Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System.
Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System.
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大肠杆菌CRISPR-CAS系统对外国DNA的监视和处理。
DOI：
10.1016/j.cell.2015.10.003
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发表时间：
2015-11-05
期刊：
影响因子：
64.5
通讯作者：
Greene EC
中科院分区：
文献类型：
--
作者：
Redding S;Sternberg SH;Marshall M;Gibb B;Bhat P;Guegler CK;Wiedenheft B;Doudna JA;Greene EC
CRISPR-Cas适应性免疫系统保护细菌和古细菌免受外来遗传因子的侵害。在大肠杆菌中，Cascade（CRISPR相关抗病毒防御复合物）是一种RNA引导的监视复合物，它结合外源DNA并招募Cas 3，Cas 3是一种用于靶向降解的反式作用核酸酶解旋酶。在这里，我们使用单分子成像来可视化Cascade和Cas 3与外源DNA靶标的结合。我们的分析揭示了两个不同的途径，由存在或不存在的原型间隔区相邻基序（PAM）。与PAM侧翼的原型间隔区的结合募集核酸酶活性Cas 3用于降解靶DNA的短单链区域，而PAM突变引发通过依赖于Cas 1和Cas 2蛋白的机制募集核酸酶失活Cas 3的替代途径。这些发现解释了Cascade的靶识别如何引起不同的结果，并支持了通过涉及沿着沿着外源DNA的进行性ATP依赖性Cas 3易位的机制获得新间隔区序列的模型。
CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease-helicase for target degradation. Here we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways, dictated by the presence or absence of a protospacer adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short singlestranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent upon the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes, and supports a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.