Mechanism of foreign DNA recognition by a CRISPR RNA-guided surveillance complex from Pseudomonas aeruginosa.
Mechanism of foreign DNA recognition by a CRISPR RNA-guided surveillance complex from Pseudomonas aeruginosa.
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DOI：
10.1093/nar/gkv094
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发表时间：
2015-02-27
影响因子：
14.9
通讯作者：
Wiedenheft B
中科院分区：
文献类型：
--
作者：
Rollins MF;Schuman JT;Paulus K;Bukhari HS;Wiedenheft B
铜绿假单胞菌中 I-F 型 CRISPR 介导的（成簇规则间隔的短回文重复序列）适应性免疫系统由两个 CRISPR 位点和六个 CRISPR 相关 (cas) 基因组成。外源 DNA 监视由 Cas 蛋白 (Csy1-4) 复合体执行，该复合体与 CRISPR RNA (crRNA) 组装成称为 Csy 复合体的 350 kDa 核糖核蛋白。在这里，我们展示了 Csy 复合物对外源核酸的识别是通过连续步骤进行的，首先是检测位于互补 DNA 靶标附近的两个连续的鸟嘌呤-胞嘧啶碱基对 (G-C/G-C)。我们证明这个基序，称为 PAM（原型间隔子相邻基序），必须是双链的，并且单链 PAM 不提供显着的区分能力。对富含 G-C/G-C 的竞争序列进行的结合测定表明，Csy 复合物直接与该二核苷酸基序相互作用，动力学分析表明，G-C/G-C 基序的识别是 crRNA 引导与靶序列结合的先决条件。总之，这些数据表明 Csy 复合物首先与 G-C/G-C 碱基对相互作用，然后对相邻的靶序列进行采样，以与 crRNA 向导互补。
The Type I-F CRISPR-mediated (clustered regularly interspaced short palindromic repeats) adaptive immune system in Pseudomonas aeruginosa consists of two CRISPR loci and six CRISPR-associated (cas) genes. Foreign DNA surveillance is performed by a complex of Cas proteins (Csy1–4) that assemble with a CRISPR RNA (crRNA) into a 350-kDa ribonucleoprotein called the Csy complex. Here, we show that foreign nucleic acid recognition by the Csy complex proceeds through sequential steps, initiated by detection of two consecutive guanine–cytosine base pairs (G–C/G–C) located adjacent to the complementary DNA target. We show that this motif, called the PAM (protospacer adjacent motif), must be double-stranded and that single-stranded PAMs do not provide significant discriminating power. Binding assays performed with G–C/G–C-rich competitor sequences indicate that the Csy complex interacts directly with this dinucleotide motif, and kinetic analyses reveal that recognition of a G–C/G–C motif is a prerequisite for crRNA-guided binding to a target sequence. Together, these data indicate that the Csy complex first interacts with G–C/G–C base pairs and then samples adjacent target sequences for complementarity to the crRNA guide.
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影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
3.2
作者：
Deveau, Helene;Barrangou, Rodolphe;Moineau, Sylvain
通讯作者：
Moineau, Sylvain
基因组分析表明，铜绿假单胞菌的毒力是组合。
影响因子：
12.3
作者：
Lee DG;Urbach JM;Wu G;Liberati NT;Feinbaum RL;Miyata S;Diggins LT;He J;Saucier M;Déziel E;Friedman L;Li L;Grills G;Montgomery K;Kucherlapati R;Rahme LG;Ausubel FM
通讯作者：
Ausubel FM
影响因子：
3.4
作者：
Bauer, Maximilian;Metzler, Ralf
通讯作者：
Metzler, Ralf