Extended Data Fig. 3: Specificity and efficiency of transposon DNA excision by TnpA.
From: Transposon-encoded nucleases use guide RNAs to promote their selfish spread
a, Schematic of heterologous E. coli transposon excision assay. Plasmids encode TnpA and a mini-transposon (Mini-Tn) substrate, whose loss is monitored by PCR using the indicated primers. The expected sizes of PCR products generated from donor joints that are produced upon religation of flanking sequences are shown, for both ISGst2 and H. pylori IS608. b, TnpA homologs do not cross-react with distinct IS elements, as assessed by analytical PCR. Cell lysates were tested after overnight expression of TnpA in combination with a mini-Tn substrate from either G. stearothermophilus (G) or H. pylori (H), and PCR products were resolved by agarose gel electrophoresis. M refers to catalytically inactive mutants. Note that HpyTnpA is substantially more active for DNA excision than GstTnpA under the tested conditions. U, unexcised; E, excised. c, Schematic of qPCR assay to quantify excision frequencies, in which one of the two primers anneals directly to the donor joint formed upon mini-Tn excision and religation. d, Comparison of simulated excision frequencies, generated by mixing clonally excised and unexcised lysate in known ratios, versus experimentally determined integration efficiencies measured by qPCR. e, qPCR-based quantification of TnpA-mediated excision of an ISGst2 mini-Tn substrate in E. coli. Excised refers to a cloned excision product; TnpAM denotes a TnpA mutant (Y125A); ND, not detected above a 0.0001% threshold. Bars indicate mean ± standard deviation (n = 3). f, Schematic of mini-Tn for ISGst3 element, highlighting the subterminal palindromic transposon ends located on the top strand (top). Transposon-adjacent and transposon-encoded motifs (TAM and TEM) are shown in yellow and orange, respectively; DNA guides are shown in orange, and their putative base-pairing interactions are indicated; dotted lines indicate transposon boundaries and thus the sites of ssDNA cleavage and religation. Sanger sequencing of excision events confirms the identity of the expected donor joint product formed upon transposon loss (bottom). Sanger sequencing results are duplicated from Fig. 2d. g, Schematic and Sanger sequencing data as in f, but for a modified ISGst3 substrate containing TEM mutations. Experimentally detected products erroneously excise at an alternative, aberrant TEM-like sequence located outside of the native transposon boundary (orange), presumably because of the need to maintain cognate base-pairing between the DNA guide (orange and TEM (blue).
