Allingham J S, Pribil P A, Haniford D B
Department of Biochemistry, University of Western Ontario, London, Ontario, N6A 5C1, Canada.
J Mol Biol. 1999 Jun 25;289(5):1195-206. doi: 10.1006/jmbi.1999.2837.
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.
Tn10/IS10转座涉及转座子从供体位点的切除以及随后切除的转座子与新靶位点的连接。这些步骤由Tn10编码的转座酶蛋白催化，并且需要合适的二价金属离子存在。与其他转座酶和逆转录病毒整合酶蛋白一样，Tn10转座酶似乎含有一个单一的活性位点，该位点包括通常称为DDE基序的酸性氨基酸残基三联体。除了其催化作用外，Tn10转座酶DDE基序在靶标捕获中也起作用，在体外，合适的二价金属离子的存在极大地促进了这一步骤。我们表明，Tn10转座酶中DDE基序残基处的半胱氨酸残基取代导致催化所需的二价金属离子发生变化，使得可以使用Mn2+而不是Mg2+。金属离子特异性的这种转变提供了证据，表明DDE基序的每个残基都直接参与金属离子结合。我们还展示了DDE突变对金属离子辅助靶标捕获的不同影响。包括双金属离子活性位点在内的许多模型被用来解释这些影响。
