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Proc Natl Acad Sci U S A. 2017 Apr 24. pii: 201616395. doi: 10.1073/pnas.1616395114. [Epub ahead of print]

Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity.

Author information

1
Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717.
2
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037.
3
Department of Microbiology and Immunology, University of California, San Francisco, CA 94158.
4
Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717; bwiedenheft@gmail.com.

Abstract

The type I-F CRISPR adaptive immune system in Pseudomonas aeruginosa (PA14) consists of two CRISPR loci and six CRISPR-associated (cas) genes. Type I-F systems rely on a CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease (i.e., Cas2/3) for target degradation. In most type I systems, Cas2 and Cas3 are separate proteins involved in adaptation and interference, respectively. However, in I-F systems, these proteins are fused into a single polypeptide. Here we use biochemical and structural methods to show that two molecules of Cas2/3 assemble with four molecules of Cas1 (Cas2/32:Cas14) into a four-lobed propeller-shaped structure, where the two Cas2 domains form a central hub (twofold axis of symmetry) flanked by two Cas1 lobes and two Cas3 lobes. We show that the Cas1 subunits repress Cas2/3 nuclease activity and that foreign DNA recognition by the Csy complex activates Cas2/3, resulting in bidirectional degradation of DNA targets. Collectively, this work provides a structure of the Cas1-2/3 complex and explains how Cas1 and the target-bound Csy complex play opposing roles in the regulation of Cas2/3 nuclease activity.

KEYWORDS:

CRISPR; Cas; Cas1; Cas2/3; type I-F

PMID:
28438998
DOI:
10.1073/pnas.1616395114
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