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Identification of residues in the Mu transposase essential for catalysis
Abstract:
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.
Insights
Mutations in Mu transposase (MuA) affect DNA cleavage and joining. These MuA variants are reactivated by manganese ions (Mn2+), suggesting conserved acidic residues coordinate metal ions in transposase active sites.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Mu transposase (MuA) catalyzes DNA transposition by cleaving and joining DNA ends.
- Specific mutations in MuA at Asp-269 or Glu-392 abolish catalytic activity but not tetramer assembly.
Purpose of the Study:
- To investigate the role of Asp-269 and Glu-392 in MuA catalysis.
- To explore the metal ion dependency of wild-type and mutant MuA.
Main Methods:
- Site-directed mutagenesis of MuA at Asp-269 and Glu-392.
- In vitro assays to assess DNA cleavage and joining activities.
- Enzyme kinetics studies with varying magnesium (Mg2+) and manganese (Mn2+) concentrations.
Main Results:
- Mutant MuA proteins (D269A, E392A) were catalytically inactive in the presence of Mg2+.
- Mutant MuA showed partial activity with Mn2+ at concentrations higher than optimal for wild-type MuA.
- Sequence analysis suggested Asp-269 and Glu-392 are analogs of the conserved D-D-35-E motif.
Conclusions:
- Asp-269 and Glu-392 are critical for MuA catalytic activity.
- The conserved acidic residues in MuA likely coordinate divalent metal ions in the active site.
- MuA's metal ion coordination mechanism may be conserved across transposases and integrases.