In type I CRISPR-Cas system, Cas3-a nuclease cum helicase-in cooperation with Cascade surveillance complex cleaves the target DNA. Unlike the Cascade/I-E, which is composed of five subunits, the Cascade/I-C is made of only three subunits lacking the CRISPR RNA processing enzyme Cas6, whose role is assumed by Cas5. How these differences in the composition and organization of Cascade subunits in type I-C influence the Cas3/I-C binding and its target cleavage mechanism is poorly understood. Here, we show that Cas3/I-C is intrinsically a single-strand specific promiscuous nuclease. Apart from the helicase domain, a constellation of highly conserved residues-which are unique to type I-C-located in the uncharacterized C-terminal domain appears to influence the nuclease activity. Recruited by Cascade/I-C, the HD nuclease of Cas3/I-C nicks the single-stranded region of the non-target strand and positions the helicase motor. Powered by ATP, the helicase motor reels in the target DNA, until it encounters the roadblock en route, which stimulates the HD nuclease. Remarkably, we show that Cas3/I-C supplants Cas3/I-E for CRISPR interference in type I-E in vivo, suggesting that the target cleavage mechanism is evolutionarily conserved between type I-C and type I-E despite the architectural difference exhibited by Cascade/I-C and Cascade/I-E.

中文翻译：

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CRISPR干扰期间，Cas3 / IC介导的目标DNA识别和切割与Cascade监视复合体的组成和架构无关。

在I型CRISPR-Cas系统中，Cas3-a核酸酶和解旋酶与Cascade监测复合物一起切割目标DNA。与由五个亚基组成的Cascade / IE不同，Cascade / IC仅由三个缺少CRISPR RNA处理酶Cas6的亚基组成，Cas5承担了该作用。对IC类型的级联亚基的组成和组织的这些差异如何影响Cas3 / IC结合及其靶标裂解机制的了解很少。在这里，我们显示Cas3 / IC本质上是单链特异性混杂核酸酶。除解旋酶结构域外，位于未表征的C末端结构域的IC类型所特有的高度保守的残基群似乎会影响核酸酶的活性。由Cascade / IC招募，Cas3 / IC的HD核酸酶切割非目标链的单链区域，并定位解旋酶马达。在由ATP提供动力的情况下，解旋酶马达释放目标DNA，直到它在途中遇到阻碍HD核酸酶的路障。值得注意的是，我们显示Cas3 / IC在IE型活体内取代了CRISPR干扰的Cas3 / IE，这表明尽管Cascade / IC和Cascade / IE在结构上存在差异，但目标裂解机制在IC型和IE型之间是进化保守的。