Fischer H, Hinkle D C
J Biol Chem. 1980 Aug 25;255(16):7956-64.
Four T7 products, DNA polymerase, gene 4 protein, RNA polymerase, and DNA binding protein, have been purified from phage-infected cells. It has been previously shown (Hinkle, D. C., and Richardson, C. C. (1975) J. Biol. Chem. 250, 5523-5529; Kolodner, R., and Richardson, C. C. (1978) J. Biol. Chem. 253, 574-584) that two T7 products, DNA polymerase and gene 4 protein, catalyze extensive synthesis on duplex T7 DNA containing single strand breaks. However, the T7 DNA polymerase purified by our procedure does not efficiently contribute in this reaction, although the preliminary evidence suggests that this enzyme may be the native form of the DNA polymerase. Such inefficient T7 DNA synthesis is greatly augmented by adding the third T7 product, namely T7 RNA polymerase. This DNA synthesis apparently requires transcription, since each of the four rNTPs must be present. The rate of synthesis is increased about 2-fold by the addition of T7 DNA binding protein. In contrast to the results obtained when DNA synthesis is initiated at single strand breaks in a duplex DNA molecule, essentially none of the DNA synthesized in the presence of T7 RNA polymerase is covalently attached to the T7 DNA template. We postulate that in this in vitro system, T7 DNA replication is initiated using an RNA primer synthesized by the T7 RNA polymerase.
已从噬菌体感染的细胞中纯化出四种T7产物，即DNA聚合酶、基因4蛋白、RNA聚合酶和DNA结合蛋白。此前已有研究表明（欣克尔，D.C.，和理查森，C.C.（1975年）《生物化学杂志》250，5523 - 5529；科洛德纳，R.，和理查森，C.C.（1978年）《生物化学杂志》253，574 - 584），两种T7产物，DNA聚合酶和基因4蛋白，可在含有单链断裂的双链T7 DNA上催化大量合成。然而，通过我们的方法纯化的T7 DNA聚合酶在该反应中贡献不大，尽管初步证据表明这种酶可能是DNA聚合酶的天然形式。添加第三种T7产物，即T7 RNA聚合酶，可大大增强这种低效的T7 DNA合成。这种DNA合成显然需要转录，因为四种核糖核苷三磷酸都必须存在。添加T7 DNA结合蛋白后，合成速率提高约2倍。与在双链DNA分子的单链断裂处起始DNA合成时获得的结果相反，在T7 RNA聚合酶存在下合成的DNA基本上没有共价连接到T7 DNA模板上。我们推测，在这个体外系统中，T7 DNA复制是使用由T7 RNA聚合酶合成的RNA引物起始的。
