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Title: Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity

Abstract

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G{sub +4} base pair for the wild-type A:T{sub +4} base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T{sub +4} were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T{sub +4} or the C:G{sub +4} base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G{sub +4} recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T{sub +4} target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G{sub +4} target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences inmore » binding affinities account for the observed -36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G{sub +4} substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.« less

Authors:
; ; ; ; ;  [1]
  1. UIUC
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
NSFNIH
OSTI Identifier:
1023651
Resource Type:
Journal Article
Journal Name:
J. Mol. Biol.
Additional Journal Information:
Journal Volume: 405; Journal Issue: (1) ; 01, 2011; Journal ID: ISSN 0022-2836
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; AMINO ACIDS; CLEAVAGE; CRYSTAL STRUCTURE; DNA; ENDONUCLEASES; ENZYMES; GENE THERAPY; IN VIVO; LIBRARIES; MUTANTS; PROTEIN ENGINEERING; PROTEINS; RESIDUES; SPECIFICITY

Citation Formats

Joshi, Rakesh, Ho, Kwok Ki, Tenney, Kristen, Chen, Jui-Hui, Golden, Barbara L., Gimble, Frederick S., and Purdue). Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity. United States: N. p., 2013. Web. doi:10.1016/j.jmb.2010.10.029.
Joshi, Rakesh, Ho, Kwok Ki, Tenney, Kristen, Chen, Jui-Hui, Golden, Barbara L., Gimble, Frederick S., & Purdue). Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity. United States. https://doi.org/10.1016/j.jmb.2010.10.029
Joshi, Rakesh, Ho, Kwok Ki, Tenney, Kristen, Chen, Jui-Hui, Golden, Barbara L., Gimble, Frederick S., and Purdue). Wed . "Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity". United States. https://doi.org/10.1016/j.jmb.2010.10.029.
@article{osti_1023651,
title = {Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity},
author = {Joshi, Rakesh and Ho, Kwok Ki and Tenney, Kristen and Chen, Jui-Hui and Golden, Barbara L. and Gimble, Frederick S. and Purdue)},
abstractNote = {Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G{sub +4} base pair for the wild-type A:T{sub +4} base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T{sub +4} were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T{sub +4} or the C:G{sub +4} base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G{sub +4} recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T{sub +4} target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G{sub +4} target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed -36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G{sub +4} substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.},
doi = {10.1016/j.jmb.2010.10.029},
url = {https://www.osti.gov/biblio/1023651}, journal = {J. Mol. Biol.},
issn = {0022-2836},
number = (1) ; 01, 2011,
volume = 405,
place = {United States},
year = {2013},
month = {9}
}