Gene A protein of bacteriophage phi X174 is a highly specific single-strand nuclease and binds via a tyrosyl residue to DNA after cleavage.

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 phi X RF DNA replication and single-stranded (ss) DNA fragments of phi X 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 phi X 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 phi X 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 phi X 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.