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TitleThe Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation.
Journal, issue, pagesElife, Vol. 8, Year 2019
Publish dateApr 30, 2019
AuthorsHayun Lee / Yukti Dhingra / Dipali G Sashital /
PubMed AbstractCRISPR 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 ...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.
External linksElife / PubMed:31021314 / PubMed Central
MethodsEM (single particle)
Resolution19.7 - 24.4 Å
Structure data

EMDB-20127:
Bacillus halodurans Cas1-Cas2 complex in presence of target CRISPR DNA
Method: EM (single particle) / Resolution: 22.1 Å

EMDB-20128:
Bacillus halodurans Cas4-Cas1-Cas2 asymmetrical complex in presence of target CRISPR DNA
Method: EM (single particle) / Resolution: 21.4 Å

EMDB-20129:
Bacillus halodurans Cas4-Cas1-Cas2 symmetrical complex in presence of target CRISPR DNA
Method: EM (single particle) / Resolution: 19.7 Å

EMDB-20130:
Bacillus halodurans Cas4-Cas1-Cas2 asymmetrical complex in presence of prespacer DNA
Method: EM (single particle) / Resolution: 24.4 Å

EMDB-20131:
Bacillus halodurans Cas4-Cas1-Cas2 symmetrical complex in presence of prespacer DNA
Method: EM (single particle) / Resolution: 21.6 Å

Source
  • Bacillus halodurans C-125 (bacteria)
  • unidentified (others)

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