Structural and Mechanistic Basis of PAM-Dependent Spacer Acquisition in CRISPR-Cas Systems
Structural and Mechanistic Basis of PAM-Dependent Spacer Acquisition in CRISPR-Cas Systems
复制标题
CRISPR-Cas 系统中 PAM 依赖性间隔区获取的结构和机制基础
DOI：
10.1016/j.cell.2015.10.008
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发表时间：
2015-11-05
期刊：
影响因子：
64.5
通讯作者：
Wang, Yanli
中科院分区：
文献类型：
--
作者：
Wang, Jiuyu;Li, Jiazhi;Wang, Yanli
细菌通过将侵入性DNA序列元素整合到宿主CRISPR基因座中，在CRISPR阵列中产生新的间隔片段，从而获得对病毒入侵者的记忆。我们报道了cas1 - cas2 -双叉DNA复合物的结构，以努力理解在插入CRISPR位点之前如何对原间隔物进行采样。我们的研究揭示了一个由酪氨酸残基包围的23bp双链组成的原间隔物DNA，以及锚定的侧翼30个悬垂片段。Cas1a催化亚基以碱基特异性的方式识别30悬垂中的pam互补序列，随后在双链边界的5 nt位置切割产生33 nt的DNA中间体，该中间体通过剪切-粘贴机制整合到CRISPR阵列中。在原间隔物结合后，Cas1-Cas2发生了显著的构象变化，产生了有利于原间隔物识别的平面。在这里，我们的研究为pam依赖的隔离剂获取提供了重要的基于结构的机制见解。
Bacteria acquire memory of viral invaders by incorporating invasive DNA sequence elements into the host CRISPR locus, generating a new spacer within the CRISPR array. We report on the structures of Cas1-Cas2-dual-forked DNA complexes in an effort toward understanding how the protospacer is sampled prior to insertion into the CRISPR locus. Our study reveals a protospacer DNA comprising a 23-bp duplex bracketed by tyrosine residues, together with anchored flanking 30 overhang segments. The PAM-complementary sequence in the 30 overhang is recognized by the Cas1a catalytic subunits in a base-specific manner, and subsequent cleavage at positions 5 nt from the duplex boundary generates a 33-nt DNA intermediate that is incorporated into the CRISPR array via a cut-and-paste mechanism. Upon protospacer binding, Cas1-Cas2 undergoes a significant conformational change, generating a flat surface conducive to proper protospacer recognition. Here, our study provides important structure-based mechanistic insights into PAM-dependent spacer acquisition.