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对一种类型V-M CRISPR-Cas效应酶的机制和进化见解
Satoshi N Omura1, Ryoya Nakagawa1, Christian Südfeld2
1Department of Biological Sciences, Graduate School of Science, the University of Tokyo, Tokyo, Japan.
Nature structural & molecular biology
|July 17, 2023
概括
Cas12m2 CRISPR-Cas效应器使用强大的DNA结合用于细菌免疫,而不是DNA裂变. 结构研究揭示了其独特的RNA引导基因组失活和适应性免疫机制.
科学领域:
- 分子生物学分子生物学
- 微生物学 微生物学
- 遗传学 遗传学 是一个
背景情况:
- 由RNA引导的CRISPR-Cas12效应器赋予了 prokaryotes 中的适应性免疫力.
- Cas12m2是一种紧的V型效应器,具有独特的RuvC活性部位,但缺乏dsDNA裂解能力.
- 它的RNA导向基因组失活和对移动遗传元素 (MGE) 的保护机制以前是未知的.
研究的目的:
- 通过Cas12m2效应器阐明RNA引导基因组失活的分子机制.
- 确定Cas12m2强大的DNA结合亲和力及其在适应性免疫中的作用的结构基础.
- 了解Cas12m2及其假定祖先TnpB之间的进化关系.
主要方法:
- 使用冷电子显微镜 (cryo-EM) 确定了Cas12m2复合物的结构.
- 解决了Cas12m2-crRNA二进制复合物的结构和Cas12m2-crRNA-目标DNA三进制复合物的两个状态.
- 在Cas12m2和TnpB之间进行了比较结构分析.
主要成果:
- 低温EM结构揭示了crRNA-目标DNA异重复形成的动态过程.
- Cas12m2 具有强烈的DNA结合,这是由于与富含氨酸的集群及其非正规的RuvC活性位点的相互作用造成的.
- 在Cas12m2中的REC2插入在结合原体空间邻的基因-远距离区域中发挥着关键作用,这对适应性免疫至关重要.
结论:
- Cas12m2采用一种独特的转录沉默机制,通过强大的DNA结合来实现适应性免疫.
- 结构洞察力澄清了Cas12m2非正规活性位点的功能及其与目标DNA的相互作用.
- 这些发现有助于理解V型CRISPR-Cas系统以及从TnpB到Cas12酶的演变.
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