Protein engineering of bacteriophage Mu transposase

Bacteriophage Mu is an ideal system to study DNA transposition. The 70-KDa protein product of the phage early gene A, termed transposase, is absolutely required for transposition. Transposase binds specifically at sites located at both ends of the phage genome, termed attL and attR, and at an enhancer-like element at the left end of the genome, called IAS (internal activation sequence). It then nicks at these ends, and nicks a random target DNA sequence in a 5 base pair staggered fashion with 5$ sp prime$ extensions and promotes strand transfer between the Mu ends and the target DNA. The transposase protein can be roughly divided into three domains. The other activities of the protein have not been mapped even at the domain level. To further define the different functional domains of this complex enzyme, a series of insertion mutants at 8 different sites along the transposase protein were constructed using TAB linker mutagenesis. In this study, 1 and 2 TAB linkers were inserted into 8 HpaII sites in the Mu A gene, generating a set of 2 and 4 amino acid insertion mutants. Examination of these mutants for specific DNA-binding activity of transposase to the ends of the phage genome in vitro revealed temperature sensitive proteins. Transpositional activity of the mutant proteins revealed that the mutant proteins, which are temperature sensitive in specific DNA-binding activity, are deficient in transpositional activity