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DNA-binding activity and subunit interaction of the mariner transposase
L Zhang1, A Dawson, D J Finnegan
1Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, UK.
Nucleic Acids Research
|August 28, 2001
Summary
Mos1 transposase interactions are crucial for its function in transposition. Mutations affecting these interactions alter DNA target specificity, offering new insights into mariner/Tc1 transposon mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Mos1 is a mariner/Tc1 family transposable element from Drosophila mauritiana.
- It utilizes a cut-and-paste transposition mechanism, inserting at TA dinucleotides, and encodes a transposase essential for this process.
- Transposase-transposase interactions in mariner/Tc1 elements remain incompletely understood.
Purpose of the Study:
- To investigate the DNA-binding properties of Mos1 transposase.
- To elucidate the role of transposase-transposase interactions in Mos1 transposition.
- To characterize a mutant transposase with altered target specificity.
Main Methods:
- Purification of Mos1 transposase.
- DNA-binding assays using terminal inverted repeats.
- Yeast two-hybrid assays to study protein-protein interactions.
- In vitro transposition assays with wild-type and mutant transposases.
Main Results:
- Mos1 transposase binds to Mos1 terminal inverted repeats via an N-terminal DNA-binding domain (residues 1-120), including a helix-turn-helix motif (residues 88-108).
- Binding and cleavage are preferential to the right terminal repeat.
- Wild-type transposase monomers interact in yeast two-hybrid assays, suggesting a role for protein-protein interactions.
- A mutant transposase with reduced interaction capability shows impaired excision and transposition, indicating the necessity of transposase-transposase interactions for synaptic complex formation and coordinate end cleavage.
- This mutant transposase exhibits altered target specificity, inserting at non-TA dinucleotides, including GC sequences.
Conclusions:
- Transposase-transposase interactions are essential for forming a synaptic complex required for efficient Mos1 transposition.
- These interactions facilitate coordinate cleavage at both ends of the transposon.
- A novel mariner/Tc1 transposase mutant demonstrates altered target specificity, expanding our understanding of transposon insertion mechanisms.