title: All three residues of the Tn 10 transposase DDE catalytic triad function in divalent metal ion binding.
abstract: Tn 10/IS 10 transposition involves excision of the transposon from a donor site and subsequent joining of the excised transposon to a new target site. These steps are catalyzed by the Tn 10 -encoded transposase protein and require the presence of a suitable divalent metal ion. Like other transposase and retroviral integrase proteins, Tn 10 transposase appears to contain a single active site which includes a triad of acidic amino acid residues generally referred to as the DDE motif. In addition to its role in catalysis, the Tn 10 transposase DDE motif also functions in target capture, a step that in vitro is greatly facilitated by the presence of a suitable divalent metal ion. We show that cysteine residue substitutions at each of the DDE motif residues in Tn 10 transposase result in a change in the divalent metal ion requirements for catalysis, such that Mn2+but not Mg2+can be used. This switch in metal ion specificity provides evidence that each of the DDE motif residues functions directly in metal ion binding. We also show differential effects of DDE mutations on metal ion-assisted target capture. A number of models, including a two metal ion active site, are considered to explain these effects.
authors.affiliation: Department of Biochemistry, University of Western Ontario, London, Ontario, N6A 5C1, Canada.
authors.forename: J S
authors.initials: JS
authors.lastname: Allingham
authors.forename: P A
authors.initials: PA
authors.lastname: Pribil
authors.forename: D B
authors.initials: DB
authors.lastname: Haniford
mesh_terms: D002118:Calcium / Q000378:metabolism*; D020134:Catalytic Domain; D002413:Cations, Divalent; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D016296:Mutagenesis; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D008345:Manganese; D019895:Transposases; D008274:Magnesium; D002118:Calcium