The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation
- Iowa State University, United States
Abstract
CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4.
Data availability
The negative-stain EM volumes for the Cas1-Cas2-target, asymmetrical Cas4-Cas1-Cas2-target, symmetrical Cas4-Cas1-Cas2-target, asymmetrical Cas4-Cas1-Cas2-prespacer and symmetrical Cas4-Cas1-Cas2-prespacer complexes have been deposited to EMDB under the accession numbers EMDB-20127, EMDB-20128, EMDB-20129, EMDB-20130 and EMDB-20131, respectively.
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Bacillus halodurans Cas1-Cas2-targetThe Electron Microscopy Data Bank, EMDB-20127.
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Bacillus halodurans Cas4-Cas1-Cas2-target asymmetricalThe Electron Microscopy Data Bank, EMDB-20128.
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Bacillus halodurans Cas4-Cas1-Cas2-target symmetricalThe Electron Microscopy Data Bank, EMDB-20129.
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Bacillus halodurans Cas4-Cas1-Cas2-prespacer asymmetricalThe Electron Microscopy Data Bank, EMDB-20130.
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Bacillus halodurans Cas4-Cas1-Cas2-prespacer symmetricalThe Electron Microscopy Data Bank, EMDB-20131.
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Crystal structure of the Cas1-Cas2 complex from Escherichia coliProtein Data Bank, 4P6I.
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E. far Cas1-Cas2/prespacer binary complexProtein Data Bank, 5XVN.
Article and author information
Author details
Funding
National Institute of General Medical Sciences (GM115874)
- Dipali G Sashital
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Lee et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation
eLife 8:e44248.
https://doi.org/10.7554/eLife.44248
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