title: DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans.
abstract: Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive γ irradiation. We have determined the crystal structures of four ISDra2 transposase/IS end complexes; combined with in vivo activity assays and fluorescence anisotropy binding measurements, these have revealed the molecular basis of strand discrimination and transposase action. The structures also show that previously established structural rules of target site recognition that allow different specific sequences to be targeted are only partially conserved among family members. Furthermore, we have captured a fully assembled active site including the scissile phosphate bound by a divalent metal ion cofactor (Cd²(+)) that supports DNA cleavage. Finally, the observed active site rearrangements when the transposase binds a metal ion in which it is inactive provide a clear rationale for metal ion specificity.
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Alison Burgess
authors.initials: AB
authors.lastname: Hickman
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: James
authors.forename: Orsolya
authors.initials: O
authors.lastname: Barabas
authors.forename: Cécile
authors.initials: C
authors.lastname: Pasternak
authors.forename: Bao
authors.initials: B
authors.lastname: Ton-Hoang
authors.forename: Michael
authors.initials: M
authors.lastname: Chandler
authors.forename: Suzanne
authors.initials: S
authors.lastname: Sommer
authors.forename: Fred
authors.initials: F
authors.lastname: Dyda
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D034301:Deinococcus / Q000737:chemistry / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D019895:Transposases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D019895:Transposases; D015032:Zinc