Fig. 2. Can1 and Can2 ancillary nucleases function as cA₄-activated nucleases on plasmid DNA, ssRNA, and ssDNA.

a Domain organization of Can1 and Can2 proteins. Can2 proteins have two domains – CARF and PD-(D/E)XK superfamily nuclease domain. Can1 is predicted to be derived from Can2 by gene duplication³². NLD – nuclease-like domain. b Maximum-likelihood phylogeny of 204 Can1 (CARF2 and PD-(D/E)XK nuclease domain) and 3121 Can2 proteins. The light gray numbers indicate bootstrap values. Previously studied effectors are marked with asterisks (*). Effectors chosen for purification and in vitro experiments are underlined. c–e Plasmid DNA (15 nM), ssRNA (425 nM), and ssDNA (15 nM) cleavage assay with TtCan1 (200 nM) in the presence of cA₄ (50 nM). The reactions were incubated 15 min at 60 °C. n – nicked, lin – linear, sc – supercoiled plasmid. f–h Cleavage assays with AaCan2 (200 nM) in the presence of cA₄ (50 nM). Assays were performed with 15 nM plasmid DNA, 425 nM ssRNA or 15 nM ssDNA for 15 min at 60 °C. n – nicked, lin – linear, sc – supercoiled plasmid. Source data are provided as a Source Data file.