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Single-stranded DNA intermediates in IS91 rolling-circle transposition
M del Pilar Garcillán-Barcia1, I Bernales, M V Mendiola
1Departamento de Biología Molecular (Unidad Asociada al CIB, CSIC), Universidad de Cantabria, C/Herrera Oria s/n, 39011 Santander, Spain.
Molecular Microbiology
|January 3, 2001
Summary
The study reveals that specific tyrosine residues in IS91 transposase are crucial for its novel rolling-circle (RC) transposition mechanism. Mutations in these tyrosines prevent the formation of key single-stranded DNA (ssDNA) intermediates, providing biochemical evidence for RC transposition in insertion sequences.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Insertion sequence (IS) elements are mobile genetic elements with diverse transposition mechanisms.
- IS91 exhibits unique characteristics suggesting a novel rolling-circle (RC) transposition pathway.
- The IS91 transposase (TnpA) shares conserved domains with RC-replicating phage A proteins, including catalytic tyrosines.
Purpose of the Study:
- To investigate the role of invariant tyrosines (Tyr-249 and Tyr-253) in IS91 transposase (TnpA) function.
- To determine if TnpA acts as a functional analog of RC replicating phage A proteins.
- To provide biochemical evidence for the proposed RC transposition mechanism of IS91.
Main Methods:
- Site-directed mutagenesis of IS91 TnpA to create Tyr-249 and Tyr-253 mutants (phenylalanine or serine).
- In vivo transposition assays using a kanamycin resistance cassette flanked by IS91 terminal sequences.
- Analysis of DNA intermediates (ssDNA and dsDNA circles) formed during transposition using mutant and wild-type transposases.
Main Results:
- Mutation of either Tyr-249 or Tyr-253 completely abolished IS91 transposition activity in vivo.
- Induction of wild-type TnpA expression led to the formation of ssDNA and dsDNA intermediates containing the transposable cassette.
- Mutant TnpA proteins failed to produce these ssDNA and dsDNA intermediates, indicating the essentiality of Tyr-249 and Tyr-253 for their formation.
Conclusions:
- The invariant tyrosines Tyr-249 and Tyr-253 are essential for IS91 transposition.
- TnpA functions as a functional analog of RC replicating phage A proteins.
- The in vivo formation of ssDNA intermediates provides the first biochemical evidence supporting a rolling-circle transposition mechanism for IS91.