Fig. 4: BbYY1 contributes to precise TIR ends (TIREs) by aiding the recognition of appropriate TIRs.
a Illustration of the LM-PCR method for detecting cleavage sites of TIR substrate after bbRAG1L/2L-mediated recombination. Right TIRE happened when cleavages occurred at the borders of both 5′ and 3′ TIRs, while short TIRE happened when cleavages occurred between the 5′ and 3′ TIRs. P5, P6, and P7 indicate specific PCR primers. The adapter and these primers are listed in Supplementary Table 1. b, c The upper gels show that fewer and shorter TIREs occurred after TIR-dependent recombination in 293TshYY1 cells. sTIRE, short TIR ends. The lower gels show the expression of the normalized control gene (Kanamycin resistance gene, indicated as Kan) on pTIR104. LM-PCRs were performed using rTaq DNA polymerase with 30 cycles (b) or KOD plus neo DNA polymerase with 45 cycles (c). The representative LM-PCR gels represent three independent duplications. For more details regarding the PCR procedures, see the Methods section. d Alignment of 5′ TIREs after TIR-dependent cleavage in 293TshYY1 cells showed a broad range of imprecise cleavage, indicating the formation of group 1 imprecise HDJs in 293TshYY1 cells. e Schematic diagram illustration of different TIR signal recognition mediated by bbRAG1L/2L in vivo. The recognition and cleavage occurring between sites 1 and 2 or between sites 1 and 3 would lead to GFP or mCherry expression, respectively. f Percentage of GFP- and mCherry-positive cells after bbRAG1L/2L-mediated recombination in 293T and 293TshYY1 cells using the pSel G-mCh construct as e indicated. The values represent 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. See also Supplementary Fig. 4E. g The DNA pulldown assay showed that greater abundances of bbRAG1L/2 L and bbYY1 were pulled down by the WT 5′ TIR (bio-5′ TIR) but not the TR5-mutated 5′ TIR (bio-5′ TIR-Mu) in the presence of increasing poly dA:dT. The blots represent three independent duplications. For b, c, f, and g, source data are provided as Source Data file.
