Abstract
Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MATα). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons.
Highlights
Hobo from Drosophila[11], Ac from maize[12] and Tam[3] from the snap dragon[13] are the founding members of the hAT-family of DNA transposons, which seems to be restricted to eukaryotes
It was shown that Hermes excise through the generation of DNA double stranded breaks (DSBs) that are hairpin-capped on the flanking DNA18
We found that several amino acids conserved among hAT-transposases were essential for Kat[1] activity both in vitro and in vivo
Summary
Hobo from Drosophila[11], Ac from maize[12] and Tam[3] from the snap dragon[13] are the founding members of the hAT-family of DNA transposons, which seems to be restricted to eukaryotes This family has several distinguishing features, including 8-bp target site duplications and a carboxyl terminal ~50 amino acid domain called the hAT-domain (IPR008906)[14]. It was shown that Hermes excise through the generation of DNA double stranded breaks (DSBs) that are hairpin-capped on the flanking DNA18. Kat[1] displays functional similarities with hAT-transposases, most notably generation of hairpin-capped DSBs. the amino acid similarity between Kat[1] and hAT-transposases is limited. We found that several amino acids conserved among hAT-transposases were essential for Kat[1] activity both in vitro and in vivo. Gas-phase electrophoretic macromolecule analysis (GEMMA) suggests that Kat[1] can form a hexamer
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