1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation

  1. Hayun Lee
  2. Yukti Dhingra
  3. Dipali G Sashital  Is a corresponding author
  1. Iowa State University, United States
https://doi.org/10.7554/eLife.44248
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  1. Hayun Lee
  2. Yukti Dhingra
  3. Dipali G Sashital
(2019)
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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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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The following previously published data sets were used

Article and author information

Author details

  1. Hayun Lee

    Roy J Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. Yukti Dhingra

    Roy J Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Dipali G Sashital

    Roy J Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, United States
    For correspondence
    sashital@iastate.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7681-6987

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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