title: Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching.
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.
authors.affiliation: Department of Molecular Biosciences, the Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden.
authors.forename: Kishore K
authors.initials: KK
authors.lastname: Chiruvella
authors.affiliation: Department of Molecular Biosciences, the Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden.
authors.forename: Naghmeh
authors.initials: N
authors.lastname: Rajaei
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
authors.forename: Venkateswara Rao
authors.initials: VR
authors.lastname: Jonna
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Hofer
authors.affiliation: Department of Molecular Biosciences, the Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden.
authors.forename: Stefan U
authors.initials: SU
authors.lastname: Åström
mesh_terms: D020816:Amino Acid Motifs; D020134:Catalytic Domain; D017124:Conserved Sequence; D053903:DNA Breaks, Double-Stranded; D053837:DNA Cleavage; D004251:DNA Transposable Elements; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D049770:Genes, Mating Type, Fungal; D055029:Inverted Repeat Sequences; D007716:Kluyveromyces / Q000201:enzymology / Q000235:genetics*; D017354:Point Mutation; D055503:Protein Multimerization; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D019895:Transposases / Q000235:genetics* / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D004271:DNA, Fungal; D005656:Fungal Proteins; D019895:Transposases