Mutational analysis of the att DNA-binding domain of phage Mu transposase.
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 approximately 135 aa region within domain I beta gamma that specifies binding to Mu att sites. This peptide was overexpressed and its properties compared with that of the larger domain I beta gamma as well as the intact Mu A protein. Extensive mutagenesis of residues around a putative helix-turn-helix DNA-binding motif within the I beta 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 gamma region are required for activity and that residues outside the beta gamma region must also influence discrimination between the multiple att sites.
D000595:Amino Acid Sequence; D001287:Attachment Sites, Microbiological; D010583:Bacteriophage mu / Q000201:enzymology*; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I / Q000378:metabolism; D017353:Gene Deletion; D019077:Helix-Turn-Helix Motifs; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics*; D011487:Protein Conformation; D019895:Transposases
D004269:DNA, Bacterial; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009713:Nucleotidyltransferases; D019895:Transposases; D003850:Deoxyribonuclease I