1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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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
Research Article
  • Cited 3
  • Views 1,358
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Cite this article as: eLife 2019;8:e44248 doi: 10.7554/eLife.44248

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.

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.

Reviewing Editor

  1. Ailong Ke, Cornell University

Publication history

  1. Received: December 9, 2018
  2. Accepted: April 19, 2019
  3. Accepted Manuscript published: April 25, 2019 (version 1)
  4. Accepted Manuscript updated: April 30, 2019 (version 2)
  5. Version of Record published: May 15, 2019 (version 3)

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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