Identification of residues in the Mu transposase essential for catalysis.
A tetramer of Mu transposase (MuA) cleaves the phage Mu DNA and joins these ends to a target DNA to catalyze transposition. Substitution mutations at Asp-269 or Glu-392 within MuA destroy both the DNA cleavage and joining activities without blocking tetramer assembly, indicating that the mutations specifically affect catalysis. Although inactive under standard reaction conditions (10 mM Mg2+), the mutant proteins are partially resuscitated by 10-20 mM Mn2+, concentrations 5- to 10-fold higher than optimal for wild-type MuA. Amino acid sequence alignment and the similar effects of mutations suggests that Asp-269 and Glu-392 of MuA may be analogs of the first Asp and final Glu of a conserved triad of acidic amino acids present in many transposases and the retroviral integrases (the D-D-35-E motif). The higher Mn2+ optima observed with MuA derivatives altered at these positions supports a role for the conserved acidic amino acids in coordinating divalent metal ions in the active sites of transposases.
Baker
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Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Luo
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D000595:Amino Acid Sequence; D001224:Aspartic Acid; D010583:Bacteriophage mu / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005971:Glutamates; D018698:Glutamic Acid; D019426:Integrases; D046911:Macromolecular Substances; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019895:Transposases
D004268:DNA-Binding Proteins; D005971:Glutamates; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D008345:Manganese; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D009713:Nucleotidyltransferases; D019895:Transposases