Abstract
CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.
Highlights
IntroductionCRISPR RNAs (crRNAs) are the key elements of a prokaryotic immune system in defending against invading mobile genetic elements termed CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated genes) [1]
CRISPR RNAs are the key elements of a prokaryotic immune system in defending against invading mobile genetic elements termed CRISPR-Cas (CRISPR-associated genes) [1]
By analyzing the genomic context of the archaeal subtype I-A CRISPR-Cas systems, Csa5 could be identified in every cascade operon, which indicates a conserved function of this subunit within the interference complex
Summary
CRISPR RNAs (crRNAs) are the key elements of a prokaryotic immune system in defending against invading mobile genetic elements termed CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated genes) [1]. This CRISPR-Cas system is the only adaptive immune system in prokaryotes known so far. A protein complex containing Cas and 2 binds the invading nucleic acid, e.g. phage DNA, and recognizes a sequence motif consisting of few nucleotides, named the protospacer-adjacent motif (or PAM-motif) [3,4,5,6]. The associated Cas protein cleaves the targeted sequence, leading to its degradation [13,14,16,17,18,19]
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