Nucleic Acids Research, 1995, Vol. 23, No. 19 3937-3943
© 1995
MOLECULAR BIOLOGY |
Mutational analysis of the att DNA-binding domain of phage Mu transposase
Department of Microbiology, University of Texas at Austin Austin, TX 78712, USA
*To whom correspondence should be addressed
Received June 16, 1995. Revised August 31, 1995. Accepted August 31, 1995.
The transposase (A protein) of phage Mu encodes binding to two families of DNA sites, att sites located at the Mu ends and enhancer sites located internally. Separate subdomains in the N-terminal domain I of Mu A protein are known to be involved in recognition of the att and enhancer sites. We have delineated an {small tilde}135 aa region within domain 1ß
that specifies binding to Mu att sites. This peptide was overexpressed and its properties compared with that of the larger domain 1ß as well as the intact Mu A protein. Extensive muta genesis of residues around a putative helix-turn-helix DNA-binding motif within the 1ßß domain identified several mutants defective in DNA transposition in vivo. Of these, Mu A(K157Q) was completely defective in att DNA-binding. Mu A(F131S) and Mu A(R146N) had a lower affinity for att DNA and low levels of transposition in vitro. Our results indicate that residues in the region are required for activity and that residues outside the ß region must also influence discrimination between the multiple att sites.