Kavli Institute of Nanoscience and Department of Bionanoscience, Delft University of Technology, Delft 2629HZ, the Netherlands.
Kavli Institute of Nanoscience and Department of Bionanoscience, Delft University of Technology, Delft 2629HZ, the Netherlands; Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Wageningen 6708WE, the Netherlands.
Mol Cell. 2018 May 3;70(3):385-394.e3. doi: 10.1016/j.molcel.2018.03.031. Epub 2018 Apr 26.
CRISPR-Cas provides RNA-guided adaptive immunity against invading genetic elements. Interference in type I systems relies on the RNA-guided Cascade complex for target DNA recognition and the Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here, we employed single-molecule Förster resonance energy transfer (FRET) to probe the helicase activity with high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling the DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of 3 bp, which underlie three successive 1-nt unwinding events. Reeling is highly repetitive, allowing Cas3 to repeatedly present its inefficient nuclease domain with single-strand DNA (ssDNA) substrate. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure controlled degradation of target DNA only.
CRISPR-Cas 提供了针对入侵遗传元件的 RNA 指导适应性免疫。I 型系统的干扰依赖于 RNA 指导的级联复合物来识别靶 DNA，以及 Cas3 解旋酶/核酸酶蛋白来靶标降解。尽管 CRISPR 干扰的生物化学已经得到了广泛的研究，但 Cas3 的解旋和核酸酶结构域的解旋和偶联的生物物理学仍然难以捉摸。在这里，我们采用单分子Förster 共振能量转移（FRET）技术以高时空分辨率探测解旋酶活性。我们表明，Cas3 在使用弹簧加载机制回收 DNA 的同时，仍然与靶标结合的级联复合物紧密相关。这种弹簧加载的回收以 3 bp 的明显爆发发生，这是三个连续的 1-nt 解旋事件的基础。回收是高度重复的，允许 Cas3 用单链 DNA（ssDNA）底物反复呈现其低效的核酸酶结构域。我们的研究表明，Cas3 的不连续解旋酶特性及其与级联复合物的紧密相互作用仅确保靶标 DNA 的受控降解。
