Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity
Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity
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DOI：
10.1073/pnas.1616395114
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发表时间：
2017-06-27
影响因子：
11.1
通讯作者：
Wiedenheft, Blake
中科院分区：
文献类型：
--
作者：
Rollins, MaryClare F.;Chowdhury, Saikat;Wiedenheft, Blake
铜绿假单胞菌（PA 14）中的I-F型CRISPR适应性免疫系统由两个CRISPR基因座和六个CRISPR相关（cas）基因组成。I-F型系统依赖于CRISPR RNA（crRNA）引导的监视复合物（Csy复合物）来结合外源DNA并募集反式作用核酸酶（即，Cas 2/3）用于靶降解。在大多数I型系统中，Cas 2和Cas 3是分别参与适应和干扰的独立蛋白质。然而，在I-F系统中，这些蛋白质融合成单个多肽。在这里，我们使用生物化学和结构方法来显示两个Cas 2/3分子与四个Cas 1分子（Cas 2/3（2）：Cas 1（4））组装成四叶螺旋桨状结构，其中两个Cas 2结构域形成一个中心枢纽（双重对称轴），两侧是两个Cas 1叶和两个Cas 3叶。我们发现Cas 1亚基抑制Cas 2/3核酸酶活性，并且Csy复合物对外源DNA的识别激活了Cas 2/3，导致DNA靶标的双向降解。总的来说，这项工作提供了Cas 1 -2/3复合物的结构，并解释了Cas 1和靶结合的Csy复合物如何在Cas 2/3核酸酶活性的调节中发挥相反的作用。
The type I-F CRISPR adaptive immune system in Pseudomonas aeruginosa (PA14) consists of two CRISPR loci and six CRISPR-associated (cas) genes. Type I-F systems rely on a CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease (i.e., Cas2/3) for target degradation. In most type I systems, Cas2 and Cas3 are separate proteins involved in adaptation and interference, respectively. However, in I-F systems, these proteins are fused into a single polypeptide. Here we use biochemical and structural methods to show that two molecules of Cas2/3 assemble with four molecules of Cas1 (Cas2/3(2):Cas1(4)) into a four-lobed propeller-shaped structure, where the two Cas2 domains form a central hub (twofold axis of symmetry) flanked by two Cas1 lobes and two Cas3 lobes. We show that the Cas1 subunits repress Cas2/3 nuclease activity and that foreign DNA recognition by the Csy complex activates Cas2/3, resulting in bidirectional degradation of DNA targets. Collectively, this work provides a structure of the Cas1-2/3 complex and explains how Cas1 and the target-bound Csy complex play opposing roles in the regulation of Cas2/3 nuclease activity.