Cas4 核酸酶定义了 CRISPR 位点整合的 DNA 片段的 PAM、长度和方向。
Cas4 Nucleases Define the PAM, Length, and Orientation of DNA Fragments Integrated at CRISPR Loci.
机构信息
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
Department of Genetics and Genome Sciences, Institute for Systems Genomics, UConn Stem Cell Institute, UConn Health, Farmington, CT 06030, USA.
出版信息
Mol Cell. 2018 Jun 7;70(5):814-824.e6. doi: 10.1016/j.molcel.2018.05.002.
To achieve adaptive and heritable immunity against viruses and other mobile genetic elements, CRISPR-Cas systems must capture and store short DNA fragments (spacers) from these foreign elements into host genomic CRISPR arrays. This process is catalyzed by conserved Cas1/Cas2 integration complexes, but the specific roles of another highly conserved protein linked to spacer acquisition, the Cas4 nuclease, are just now emerging. Here, we show that two Cas4 nucleases (Cas4-1 and Cas4-2) play critical roles in CRISPR spacer acquisition in Pyrococcus furiosus. The nuclease activities of both Cas4 proteins are required to process protospacers to the correct size. Cas4-1 specifies the upstream PAM (protospacer adjacent motif), while Cas4-2 specifies the conserved downstream motif. Both Cas4 proteins ensure CRISPR spacer integration in a defined orientation leading to CRISPR immunity. Collectively, these findings provide in vivo evidence for critical roles of Cas4 nucleases in protospacer generation and functional spacer integration at CRISPR arrays.
为了实现对病毒和其他移动遗传元件的适应性和遗传性免疫，CRISPR-Cas 系统必须将这些外源元件的短 DNA 片段（间隔物）捕获并存储到宿主基因组的 CRISPR 阵列中。这个过程是由保守的 Cas1/Cas2 整合复合物催化的，但与间隔物获取相关的另一种高度保守的蛋白质 Cas4 核酸酶的具体作用才刚刚显现出来。在这里，我们表明，两种 Cas4 核酸酶（Cas4-1 和 Cas4-2）在 Pyrococcus furiosus 的 CRISPR 间隔物获取中发挥关键作用。两种 Cas4 蛋白的核酸酶活性都需要将原间隔物加工成正确的大小。Cas4-1 指定上游 PAM（原间隔子相邻基序），而 Cas4-2 指定保守的下游基序。这两种 Cas4 蛋白都确保了 CRISPR 间隔子以确定的方向整合，从而实现 CRISPR 免疫。总的来说，这些发现为 Cas4 核酸酶在 CRISPR 阵列中原间隔子产生和功能间隔子整合中的关键作用提供了体内证据。
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