TY - JOUR TI - The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation AU - Lee, Hayun AU - Dhingra, Yukti AU - Sashital, Dipali G A2 - Ke, Ailong A2 - Wolberger, Cynthia A2 - White, Malcolm F VL - 8 PY - 2019 DA - 2019/04/25 SP - e44248 C1 - eLife 2019;8:e44248 DO - 10.7554/eLife.44248 UR - https://doi.org/10.7554/eLife.44248 AB - 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. KW - B. halodurans KW - CRISPR-Cas KW - electron microscopy KW - DNA-protein interaction KW - PAM KW - nuclease JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -