Fig. 5: BbYY1 improves TTJ formation and suppresses TIR-dependent transposition.
a Schematic of the TTJ detection assay. Quantities of GFP-positive cells were used to indicate TTJ formation by transfecting a pair of reversed TIRs containing substrate pTIRG8-ivt with bbRAG1L/2L expression vectors into 293T or 293TshYY1 cells. b The flow cytometry assay showed that bbYY1 promoted TTJ formation based on an analysis of the quantification of GFP-positive cells in 293T and 293TshYY1 cells. See also Supplementary Fig. 4F. c Schematic diagram of intermolecular transposition. A donor plasmid (pTet-dTIR) containing the tetracycline resistance gene (Tet) flanked by the full-length TIR (TIR-FL) pairs and a target plasmid (pEGFP-N1) containing the kanamycin resistance gene were co-transfected with bbRAG1L/2L expression vectors into 293T or 293TshYY1 cells. When bbRAG1L/2L-mediated DNA cleavage and transposition occurred, the Tet was removed from the donor plasmid (pTet-dTIR) and transposed into the target plasmid pEGFP-N1. The transposition efficiency could be quantified by survival rates of colonies on Kanamycin-Tetracycline-Streptomycin (KTS) resistance plates after bacterial transformation. Streptomycin (str) resistance was reduced by the rpsL gene in donor plasmid. TS, target site; TSD, target site duplication; Tet, tetracycline resistance gene. d Ex vivo intermolecular transposition assays showed that bbYY1 could suppress transposon efficiency according to the quantification analysis of transposition frequency of indicated plasmids in 293T and 293TshYY1 cells. For more details regarding intermolecular transposition, also see the Methods section. e TSD types of intermolecular transposition products occurred in 293T and 293TshYY1 cells. f The distribution of transposition sites generated by plasmid-to-plasmid transposition mediated by bbRAG1L/2L. The values shown in b and d are the means ± s.d., with n = 3 biologically independent experiments. A two-tailed, unpaired Student’s t test was used for comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant. For b and d–f, source data are provided as Source Data file.
