Isolation and Characterization of New Homing Endonuclease Specificities at Individual Target Site Positions

Homing endonucleases are highly specific DNA endonucleases, encoded within mobile introns or inteins, that induce targeted recombination, double-strand repair and gene conversion of their cognate target sites. Due to their biological function and high level of target specificity, these enzymes are u...

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Bibliographic Details
Published in:Journal of molecular biology 2004-09, Vol.342 (1), p.31-41
Main Authors: Sussman, Django, Chadsey, Meg, Fauce, Steve, Engel, Alex, Bruett, Anna, Monnat, Ray, Stoddard, Barry L., Seligman, Lenny M.
Format: Article
Language:English
Subjects:
Base Pairing
Base Sequence
Binding Sites
crystal structure
Crystallography, X-Ray
DNA - chemistry
DNA - metabolism
endonuclease
Endonucleases - genetics
Endonucleases - isolation & purification
Endonucleases - metabolism
engineering
Gene Targeting
Models, Molecular
Protein Binding
Protein Conformation
specificity
Substrate Specificity
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Summary:Homing endonucleases are highly specific DNA endonucleases, encoded within mobile introns or inteins, that induce targeted recombination, double-strand repair and gene conversion of their cognate target sites. Due to their biological function and high level of target specificity, these enzymes are under intense investigation as tools for gene targeting. These studies require that naturally occurring enzymes be redesigned to recognize novel target sites. Here, we report studies in which the homodimeric LAGLIDADG homing endonuclease I- CreI is altered at individual side-chains corresponding to contact points to distinct base-pairs in its target site. The resulting enzyme constructs drive specific elimination of selected DNA targets in vivo and display shifted specificities of DNA binding and cleavage in vitro. Crystal structures of two of these constructs demonstrate that substitution of individual side-chain/DNA contact patterns can occur with almost no structural deformation or rearrangement of the surrounding complex, facilitating an isolated, modular redesign strategy for homing endonuclease activity and specificity.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2004.07.031