3KO2

I-MsoI re-designed for altered DNA cleavage specificity (-7C)

3KO2 の概要

• エントリーDOI: 10.2210/pdb3ko2/pdb
• 分子名称: Site-specific DNA endonuclease I-MsoI, 5'-D(*GP*CP*AP*GP*AP*CP*CP*GP*TP*CP*GP*TP*GP*AP*GP*AP*CP*AP*GP*TP*TP*CP*CP*G)-3', 5'-D(*CP*GP*GP*AP*AP*CP*TP*GP*TP*CP*TP*CP*AP*CP*GP*AP*CP*GP*GP*TP*CP*TP*GP*C)-3', ... (5 entities in total)
• 機能のキーワード: redesign, protein-dna complex, chloroplast, endonuclease, hydrolase-dna complex, hydrolase/dna
• 由来する生物種: Monomastix sp.; 詳細
• タンパク質・核酸の鎖数: 8
• 化学式量合計: 108131.54

構造登録者

Taylor, G.K.,Stoddard, B.L. (登録日: 2009-11-13, 公開日: 2010-05-19, 最終更新日: 2023-09-06)

主引用文献

Ashworth, J.,Taylor, G.K.,Havranek, J.J.,Quadri, S.A.,Stoddard, B.L.,Baker, D.
Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs.
Nucleic Acids Res., 38:5601-5608, 2010

PubMed Abstract: Site-specific homing endonucleases are capable of inducing gene conversion via homologous recombination. Reprogramming their cleavage specificities allows the targeting of specific biological sites for gene correction or conversion. We used computational protein design to alter the cleavage specificity of I-MsoI for three contiguous base pair substitutions, resulting in an endonuclease whose activity and specificity for its new site rival that of wild-type I-MsoI for the original site. Concerted design for all simultaneous substitutions was more successful than a modular approach against individual substitutions, highlighting the importance of context-dependent redesign and optimization of protein-DNA interactions. We then used computational design based on the crystal structure of the designed complex, which revealed significant unanticipated shifts in DNA conformation, to create an endonuclease that specifically cleaves a site with four contiguous base pair substitutions. Our results demonstrate that specificity switches for multiple concerted base pair substitutions can be computationally designed, and that iteration between design and structure determination provides a route to large scale reprogramming of specificity.

PubMed: 20435674
DOI: 10.1093/nar/gkq283

実験手法

X-RAY DIFFRACTION (2.9 Å)