title: A bend, flip and trap mechanism for transposon integration.
abstract: Cut-and-paste DNA transposons of the mariner/Tc1 family are useful tools for genome engineering and are inserted specifically at TA target sites. A crystal structure of the mariner transposase Mos1 (derived from Drosophila mauritiana), in complex with transposon ends covalently joined to target DNA, portrays the transposition machinery after DNA integration. It reveals severe distortion of target DNA and flipping of the target adenines into extra-helical positions. Fluorescence experiments confirm dynamic base flipping in solution. Transposase residues W159, R186, F187 and K190 stabilise the target DNA distortions and are required for efficient transposon integration and transposition in vitro. Transposase recognises the flipped target adenines via base-specific interactions with backbone atoms, offering a molecular basis for TA target sequence selection. Our results will provide a template for re-designing mariner/Tc1 transposases with modified target specificities.
authors.affiliation: Institute of Quantitative Biology, Biochemistry and Biotechnology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Elizabeth R
authors.identifier: 0000-0003-1893-7515
authors.initials: ER
authors.lastname: Morris
authors.affiliation: Institute of Quantitative Biology, Biochemistry and Biotechnology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Heather
authors.initials: H
authors.lastname: Grey
authors.affiliation: EaStCHEM School of Chemistry, Edinburgh, United Kingdom.
authors.forename: Grant
authors.initials: G
authors.lastname: McKenzie
authors.affiliation: EaStCHEM School of Chemistry, Edinburgh, United Kingdom.
authors.forename: Anita C
authors.initials: AC
authors.lastname: Jones
authors.affiliation: Institute of Quantitative Biology, Biochemistry and Biotechnology, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Julia M
authors.identifier: 0000-0002-1547-3009
authors.initials: JM
authors.lastname: Richardson
mesh_terms: D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D011995:Recombination, Genetic; D019895:Transposases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; C111652:mariner transposases; D004247:DNA; D019895:Transposases
keywords: DNA integration; E. coli; X-ray crystallography; base analogues; base flipping; biophysics; structural biology; time-resolved fluorescence; transposition