Van Mansfeld A D, Baas P D, Jansz H S
Adv Exp Med Biol. 1984;179:221-30. doi: 10.1007/978-1-4684-8730-5_23.
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.
利用含有φX RF DNA复制起点序列（部分）的合成寡核苷酸以及φX和G4的单链（ss）DNA片段，研究了基因A蛋白和A蛋白内切核酸酶活性的序列特异性。通过对被切割序列的比较，推导了基因A蛋白切割ssDNA的共有序列。该共有序列存在于φX和G4的ssDNA的起点以及另一个位点。这很令人惊讶，因为滚环机制要求基因A蛋白仅在起点处切割。然而，可以证明，在单链结合蛋白（SSB蛋白）存在的情况下，φX和G4的ssDNA仅在起点处被切割，这可能是由于一个强基因A蛋白结合位点，即关键序列，它是φX及相关噬菌体30 bp起点区域的一部分。基因A蛋白和A蛋白在切割位点的5'-端与DNA共价结合。使用一种独特的、内部用32P标记的寡核苷酸作为底物，结果表明基因A蛋白和A*蛋白通过一个酪氨酰残基与被切割的磷酸二酯键的5'-磷酸基团结合。
