A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction.
A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction.
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DOI：
10.1093/nar/gkw1274
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发表时间：
2017-02-28
影响因子：
14.9
通讯作者：
She Q
中科院分区：
文献类型：
--
作者：
Han W;Li Y;Deng L;Feng M;Peng W;Hallstrøm S;Zhang J;Peng N;Liang YX;White MF;She Q
CRISPR（成簇的规则间隔短回文重复序列）系统通过以crRNA引导的方式消除核酸入侵者来保护古细菌和细菌。冰岛硫化叶菌III-B型Cmr-α系统在RNA和DNA水平上靶向入侵核酸，DNA靶向依赖于原型间隔区在体内的定向转录。为了进一步了解所涉及的机制，我们从S. islandicus，并在体外对其进行了表征。Cmr-α切割复合物中存在的与crRNA互补的RNA，并且其ssDNA破坏活性被靶RNA激活。ssDNA切割需要crRNA的5 ′-标签和靶RNA的3 ′-侧翼区域之间的错配。侵入质粒分析表明，Cmr-2α蛋白的组氨酸-天冬氨酸（HD）结构域（四重突变）或GGDD基序中的突变导致体内DNA干扰减弱。然而，HD基序的双突变仅在体外消除DNase活性。此外，激活的Cmr-α二元复合物作为一种高活性的DNA酶，可以破坏大量过量的DNA底物，这可能提供了一种强有力的手段，快速降解复制的病毒DNA。
The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr–α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of III-B Cmr–α from S. islandicus and characterized it in vitro. Cmr–α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5΄-tag of crRNA and the 3΄-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr–2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr–α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA.