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Tn552 transposase purification and in vitro activities

S J Rowland1, D J Sherratt, W M Stark

  • 1Genetics Laboratory, University of Glasgow, UK.

The EMBO Journal
|January 3, 1995
PubMed
Summary

The Staphylococcus aureus transposon Tn552 transposase (p480) binds to specific DNA sites and mediates strand transfer, crucial for its transposition mechanism. This study elucidates key in vitro activities of p480, supporting its role in Tn552 mobility.

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Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • The Staphylococcus aureus transposon Tn552 encodes a protein, p480.
  • p480 shares the 'D,D(35)E' motif with retroviral integrases and other bacterial transposases.
  • This motif suggests a conserved catalytic mechanism for DNA transposition.

Purpose of the Study:

  • To characterize the biochemical activities of the p480 protein.
  • To investigate the DNA binding and strand transfer capabilities of p480 in vitro.
  • To understand the molecular basis of Tn552 transposition.

Main Methods:

  • Overexpression and purification of p480 and its histidine-tagged derivative in Escherichia coli.
  • DNase I footprinting and gel binding assays to study DNA-protein interactions.
  • In vitro strand transfer assays using transposon end fragments and target DNA.

Main Results:

  • p480 binds to two adjacent, directly repeated 23 bp motifs at the ends of Tn552.
  • In vitro conditions were established for p480 strand transfer activity with pre-cleaved 3' termini.
  • Strand transfer is Mn(2+)-dependent and joins transposon ends to target DNA.
  • The terminal 3'-CA dinucleotide is essential for activity.

Conclusions:

  • The in vitro biochemical activities of p480 are consistent with its function as the Tn552 transposase.
  • p480 plays a direct role in the integration of Tn552 into target DNA.
  • This research provides insights into the mechanism of bacterial transposon mobility.