Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase
Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase
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新型 III 型 CRISPR-Cas 效应子的表征为 Cas10 DNase 的变构激活和抑制提供了新的见解
DOI：
10.1038/s41421-020-0160-4
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发表时间：
2020-05-12
期刊：
影响因子：
33.5
通讯作者：
She, Qunxin
中科院分区：
文献类型：
--
作者：
Lin, Jinzhong;Feng, Mingxia;She, Qunxin
III型CRISPR-Cas系统的抗病毒防御依赖于其效应物的两个不同的活性：RNA激活的DNA裂解和环寡腺苷的合成。这两种活动都是以不分青红皂白的核酸裂解为特征的，并受到时空调节。为了进一步了解相关的机制，我们在大肠杆菌中重组了LdCsm，这是一个乳杆菌III-A系统。一旦被靶RNA激活，这种免疫系统就会介导强劲的DNA降解，但缺乏环状寡腺苷的合成。对Csm3和Cas10保守残基的突变表明，Csm3和Cas10中的多个结构域在变构调节中发挥作用，以产生活性酶。设计并测试了3个ʹ抗标签中携带不同截短片段的靶RNA对LdCsm结合和切割的影响。确定了LdCsm三元络合物的三种不同状态。特别是，将携带3ʹ抗标签中单个核苷酸的靶RNA与LdCsm结合可产生活性的LdCsm DNA酶，无论核苷酸与crRNA5‘标签是否错配，如同源靶RNA，还是匹配，如非同源靶RNA5’标签。此外，进一步增加CTR中3ʹ反义TAG的数目，促进底物结合，促进底物降解，而在正常情况下，底物结合逐渐减少，最终切断酶的切割作用。综上所述，这些结果为LdCsm酶的变构激活和抑制提供了机械性的见解。
Antiviral defense by type III CRISPR-Cas systems relies on two distinct activities of their effectors: the RNA-activated DNA cleavage and synthesis of cyclic oligoadenylate. Both activities are featured as indiscriminate nucleic acid cleavage and subjected to the spatiotemporal regulation. To yield further insights into the involved mechanisms, we reconstituted LdCsm, a lactobacilli III-A system inEscherichia coli. Upon activation by target RNA, this immune system mediates robust DNA degradation but lacks the synthesis of cyclic oligoadenylates. Mutagenesis of the Csm3 and Cas10 conserved residues revealed that Csm3 and multiple structural domains in Cas10 function in the allosteric regulation to yield an active enzyme. Target RNAs carrying various truncations in the 3ʹ anti-tag were designed and tested for their influence on DNA binding and DNA cleavage of LdCsm. Three distinct states of ternary LdCsm complexes were identified. In particular, binding of target RNAs carrying a single nucleotide in the 3ʹ anti-tag to LdCsm yielded an active LdCsm DNase regardless whether the nucleotide shows a mismatch, as in the cognate target RNA (CTR), or a match, as in the noncognate target RNA (NTR), to the 5′ tag of crRNA. In addition, further increasing the number of 3ʹ anti-tag in CTR facilitated the substrate binding and enhanced the substrate degradation whereas doing the same as in NTR gradually decreased the substrate binding and eventually shut off the DNA cleavage by the enzyme. Together, these results provide the mechanistic insights into the allosteric activation and repression of LdCsm enzymes.