Sanhueza S, Eisenberg S
J Virol. 1985 Feb;53(2):695-7. doi: 10.1128/JVI.53.2.695-697.1985.
The phi X174 A protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated end. To determine the nature of the covalent linkage and the amino acid involved, we used the A protein to cleave DNA synthesized in vitro with [alpha-32P]dATP to form the complex of A protein covalently linked to single-stranded DNA. The complex was then digested with DNase I, and the 32P-labeled A protein was isolated by electrophoresis on polyacrylamide gels. The isolated complex was treated extensively with trypsin, and the released peptide-oligonucleotide complexes were incubated with formic acid and diphenylamine (Burton reaction). The Burton reaction caused a transfer of the labeled phosphate from dAMP to the peptide. The labeled phosphopeptides were isolated and hydrolyzed, revealing a linkage of the phosphate to a tyrosine. These results indicate that the A protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated terminus by a tyrosyl-dAMP phosphodiester bond.
噬菌体φX174 A蛋白可切割单链DNA，并与5'-磷酸化末端共价结合。为了确定共价连接的性质以及所涉及的氨基酸，我们使用A蛋白切割用[α-32P]dATP体外合成的DNA，以形成与单链DNA共价连接的A蛋白复合物。然后用DNase I消化该复合物，并通过聚丙烯酰胺凝胶电泳分离出32P标记的A蛋白。将分离出的复合物用胰蛋白酶进行充分处理，释放出的肽-寡核苷酸复合物与甲酸和二苯胺一起孵育（伯顿反应）。伯顿反应导致标记的磷酸基团从dAMP转移到肽上。分离并水解标记的磷酸肽，结果显示磷酸基团与酪氨酸相连。这些结果表明，A蛋白通过酪氨酰-dAMP磷酸二酯键切割单链DNA并与5'-磷酸化末端共价结合。
