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Structure and mechanism of the CRISPR associated complex for antiviral defense (CASCADE)
Author(s) -
Lawrence C. Martin,
Lintner Nathanael G.,
Kerou Melina,
White Malcolm F.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.941.1
Subject(s) - trans activating crrna , crispr , rna , biology , dna , genetics , ribonuclease , computational biology , microbiology and biotechnology , cas9 , gene
The CRISPR/Cas prokaryotic adaptive immune system archives fragments of virus‐derived DNA within a hosts CRISPR loci. The CRISPR loci are then transcribed, processed, and loaded into 2 distinct complexes, CMR and CASCADE, that respectively target destruction of complementary invading RNA and DNA in subsequent infections. We report structural and functional characterization of the evolutionarily conserved components of CASCADE. Cas7 (aka Csa2), the dominant CASCADE protein, co‐purifies with Cas6‐processed CRISPR‐RNA (crRNA) and smaller amounts of Cas5a. TEM studies of the nucleoprotein complex reveal that Cas7 assembles along crRNA with right handed helical symmetry. The crystal structure of Cas7 unexpectedly revealed a decorated RNA‐recognition motif (RRM) and two additional domains present as insertions in the RRM. Biochemical and ribonuclease protection assays, mutational analysis, particle symmetry and modeling studies collectively indicate that the crRNA is bound nonspecifically along the inside surface of this central Cas7 helix, which is capped at the 5′ and 3′ ends by Cas5a and Cas6, respectively. Importantly, we identify similarities between CASCADE and the RecA/DNA complex that strongly suggest mechanistic similarities between CASCADE mediated recognition of invading DNA by crRNA, and RecA catalyzed strand invasion and D‐loop formation during DNA double strand break repair. Supported by the National Science Foundation (MCB‐0920312).