Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli.
Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli.
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DOI：
10.1038/nature16995
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发表时间：
2016-02-25
期刊：
影响因子：
64.8
通讯作者：
Ke, Ailong
中科院分区：
文献类型：
--
作者：
Hayes, Robert P.;Xiao, Yibei;Ding, Fran;van Erp, Paul B. G.;Rajashankar, Kanagalaghatta;Bailey, Scott;Wiedenheft, Blake;Ke, Ailong
规则间隔的短回文重复序列（CRISPR）和cas（CRISPR相关）操纵子的簇形成了针对原核生物中外源遗传元件的基于RNA的适应性免疫系统。I型占CRISPR系统的95%，并已被用于控制基因表达和细胞命运。在CRISPR RNA（crRNA）引导的干扰过程中，Cascade（用于抗病毒防御的CRISPR相关复合物）促进crRNA引导的双链DNA（dsDNA）入侵，与靶DNA链互补碱基配对，同时置换非靶链，形成R环。随后募集Cas 3核酸酶/解旋酶以降解两条DNA链。靶DNA侧翼的原间隔区邻近基序（PAM）对于自身与外来区分是至关重要的。本文报道了E. coli Cascade与外源dsDNA靶标结合。5′-ATG PAM以双链形式从小沟侧被Cse 1中的三个结构特征识别。小沟DNA识别所固有的混杂性使一个Cascade可以响应几个不同的PAM序列的令人困惑的观察合理化。最佳PAM识别与楔形插入一致，启动定向靶DNA链解旋以与crRNA进行分段碱基配对。非靶链被沿着平行路径25隔开地引导，并且R环结构通过将该链锁定在Cse 2二聚体之后而进一步稳定。这些观测结果为理解PAM依赖的定向R环形成过程提供了结构基础。
Clusters of regularly interspaced short palindromic repeats (CRISPRs) and cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I account for 95% of CRISPR systems, and have been utilized to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defense) facilitates crRNA-guided invasion of double-stranded DNA (dsDNA) for complementary base-pairing with the target DNA strand, while displacing the non-target strand, forming an R-loop. Cas3 nuclease/helicase is recruited subsequently to degrade two DNA strands. Protospacer adjacent motif (PAM) flanking target DNA is crucial for self vs. foreign discrimination. Here we present a 2.45 Å crystal structure of E. coli Cascade bound to a foreign dsDNA target. The 5′-ATG PAM is recognized in double-stranded form, from the minor groove side, by three structural features in Cse1. The promiscuity inherent to minor groove DNA recognition rationalizes the puzzling observation that a single Cascade can respond to several distinct PAM sequences. Optimal PAM recognition coincides with a wedge insertion, initiating the directional target DNA strand unwinding for segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind Cse2 dimer. These observations provide the structure basis for understanding the PAM-dependent directional R-loop formation process.
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