Pritchard C G, DePamphilis M L
J Biol Chem. 1983 Aug 25;258(16):9801-9.
Essentially all of the DNA polymerase alpha activity in CV-1 monkey cells could be extracted as an enzyme complex that used DNA substrates with a low primer:template ratio, such as denatured DNA, at least 25 times more efficiently than did purified alpha polymerase. This form of the enzyme was rapidly dissociated either by the nonionic detergent Triton X-100 or by chromatography on phosphocellulose to generate alpha polymerase and its protein cofactor complex, C1C2. Both alpha polymerase and C1C2 were then independently purified free of deoxyribonuclease, RNA polymerase, DNA ligase, and ATPase activities, and the C1C2 complex was shown to consist of at least two proteins. Purified C1C2, which exhibited no DNA polymerase activity, completely restored the ability of alpha polymerase to use denatured DNA. Although high concentrations of denatured DNA inhibited the activity of C1C2, which binds tightly to single-stranded but not double-stranded DNA, low concentrations catalyzed reconstitution of alpha polymerase with C1C2. The resulting enzyme complex was chromatographically distinct from alpha polymerase on DEAE-Bio-Gel, was no longer dependent upon addition of C1C2 in order to utilize denatured DNA as effectively as DNase I-activated DNA, and was not inhibited by high concentrations of denatured DNA. These properties of the purified reconstituted enzyme were indistinguishable from those native alpha X C1C2-polymerase.
基本上，CV - 1猴细胞中所有的DNA聚合酶α活性都可以作为一种酶复合物被提取出来，该复合物使用低引物:模板比的DNA底物，如变性DNA，其效率至少是纯化的α聚合酶的25倍。这种酶形式可通过非离子去污剂 Triton X - 100或通过磷酸纤维素柱层析快速解离，以产生α聚合酶及其蛋白质辅因子复合物C1C2。然后，α聚合酶和C1C2都被独立纯化，去除脱氧核糖核酸酶、RNA聚合酶、DNA连接酶和ATP酶活性，并且C1C2复合物被证明至少由两种蛋白质组成。纯化的C1C2没有DNA聚合酶活性，但能完全恢复α聚合酶使用变性DNA的能力。虽然高浓度的变性DNA会抑制C1C2的活性（C1C2紧密结合单链DNA而非双链DNA），但低浓度时能催化α聚合酶与C1C2的重组。所得的酶复合物在DEAE - Bio - Gel上的色谱行为与α聚合酶不同，不再依赖添加C1C2就能像利用DNase I激活的DNA一样有效地利用变性DNA，并且不受高浓度变性DNA的抑制。纯化重组酶的这些特性与天然的αX C1C2 - 聚合酶无法区分。
