Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities.
Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities.
复制标题
限制性核酸内切酶BMRI的催化结构域作为具有新型底物特异性的工程核酸内切酶的切割模块。
DOI：
10.1093/nar/gkm665
复制
发表时间：
2007
影响因子：
14.9
通讯作者：
Xu, Shuang-yong
中科院分区：
文献类型：
--
作者：
Chan, Siu-hong;Bao, Yongming;Ciszak, Ewa;Laget, Sophie;Xu, Shuang-yong
创造具有新颖序列特异性的核酸内切酶为操纵 DNA 提供了更多可能性。我们创建了一种嵌合核酸内切酶（CH-核酸内切酶），由 BmrI 限制性核酸内切酶的 DNA 切割结构域和 C.BclI（BclI 限制性核酸内切酶的控制蛋白）组成。纯化的嵌合核酸内切酶 BmrI198-C.BclI 在 C.BclI 识别序列附近的特定位点切割 DNA。在两个位点观察到双链 (ds) 断裂：上游 8 bp 和 C 盒序列内的 18 bp。使用删除了 C 盒序列的 DNA 底物，我们发现嵌合核酸内切酶仅需要 C 盒的 5' 部分来进行特异性切割。提出了蛋白质-DNA 结合和 DNA 切割模式的示意性模型。本研究表明，BmrI 切割结构域可用于创建组合核酸内切酶，在 DNA 结合伴侣指定的特定序列处切割 DNA。所得核酸内切酶可在体外和体内用于在特定位点产生双链断裂并产生缺失。
Creating endonucleases with novel sequence specificities provides more possibilities to manipulate DNA. We have created a chimeric endonuclease (CH-endonuclease) consisting of the DNA cleavage domain of BmrI restriction endonuclease and C.BclI, a controller protein of the BclI restriction-modification system. The purified chimeric endonuclease, BmrI198-C.BclI, cleaves DNA at specific sites in the vicinity of the recognition sequence of C.BclI. Double-strand (ds) breaks were observed at two sites: 8 bp upstream and 18 bp within the C-box sequence. Using DNA substrates with deletions of C-box sequence, we show that the chimeric endonuclease requires the 5′ half of the C box only for specific cleavage. A schematic model is proposed for the mode of protein–DNA binding and DNA cleavage. The present study demonstrates that the BmrI cleavage domain can be used to create combinatorial endonucleases that cleave DNA at specific sequences dictated by the DNA-binding partner. The resulting endonucleases will be useful in vitro and in vivo to create ds breaks at specific sites and generate deletions.