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Molecular and General Genetics MGG

, Volume 237, Issue 1–2, pp 206–214 | Cite as

Purification and DNA binding of the D protein, a putative resolvase of the F-factor of Escherichia coli

  • Claudia Disqué-Kochem
  • Rudolf Eichenlaub
Article

Summary

The D protein encoded by plasmid mini-F promotes resolution of plasmid cointegrates or dimers of the F-factor or mini-F. In addition, two rfsF sequences are essential for this site-specific, recA-independent recombination event. The D gene was cloned into an expression vector and the gene product was overproduced in Escherichia coli and purified to homogeneity. The sequence of the N-terminus of the D protein was determined, thus permitting identification of the correct translational start codon in the nucleotide sequence that results in a 29.6 kDa protein. The binding site for the purified D protein is located within the mini-F NcoIHpaI DNA fragment (192 bp). Binding seems to be affected by DNA methylation, since the protein did not bind to DNA isolated from a dam mutant of E. coli. The binding site, which is a region of approximately 28 bp and is located 160 by downstream of the rfsF site, was identified by DNase I footprinting using fluorescence labelled DNA.

Key words

Mini-F Site-specific recombination Resolvase Dam methylase 

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Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Claudia Disqué-Kochem
    • 1
  • Rudolf Eichenlaub
    • 1
  1. 1.Fakultät für Biologie, Lehrstuhl für Gentechnologie/MikrobiologieUniversität BielefeldBielefeld 1Germany

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