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. 2016 Dec 16;45(4):1983–1993. doi: 10.1093/nar/gkw1274

Figure 6.

Figure 6.

Functional analyses of Cmr-2α. (A) In vivo DNA interference activity of cmr-2α mutants carrying substitutions in the HD or Palm domain. pSe-Rp—a repeat cloning vector for Sulfolobus, pS10i—an invader plasmid carrying a target sequence of spacer 10 in CRISPR locus 2 in S. islandicus REY15A. Following Cmr2α mutants were used: HD-M1—a double mutation (H14N, D15N) in the HD domain; HD-M2—a quadruple HD mutation (H14N, D15N, K19A, I23A), Palm-M1—a double mutation (G666K, D667K) in the GGDD motif and Palm-M2—an octal PALM mutation (662—IYlGGDDiLA—671 to AAlAAAAiAS). (B) In vitro RNA/DNA cleavage by the Cmr-2HDM1 effector complex containing Cmr-2 HDM1 mutant protein. RNA cleavage assay was conducted with 25 nM labeled SS1 ssRNA, 50 nM Cmr–α complex of either the wild-type (WT) or the mutant complex (HD-M1) harboring Cmr2αH14N, D15N and incubated for 20 min. DNA cleavage was conducted with 25 nM labeled S32T ssDNA substrate, 200 nM SS1 RNA and 50 nM WT or HD-M1 Cmr–α and incubated for 1 h.