2O7M

The C-terminal loop of the homing endonuclease I-CreI is essential for DNA binding and cleavage. Identification of a novel site for specificity engineering in the I-CreI scaffold

2O7M の概要

• エントリーDOI: 10.2210/pdb2o7m/pdb
• 分子名称: DNA endonuclease I-CreI (2 entities in total)
• 機能のキーワード: homing endonuclease, dna, hydrolase
• 由来する生物種: Chlamydomonas reinhardtii
• 細胞内の位置: Plastid, chloroplast: P05725
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 35869.26

構造登録者

Prieto, J.,Redondo, P.,Padro, D.,Blanco, F.J.,Paques, F.,Montoya, G. (登録日: 2006-12-11, 公開日: 2007-10-23, 最終更新日: 2023-10-25)

主引用文献

Prieto, J.,Redondo, P.,Padro, D.,Arnould, S.,Epinat, J.C.,Paques, F.,Blanco, F.J.,Montoya, G.
The C-terminal loop of the homing endonuclease I-CreI is essential for site recognition, DNA binding and cleavage
Nucleic Acids Res., 35:3262-3271, 2007

PubMed Abstract: Meganucleases are sequence-specific endonucleases with large cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These enzymes open novel perspectives for genome engineering in a wide range of fields, including gene therapy. A new crystal structure of the I-CreI dimer without DNA has allowed the comparison with the DNA-bound protein. The C-terminal loop displays a different conformation, which suggests its implication in DNA binding. A site-directed mutagenesis study in this region demonstrates that whereas the C-terminal helix is negligible for DNA binding, the final C-terminal loop is essential in DNA binding and cleavage. We have identified two regions that comprise the Ser138-Lys139 and Lys142-Thr143 pairs whose double mutation affect DNA binding in vitro and abolish cleavage in vivo. However, the mutation of only one residue in these sites allows DNA binding in vitro and cleavage in vivo. These findings demonstrate that the C-terminal loop of I-CreI endonuclease plays a fundamental role in its catalytic mechanism and suggest this novel site as a region to take into account for engineering new endonucleases with tailored specificity.

PubMed: 17452357
DOI: 10.1093/nar/gkm183

実験手法

X-RAY DIFFRACTION (2 Å)