Biochemical analysis of the Cas6-1 RNA endonuclease associated with the subtype I-D CRISPR-Cas system in Synechocystis sp. PCC 6803

摘要: Specialized RNA endonucleases are critical for efficient activity of the CRISPR-Cas defense mechanisms against invading DNA or RNA. Cas6-type enzymes in many type I and III systems. These diverse residues involved recognition cleavage substrates not universally conserved. Cas6 associated with subtypes I-A, I-B, I-C, I-E I-F, as well III-B have been studied from three archaea four bacteria thus far. However, until now information about subtype I-D specific has remained scarce. Here, we report biochemical analysis Cas6-1, which is crRNA maturation system Synechocystis sp. PCC 6803. Assays turnover kinetics suggest a single mechanism Cas6-1. The mutation conserved amino acids R29A, H32A-S33A H51A revealed these essential, whereas parallel R175A-R176A led to pronounced K155A slight reduction enzymatic activity. In contrast, mutations R67A, R81A K231A left unchanged. results accordance predominant role histidine active site positively charged binding. Nevertheless, protein-RNA interaction seems differ other known systems, since imidazole could restore mutated site.