TN552 TRANSPOSASE PURIFICATION AND IN-VITRO ACTIVITIES

The Staphylococcus aureus transposon Tn552 encodes a protein (p480) co ntaining the 'D,D(35)E' motif common to retroviral integrases and the transposases of a number of bacterial elements, including phage Mu, th e integron-containing element Tn5090, Tn7 and IS3. p480 and a histidin e-tagged derivative were overexpressed in Escherichia coli and purifie d by methods involving denaturation and renaturation. DNase I footprin ting and gel binding assays demonstrated that p480 binds to two adjace nt, directly repeated 23 bp moths at each end of Tn552. Although donor strand cleavage by p480 was not detected, in vitro conditions were de fined for strand transfer activity with transposon end fragments havin g pre-cleaved 3' termini. Strand transfer was Mn2+-dependent and appea red to join a single left or right end fragment to target DNA. The imp ortance of the terminal dinucleotide CA-3' was demonstrated by mutatio n. The in vitro activities of p480 are consistent with its proposed fu nction as the Tn552 transposase.