1. Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex.
- Author
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Dillard KE, Brown MW, Johnson NV, Xiao Y, Dolan A, Hernandez E, Dahlhauser SD, Kim Y, Myler LR, Anslyn EV, Ke A, and Finkelstein IJ
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins metabolism, CRISPR-Associated Proteins chemistry, DNA, Viral metabolism, Protein Multimerization, Single Molecule Imaging, Actinomycetales enzymology, CRISPR-Associated Proteins metabolism, CRISPR-Cas Systems
- Abstract
CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In type I CRISPR-Cas systems, efficient "primed" spacer acquisition and viral degradation (interference) require both the Cascade complex and the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
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