Two distinct DNA binding modes guide dual roles of a CRISPR-Cas protein complex.
Two distinct DNA binding modes guide dual roles of a CRISPR-Cas protein complex.
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DOI：
10.1016/j.molcel.2015.01.028
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发表时间：
2015-04-02
期刊：
影响因子：
16
通讯作者：
Joo, Chirlmin
中科院分区：
文献类型：
--
作者：
Blosser, Timothy R.;Loeff, Luuk;Westra, Edze R.;Vlot, Marnix;Kunne, Tim;Sobota, Malgorzata;Dekker, Cees;Brouns, Stan J. J.;Joo, Chirlmin
小RNA引导的蛋白质复合物在原核生物中CRISPR介导的免疫中起着至关重要的作用。虽然这些复合物通过标记同源入侵者DNA进行破坏来启动干扰，但最近的证据表明它们参与了针对突变入侵者序列的新CRISPR记忆形成，称为启动。目标识别复合体介导这些不同反应-干扰和启动-的机制仍然知之甚少。使用单分子FRET，我们可视化如何真正的和突变的目标是不同的探测E。coli Cascade.我们观察到，真正的目标的识别是一个有序的过程，是严格控制的高保真度。突变的靶标以低保真度被识别，其特征在于crRNA的任何区段的短寿命和PAM和种子独立性结合。Cascade在免疫中的这些双重作用具有不同的特异性，这支持了CRISPR-Cas的稳健性，允许有效降解真正的靶标并引发突变的DNA靶标。
Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses—interference and priming—remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets.
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