Michael Daume, André Plagens, Lennart Randau
2014 vol: 9(8): e105716 doi: 10.1371/journal.pone.0105716
RISPR-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.
Topics: Sequence motif analysis, DNA-binding proteins, Electrophoretic mobility shift assay, Genetic interference, Multiple alignment calculation, Nucleic acids, Phylogenetic analysis, Sequence alignment, Monolith–MicroScale Thermophoresis, MST, Proteins, Publications