Unknown

Dataset Information

0

Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex.


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.

SUBMITTER: Dillard KE 

PROVIDER: S-EPMC6441324 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

altmetric image

Publications


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  ...[more]

Similar Datasets

| S-EPMC5059440 | biostudies-literature
| S-EPMC4075942 | biostudies-literature
| S-EPMC4117759 | biostudies-literature
| S-EPMC4359072 | biostudies-literature
| S-EPMC5930301 | biostudies-literature
| S-EPMC6867608 | biostudies-literature
| S-EPMC4005679 | biostudies-literature
| S-EPMC4678826 | biostudies-literature
| S-EPMC6607411 | biostudies-literature
| S-EPMC7515739 | biostudies-literature