019},
month = {10}
}

Journal Article:
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DOI: 10.1093/nar/gkz866

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