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Step-arrest mutants of phage Mu transposase. Implications in DNA-protein assembly, Mu end cleavage, and strand
K Kim1, S Y Namgoong, M Jayaram
1Department of Microbiology, University of Texas at Austin 78712.
Abstract:
We describe the isolation and characterization of Mu A variants arrested at specific steps of transposition. Mutations at 13 residues within the Mu A protein were analyzed for precise excision of Mu DNA in vivo. A subset of the defective variants (altered at Asp269, Asp294, Gly348, and Glu392) were tested in specific steps of transposition in vitro. It is possible that at least some residues of the Asp269-Asp294-Glu392 triad may have functional similarities to those of the conserved Asp-Asp-Glu motif found in several transposases and retroviral integrases. Mu A(D269V) is defective in high-order DNA-protein assembly, Mu end cleavage, and strand transfer. The assembly defect, but not the catalytic defect, can be overcome by precleavage of Mu ends. Mu A(E392A) can assemble the synaptic complex, but cannot cleave Mu ends. A mutation of Gly348 to aspartic acid within Mu A permits the uncoupling of cleavage and strand transfer activities. This mutant is completely defective in synaptic assembly and Mu end cleavage in presence of Mg2+. The assembly defect is alleviated by replacing Mg2+ with Ca2+. Some Mu end cleavage is observed with this mutant in the presence of Mn2+. When presented with precleaved Mu ends, Mu A(G348D) exhibits efficient strand transfer activity.
Insights
Investigating Mu A variants reveals key residues essential for DNA transposition. Specific mutations disrupt DNA-protein assembly, Mu end cleavage, and strand transfer, offering insights into transposase function.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Bacterial insertion sequences like bacteriophage Mu are crucial genetic elements.
- The Mu A protein is essential for the initiation and regulation of Mu DNA transposition.
- Understanding Mu A's mechanism provides insights into related DNA recombination systems.
Purpose of the Study:
- To characterize Mu A variants with mutations at specific residues.
- To elucidate the roles of individual amino acids in Mu DNA transposition steps.
- To investigate potential functional similarities with other catalytic triads in DNA processing enzymes.
Main Methods:
- Isolation and characterization of Mu A protein variants.
- In vivo analysis of Mu DNA precise excision.
- In vitro assays to assess DNA-protein assembly, DNA cleavage, and strand transfer activities.
- Site-directed mutagenesis to create specific Mu A variants.
Main Results:
- Mutations at Asp269, Asp294, Gly348, and Glu392 significantly impair transposition.
- Mu A(D269V) shows defects in assembly, cleavage, and strand transfer, with assembly defects overcome by precleaved DNA.
- Mu A(E392A) can form synaptic complexes but fails to cleave Mu ends.
- Mu A(G348D) uncouples cleavage and strand transfer, with assembly/cleavage dependent on divalent cations (Mg2+, Ca2+, Mn2+).
Conclusions:
- Specific residues, including those in the D269-D294-E392 triad, are critical for distinct Mu transposition steps.
- The study highlights the complex interplay between DNA-protein assembly, cleavage, and strand transfer.
- Mu A's catalytic residues may share functional similarities with conserved motifs in other transposases and integrases.