Functional characterization of arginine 30, lysine 40, and arginine 62 in Tn5 transposase

Three N-terminal basic residues of Tn5 transposase, which are associated wi th proteolytic cleavages by Escherichia coil proteinases, were mutated to g lutamine residues with the goal of producing more stable transposase molecu les. Mutation of either arginine 30 or arginine 62 to glutamine produced tr ansposase molecules that were more stable toward E. coil proteinases than t he parent hyperactive Tn5 transposase, however, they were inactive in vivo. In vitro analysis revealed these mutants were inactive, because both Arg(3 0) and Arg(62) are required for formation of the paired ends complexes when the transposon is attached to the donor backbone. These results suggest Ar g(30) and Arg(62) play critical roles in DNA binding and/or synaptic comple x formation. Mutation of lysine 40 to glutamine did not increase the overal l stability of the transposase to E. coil proteinases. This mutant transpos ase was only about 1% as active as the parent hyperactive transposase in vi vo; however, it retained nearly full activity in vitro, These results sugge st that lysine 40 is important for a step in the transposition mechanism th at is bypassed in the in vitro assay system, such as the removal of the tra nsposase molecule from DNA following strand transfer.