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
Share this article
Cite this article
  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
  2. Download BibTeX
  3. Download .RIS

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.

The following data sets were generated
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.

Metrics

  • 6,531
    views
  • 666
    downloads
  • 63
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Share this article

https://doi.org/10.7554/eLife.44248

Categories and tags

Further reading

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

    A conformational fingerprint for amyloidogenic light chains

    Cristina Paissoni, Sarita Puri ... Carlo Camilloni
    Research Article

    Both immunoglobulin light-chain (LC) amyloidosis (AL) and multiple myeloma (MM) share the overproduction of a clonal LC. However, while LCs in MM remain soluble in circulation, AL LCs misfold into toxic-soluble species and amyloid fibrils that accumulate in organs, leading to distinct clinical manifestations. The significant sequence variability of LCs has hindered the understanding of the mechanisms driving LC aggregation. Nevertheless, emerging biochemical properties, including dimer stability, conformational dynamics, and proteolysis susceptibility, distinguish AL LCs from those in MM under native conditions. This study aimed to identify a2 conformational fingerprint distinguishing AL from MM LCs. Using small-angle X-ray scattering (SAXS) under native conditions, we analyzed four AL and two MM LCs. We observed that AL LCs exhibited a slightly larger radius of gyration and greater deviations from X-ray crystallography-determined or predicted structures, reflecting enhanced conformational dynamics. SAXS data, integrated with molecular dynamics simulations, revealed a conformational ensemble where LCs adopt multiple states, with variable and constant domains either bent or straight. AL LCs displayed a distinct, low-populated, straight conformation (termed H state), which maximized solvent accessibility at the interface between constant and variable domains. Hydrogen-deuterium exchange mass spectrometry experimentally validated this H state. These findings reconcile diverse experimental observations and provide a precise structural target for future drug design efforts.

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

    Electrostatics of salt-dependent reentrant phase behaviors highlights diverse roles of ATP in biomolecular condensates

    Yi-Hsuan Lin, Tae Hun Kim ... Hue Sun Chan
    Research Article

    Liquid-liquid phase separation (LLPS) involving intrinsically disordered protein regions (IDRs) is a major physical mechanism for biological membraneless compartmentalization. The multifaceted electrostatic effects in these biomolecular condensates are exemplified here by experimental and theoretical investigations of the different salt- and ATP-dependent LLPSs of an IDR of messenger RNA-regulating protein Caprin1 and its phosphorylated variant pY-Caprin1, exhibiting, for example, reentrant behaviors in some instances but not others. Experimental data are rationalized by physical modeling using analytical theory, molecular dynamics, and polymer field-theoretic simulations, indicating that interchain ion bridges enhance LLPS of polyelectrolytes such as Caprin1 and the high valency of ATP-magnesium is a significant factor for its colocalization with the condensed phases, as similar trends are observed for other IDRs. The electrostatic nature of these features complements ATP’s involvement in π-related interactions and as an amphiphilic hydrotrope, underscoring a general role of biomolecular condensates in modulating ion concentrations and its functional ramifications.

    1. Biochemistry and Chemical Biology
    2. Neuroscience

    Neurodegeneration: A role for RNA knots in Alzheimer’s disease

    Silvia Galli, Marco Di Antonio
    Insight

    The buildup of knot-like RNA structures in brain cells may be the key to understanding how uncontrolled protein aggregation drives Alzheimer’s disease.