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PDBsum entry 1u0c
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Hydrolase/DNA
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PDB id
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1u0c
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References listed in PDB file
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Key reference
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Title
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Isolation and characterization of new homing endonuclease specificities at individual target site positions.
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Authors
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D.Sussman,
M.Chadsey,
S.Fauce,
A.Engel,
A.Bruett,
R.Monnat,
B.L.Stoddard,
L.M.Seligman.
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Ref.
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J Mol Biol, 2004,
342,
31-41.
[DOI no: ]
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PubMed id
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Abstract
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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.
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Figure 1.
Figure 1. Structure of I-CreI and DNA target sites used in
this study. (a) Structure of wild-type I-CreI bound to DNA. The
positions of residues that are targeted for selection are
indicated in the homodimer by red labels and arrows. (b)
Wild-type and mutant enzyme DNA-binding sites. Base-pairs
±6 and ±10, that interact with Q26/Y66 and Y33,
respectively, are colored to correspond to the scheme in the top
panel. Points of cleavage are noted with blue triangles and red
cleavage patterns.
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Figure 4.
Figure 4. Structures and interactions of cognate pairs at
residue 33 and base-pair 10. Protein-DNA contacts in the
vicinity of base-pair ±10, in bound complexes containing
either wild-type enzyme and DNA target site (left), or Y33C and
Y33H mutant enzymes bound to their cognate target site (middle
and right, respectively). The sequence of wild-type I-CreI
target sequence (left) and alternate target sequences (middle
and right) targeted for selection are shown below their
corresponding structures.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2004,
342,
31-41)
copyright 2004.
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