Eisenberg S, Kornberg A
J Biol Chem. 1979 Jun 25;254(12):5328-32.
Synthesis of phiX174 viral (+) strand circles in vitro requires gene A protein, rep protein, DNA binding protein, and DNA polymerase III holoenzyme (Eisenberg, S., Scott, J. F., and Kornberg, A., (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3151-3155). We have used this reaction as an assay to isolate gene A protein in greater than 90% purity. Its molecular weight under denaturing conditions is 59,000. The protein tends to aggregate and lose activity at low ionic strength. Tritium-labeled gene A protein cleaves the phiX174 duplex replicative form and is bound to it in a 1:1 ratio as part of an active replication complex. The attachment, at the 5' phosphoryl end of the cleavage point, is apparently covalent. The complex was not dissociated by: (i) banding in CsCl, (ii) treatment with 0.2 M NaOH, or (iii) boiling in 1% sodium dodecyl sulfate and electrophoresis on a sodium dodecyl sulfate-acrylamide gel; only micrococcal nuclease digestion of the DNA released the protein.
在体外合成φX174病毒（+）链环需要基因A蛋白、复制蛋白、DNA结合蛋白和DNA聚合酶III全酶（艾森伯格，S.，斯科特，J.F.，和科恩伯格，A.，（1976年）美国国家科学院院刊73，3151 - 3155）。我们利用这个反应作为一种测定方法，以分离出纯度高于90%的基因A蛋白。在变性条件下其分子量为59,000。该蛋白在低离子强度下容易聚集并失去活性。氚标记的基因A蛋白切割φX174双链复制型，并以1:1的比例与之结合，作为活性复制复合体的一部分。在切割点的5'磷酸末端的附着显然是共价的。该复合体不会被以下操作解离：（i）在CsCl中密度梯度离心，（ii）用0.2 M NaOH处理，或（iii）在1%十二烷基硫酸钠中煮沸并在十二烷基硫酸钠 - 丙烯酰胺凝胶上进行电泳；只有微球菌核酸酶对DNA的消化才能释放该蛋白。
