Bedinger P, Alberts B M
J Biol Chem. 1983 Aug 25;258(16):9649-56.
The bacteriophage T4 DNA polymerase has an intrinsic 3'-5' proofreading exonuclease activity that plays a central role in determining the fidelity of T4 DNA replication. In order to monitor this activity, we have measured the rate at which the polymerase decreases the size of a double-stranded DNA substrate in the absence of deoxyribonucleoside triphosphates. With this assay, we find that the addition of the polymerase accessory proteins, 45 protein and 44/62 protein, increases the rate at which the polymerase-associated exonuclease digests the DNA substrate 3- to 4-fold. This stimulation requires the continuous hydrolysis of ATP catalyzed by the accessory protein complex. When added alone, the T4 helix-destabilizing protein, 32 protein, inhibits the exonuclease rate at high concentrations (greater than 100 micrograms/ml), while stimulating about 3-fold at low concentrations. The 32 protein and the accessory proteins together increase the exonuclease rate 8- to 10-fold above that found for the polymerase alone. The bacteriophage T7 DNA polymerase displays a similar 3'-5' exonuclease activity, but this exonuclease is not stimulated by any of the T4 replication proteins. It therefore appears that specific protein-protein interactions are involved.
噬菌体T4 DNA聚合酶具有内在的3'-5'校对核酸外切酶活性，这在决定T4 DNA复制的保真度方面起着核心作用。为了监测这种活性，我们测量了在没有脱氧核糖核苷三磷酸的情况下，聚合酶使双链DNA底物大小减小的速率。通过这种测定方法，我们发现添加聚合酶辅助蛋白45蛋白和44/62蛋白后，与聚合酶相关的核酸外切酶消化DNA底物的速率提高了3至4倍。这种刺激需要辅助蛋白复合物催化ATP的持续水解。单独添加时，T4解旋不稳定蛋白32蛋白在高浓度（大于100微克/毫升）时会抑制核酸外切酶的速率，而在低浓度时会刺激约3倍。32蛋白和辅助蛋白一起使核酸外切酶的速率比单独的聚合酶提高了8至10倍。噬菌体T7 DNA聚合酶表现出类似的3'-5'核酸外切酶活性，但这种核酸外切酶不受任何T4复制蛋白的刺激。因此，似乎涉及特定的蛋白质-蛋白质相互作用。
