Characterization of the endoribonucleolytic activity of the Pyrococcus furiosus CRISPR-associated CARF protein Csx1
Sheppard, Nolan Filip
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Prokaryotes rely on varied mechanisms to protect themselves from viruses and other mobile genetic elements, including restriction modification, abortive infection, toxin/antitoxin, and CRISPR-Cas systems. The CRISPR-associated protein Csx1 contains a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain and a highly conserved motif that is predicted to have ribonuclease activity. This motif is found in various ribonucleases associated with prokaryotic defense systems. Biochemical analysis of Csx1 revealed that it is a single strand-specific endoribonuclease that cleaves specifically after adenosines on the 3’ side of the phosphodiester bond. Furthermore, mutation of the highly conserved residues of the predicted active site significantly decreased the observed cleavage activity of Csx1.