Supplementary Figure 4: A comparison of RNase H insertion domains among different transposases and RNase H catalytic mutants are inactive.

a, Architectures of insertion domains found in other DNA transposases. The RNase H domains (grey) of other structurally characterized DNA transposases (or the transposase-related RAG1 protein) were aligned by their respective catalytic residues (indicated in red) and ordered by increasing insertion domain size (blue). Insertion domain sizes (indicated below) were determined by approximate start and end insertion positions. The PDB numbers from which these structures were derived are in parentheses. b, Structural alignment of the P element transposase insertion domain, the Hermes insertion domain (1DWY) and the RAG1–RAG2 insertion domain (6CIK) reveals structural similarities at the fold level. c, Bar graph of relative in vivo P element excision activity of alanine-substituted catalytic mutants (D230, D303 and E531). Cell-based excision assays were performed as previously described (Rio, D. C. et al., Cell. 44, 21–32, 1986)(Beall, E. L. et al., Genes Dev. 10, 921–933, 1996). Single alanine mutants were generated by site-directed mutagenesis of pPBSKS (+) pAc-TNP and verified by sequencing over the entire coding sequence. The assay was conducted in triplicate (n = 3). Error bars indicate standard deviations. (WT, wild type). d, Representative immunoblot of wild type transposase and catalytic mutant protein expression levels. Cells were harvested 24hr after transfection and lysates were normalized to cell number. Membrane was cut and then immunoblotted with anti-transposase antibodies (α-TNP) or a loading control (α-HRP48).