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PDBsum entry 2vs7
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DNA binding protein
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PDB id
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2vs7
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References listed in PDB file
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Key reference
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Title
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Crystal structure of I-Dmoi in complex with its target DNA provides new insights into meganuclease engineering.
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Authors
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M.J.Marcaida,
J.Prieto,
P.Redondo,
A.D.Nadra,
A.Alibés,
L.Serrano,
S.Grizot,
P.Duchateau,
F.Pâques,
F.J.Blanco,
G.Montoya.
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Ref.
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Proc Natl Acad Sci U S A, 2008,
105,
16888-16893.
[DOI no: ]
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PubMed id
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Abstract
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Homing endonucleases, also known as meganucleases, are sequence-specific enzymes
with large DNA recognition sites. These enzymes can be used to induce efficient
homologous gene targeting in cells and plants, opening perspectives for genome
engineering with applications in a wide series of fields, ranging from
biotechnology to gene therapy. Here, we report the crystal structures at 2.0 and
2.1 A resolution of the I-DmoI meganuclease in complex with its substrate DNA
before and after cleavage, providing snapshots of the catalytic process. Our
study suggests that I-DmoI requires only 2 cations instead of 3 for DNA
cleavage. The structure sheds light onto the basis of DNA binding, indicating
key residues responsible for nonpalindromic target DNA recognition. In silico
and in vivo analysis of the I-DmoI DNA cleavage specificity suggests that
despite the relatively few protein-base contacts, I-DmoI is highly specific when
compared with other meganucleases. Our data open the door toward the generation
of custom endonucleases for targeted genome engineering using the monomeric
I-DmoI scaffold.
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Figure 2.
Detailed view of the I-DmoI active site. (A and B) Anomalous
difference maps illustrate the presence of only 1 atom of
calcium in the DNA bound structure (A) and 2 manganese ions in
the structure of the I-DmoI product complex (B). The protein is
shown in yellow and the DNA in green. (C) Schematic diagram of
the hypothetical enzymatic mechanism proposed for I-DmoI.
Hydrolysis of the phosphodiester bonds would follow a 2-metal
ion mechanism. The first metal ion (site1, colored in yellow in
1) is bound in 1 active site and the water nucleophile (colored
in red) is positioned in the central site and can attack the
coding strand (in 2). The regeneration of the central water
together with a second metal ion in the second site (site2,
colored in cyan in 3) would enable the second attack. The D21,
G20 and E117, A116 are contributed by the LAGLIDADG motifs of
the enzyme.
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Figure 4.
Comparison of H-DreI and I-DmoI structures. (A) Cα trace
superposition of I-DmoI and H-DreI in complex with their DNA.
(B) Detailed view of the DNA moieties. (C and D) Detailed views
of the loop1a and loop2a protein-DNA interactions. (E)
Comparison of the helical and propeller twist values of I-DmoI
and H-DreI DNAs.
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