title: Biochemical characterization and comparison of two closely related active mariner transposases.
abstract: Most DNA transposons move from one genomic location to another by a cut-and-paste mechanism and are useful tools for genomic manipulations. Short inverted repeat (IR) DNA sequences marking each end of the transposon are recognized by a DNA transposase (encoded by the transposon itself). This enzyme cleaves the transposon ends and integrates them at a new genomic location. We report here a comparison of the biophysical and biochemical properties of two closely related and active mariner/Tc1 family DNA transposases: Mboumar-9 and Mos1. We compared the in vitro cleavage activities of the enzymes on their own IR sequences, as well as cross-recognition of their inverted repeat sequences. We found that, like Mos1, untagged recombinant Mboumar-9 transposase is a dimer and forms a stable complex with inverted repeat DNA in the presence of Mg(2+) ions. Mboumar-9 transposase cleaves its inverted repeat DNA in the manner observed for Mos1 transposase. There was minimal cross-recognition of IR sequences between Mos1 and Mboumar-9 transposases, despite these enzymes having 68% identical amino acid sequences. Transposases sharing common biophysical and biochemical properties, but retaining recognition specificity toward their own IR, are a promising platform for the design of chimeric transposases with predicted and improved sequence recognition.
authors.affiliation: School of Biological Sciences, University of Edinburgh , The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, United Kingdom.
authors.forename: Maryia
authors.initials: M
authors.lastname: Trubitsyna
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Morris
authors.forename: David J
authors.initials: DJ
authors.lastname: Finnegan
authors.forename: Julia M
authors.initials: JM
authors.lastname: Richardson
mesh_terms: D002413:Cations, Divalent; D004247:DNA / Q000737:chemistry; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000737:chemistry*; D055029:Inverted Repeat Sequences; D008274:Magnesium / Q000737:chemistry; D010957:Plasmids; D055503:Protein Multimerization; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions; D013696:Temperature; D019895:Transposases / Q000737:chemistry*
chemical_list: D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D012996:Solutions; C111652:mariner transposases; D004247:DNA; D019895:Transposases; D008274:Magnesium