Fig. 3: The CARF domain of EiCsm6 catalyzes cA6 hydrolysis. | Nature Communications

Fig. 3: The CARF domain of EiCsm6 catalyzes cA6 hydrolysis.

From: Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

Fig. 3

a Zoom-in view of nucleotides A1 and A6 of cFA6 bound to the EiCsm6 CARF domain. Residues implicated in the cleavage are represented as sticks; hydrogen-bonding interactions are depicted as black dotted lines. The red arrows represent the proposed reaction mechanism of cA6 cleavage involving nucleophilic attack of the 2’-hydroxyl group of A6 onto the phosphodiester group connecting A6 and A1. b RNase activity assay comparing the activity of WT and T11A EiCsm6 proteins in the presence of 500 nM cA6. The black arrows represent spike-ins of 0.8 pmol of RNaseAlert substrate at 5 min intervals. AU, arbitrary units. c Growth curves (optical density at a wavelength of 600 nm) of Staphylococcus aureus strains harboring pTarget and pCRISPR plasmids. The pCRISPR plasmid expresses wild-type or CARF domain mutants of EiCsm6 and the Staphylococcus epidermidis Cas10–Csm effector complex containing an inactivating point mutation in the Csm3 subunit (D32A; dCsm3). Data points in b, c represent the mean of three replicates; error bars represent the standard error of the mean (s.e.m.). Source data are provided as a Source Data file.

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