THAP9 transposase cleaves DNA via conserved acidic residues in an RNaseH-like domain

The catalytic domain of most cut and paste DNA transposases have the canonical RNase-H fold which is also shared by other polynucleotidyl transferases like retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp - DDE/D) that are involved in metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons. In this study we have modelled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity, led to the identification of candidate Ds and Es in the RNaseH fold that appear to constitute the catalytic triad of hTHAP9. Significance statementMany DNA transposases execute DNA excision via a catalytic domain, which has a canonical RNase-H fold. Despite the similar nature of the catalytic domain, these transposases exhibit mechanistically different strategies of transposition. We identify a potential RNase-H fold in hTHAP9 with a conserved DDE motif required for cutting DNA. Additionally, we have found a residue, which when mutated, leads to an increase in hTHAP9s transposition activity. Such hyperactive transposase mutants can be exploited as tools in genome engineering and gene therapy. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterized transposon-derived gene in the human genome.