skip to main content

SciTech ConnectSciTech Connect

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

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] ;  [2]
  1. (UIUC)
  2. (
Publication Date:
OSTI Identifier:
1023651
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Mol. Biol.; Journal Volume: 405; Journal Issue: (1) ; 01, 2011
Research Org:
Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)
Sponsoring Org:
NSFNIH
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