DEFINING FUNCTIONAL REGIONS OF THE IS903 TRANSPOSASE

The insertion sequence IS903 encodes a 307 amino acid residue protein, transposase, that is essential for transposition It is a multi-functi onal DNA-binding protein that specifically recognizes the 18 bp invert ed repeats at the ends of the element and also recognizes DNA non-spec ifically when it captures a target site. In addition, transposase perf orms catalytic functions when it mediates the cleavage and religation steps of transposition. We have carried out deletion and mutational an alyses to define functional domains of the transposase protein. The de letion studies delineate a 99 residue region of the protein (residues 31 to 129) that specifies binding to the inverted repeat. A slightly l arger maltose-binding protein-transposase fusion that includes residue s 22 to 139 (Tnp 22-139) binds as efficiently and with the same specif icity as the full-length transposase protein. Tnp 22-139 also induces a DNA bend similar to that of the wild-type protein, and so we conclud e that all binding and bending specificity is contained within the N-t erminal domain of the protein. Unlike full-length transposase,Tnp 22-1 39 forms additional higher-order complexes in band-shift gels suggesti ng that the deletion has exposed a surface(s) capable of participating in protein-protein interactions. Six highly conserved residues in the C-terminal portion of the protein were mutated to alanine. Each mutan t protein was binding-proficient but defective in transposition. The p henotype of these substitutions, and their alignment with residues sho wn to abolish catalysis of other transposases and integrases, suggest that these are residues responsible for catalytic steps in transpositi on of IS903; we believe three of these residues comprise the DDE motif , conserved in transposases and integrases. Our data are consistent wi th IS903 transposase being composed of two domains: an N-terminal doma in primarily involved in DNA binding and a C-terminal domain tl-lat is involved in catalysis. (C) 1997 Academic Press Limited.