title: DNA binding properties of the small cascade subunit Csa5.
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.
authors.affiliation: Prokaryotic Small RNA Biology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Daume
authors.affiliation: Prokaryotic Small RNA Biology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Plagens
authors.affiliation: Prokaryotic Small RNA Biology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
authors.forename: Lennart
authors.initials: L
authors.lastname: Randau
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000502:physiology*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000502:physiology*; D019641:DNA, Archaeal / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D041542:Thermoproteus / Q000235:genetics / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004277:DNA, Single-Stranded