Rational design of a chimeric endonuclease targeted to NotI recognition site.
Rational design of a chimeric endonuclease targeted to NotI recognition site.
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针对 NotI 识别位点的嵌合核酸内切酶的合理设计。
DOI：
10.1093/protein/gzm049
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Xu,Shuang-yong
中科院分区：
文献类型：
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作者：
Zhang,Penghua;Bao,Yongming;Higgins,Lauren;Xu,Shuang-yong
通过thenotIR基因的随机诱变分离出NotI限制性内切酶(GCGGCCGC)的切割缺陷变体。 NotI 变体 D160N 可以结合 DNA 并保护质粒 DNA 免遭 EagI (CGGCCG) 和 NotI 消化。 EDTA 抗性 BmrI 限制性内切酶可切割 DNA 序列 ACTGGG N5/N4。具有非特异性核酸酶活性的 BmrI 的 N 末端切割结构域（残基 1-198）通过短接头与 NotI 变体 D160N 融合。工程嵌合核酸内切酶（CH-核酸内切酶）在高盐（100-150 mM NaCl）和二价阳离子 Mg++ 或 Ca++ 存在的情况下特异性识别 NotI 位点。与在其识别序列内进行切割的野生型 NotI 不同，BmrI198-NotI (D160N) 在 NotI 位点之外切割 DNA，导致 NotI 位点和相邻序列的删除。 BmrI 裂解结构域与 II 型限制酶的裂解缺陷变体融合以产生新的裂解位点将为 DNA 操作提供有用的工具。
A cleavage-deficient variant of NotI restriction endonuclease (GCGGCCGC) was isolated by random mutagenesis of thenotIRgene. The NotI variant D160N was shown to bind DNA and protect plasmid DNA from EagI (CGGCCG) and NotI digestions. The EDTA-resistant BmrI restriction endonuclease cleaves DNA sequence ACTGGG N5/N4. The N-terminal cleavage domain of BmrI (residues 1–198) with non-specific nuclease activity was fused to the NotI variant D160N with a short linker. The engineered chimeric endonuclease (CH-endonuclease) recognizes NotI sites specifically in the presence of high salt (100–150 mM NaCl) and divalent cations Mg++or Ca++. In contrast to wild-type NotI, which cuts within its recognition sequence, BmrI198-NotI (D160N) cleaves DNA outside of NotI sites, resulting in deletion of the NotI site and the adjacent sequences. The fusion of the BmrI cleavage domain to cleavage-deficient variants of Type II restriction enzymes to generate novel cleavage sites will provide useful tools for DNA manipulation.