Fig. 2: IS607 TnpBs are RNA-guided DNA endonucleases.
From: Discovery and structural mechanism of DNA endonucleases guided by RAGATH-18-derived RNAs
a Mapping of small RNA sequencing data to the IS607 TnpB non-coding region in Firmicutes bacterium. b Scheme of the biochemical assay used to discover the TAM position and identity. c Weblogo of the identified ISFba1 TAM sequence (left panel); in vitro cleavage assay of ISFba1 TnpB using linearized dsDNA substrates (right panel). The agarose gel was visualized by ethidium bromide (EB) staining. TAM, Target adjacent motif; 2.7 kb, dsDNA substrate; 1.7 kb and 1 kb, cleavage product. Data shown are representative of three independent experiments. d Quantification of the in vitro cleavage assays mediated by ISFba1 TnpB using linearized dsDNA substrates with different TAM sequences. Data represent means ± SD of three biological replicates. e Run-off Sanger sequencing for the ISFba1 TnpB-cleaved plasmid. NTS, the non-targeted strand; TS, the target strand. The major and minor cleavage sites are indicated by red and green triangles. f The proportion of the non-target strand (left panel) and target strand (right panel) cleavage sites quantified by the high-throughput sequencing of the ISFba1 TnpB-cleaved results. Data represent means ± SD of three biological replicates. g In vitro cleavage assay of the wild-type ISFba1 TnpB (WT) and RuvC active site mutated ISFba1 TnpB (D194A, E290A, D371A) using linearized dsDNA substrates. The agarose gel was visualized by EB staining. Data shown are representative of three independent experiments.
