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Gene A Protein of Bacteriophage ΦX174 is a Highly Specific Single-Strand Nuclease and Binds Via a Tyrosyl Residue to DNA After Cleavage

  • A. D. M. Van Mansfeld
  • P. D. Baas
  • H. S. Jansz
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 179)

Abstract

The sequence specificity of the endonuclease activity of gene A protein and A* protein was studied using synthetic oligonucleotides containing (part of) the sequence of the origin of ΦX RF DNA replication and single-stranded (ss) DNA fragments of ΦX and G4. From a comparison of the sequences that are cleaved a consensus sequence for cleavage of ssDNA by gene A protein has been deduced. This consensus sequence occurs in ssDNA of both ΦX and G4 at the origin and at one additional site. This is surprising since the rolling circle mechanism demands that gene A protein cleaves at the origin only. However, it could be shown that in the presence of SSB protein the ssDNAs of ΦX and G4 are only cleaved at the origin, which is probably due to a strong gene A protein binding site, the key sequence, which forms part of the 30 b. p. origin region of ΦX and related bacteriophages.

Gene A protein and A* protein bind covalently to the DNA at the 5’-end of the cleavage site. Using a uniquely, internally 32P- labelled oligonucleotide as a substrate, it was shown that gene A protein and A* protein are bound via a tyrosyl residue to the 5’-phosphate of the phosphodiester bond which is cleaved.

Keywords

Protein Binding Site Synthetic Oligonucleotide Phosphodiester Bond Origin Region Rolling Circle 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media New York 1984

Authors and Affiliations

  • A. D. M. Van Mansfeld
    • 1
  • P. D. Baas
    • 1
  • H. S. Jansz
    • 1
  1. 1.Institute of Molecular Biology and Laboratory for Physiological ChemistryState University of UtrechtUtrechtThe Netherlands

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