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Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities

机译:限制性内切酶BmrI的催化结构域作为具有新型底物特异性的工程内切酶的切割模块

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摘要

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.
机译:创建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。我们创建了一个嵌合核酸内切酶(CH-核酸内切酶),该核酸内切酶由BmrI限制性内切核酸酶的DNA切割域和C.BclI(BclI限制性修饰系统的控制蛋白)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,C-box序列内18 bp。使用具有C-box序列缺失的DNA底物,我们显示嵌合核酸内切酶仅需要C盒5'的一半才能进行特异性切割。对于蛋白质-DNA结合和DNA切割的模式,提出了一个示意性模型。本研究表明BmrI切割域可用于创建组合核酸内切酶,该组合核酸内切酶可在DNA结合配偶体指示的特定序列上切割DNA。所得的核酸内切酶将在体外和体内用于在特定位点产生ds断裂并产生缺失。

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