Prelich G, Tan C K, Kostura M, Mathews M B, So A G, Downey K M, Stillman B
Nature. 1987;326(6112):517-20. doi: 10.1038/326517a0.
The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.
猿猴病毒40（SV40）基因组的复制机制与细胞染色体的复制机制极为相似，因此为研究DNA复制所需的酶提供了一个极佳的模型系统。病毒复制仅需要一种病毒基因产物，即大肿瘤抗原（大T抗原），所以大多数复制酶必定是细胞来源的。实际上，感染SV40后会诱导许多与复制及细胞周期S期相关的酶活性。用人细胞制备的无细胞提取物，在补充免疫纯化的SV40大T抗原后，能支持含有SV40 DNA复制起点的质粒高效复制。利用该系统，已纯化并鉴定出一种相对分子质量为36,000（Mr = 36K）的细胞蛋白，它是体外SV40 DNA复制延伸阶段所必需的，该蛋白是一种已知的细胞周期调控蛋白，也被称为增殖细胞核抗原（PCNA）或细胞周期蛋白。人们注意到，PCNA在物理特性上与一种调节小牛胸腺DNA聚合酶δ活性的蛋白极为相似。在此我们表明，PCNA与聚合酶δ辅助蛋白具有相似的电泳行为，且都能被抗PCNA人自身抗体识别。更重要的是，这两种蛋白在功能上是等效的；它们在体外刺激SV40 DNA复制，并提高小牛胸腺DNA聚合酶δ的持续合成能力。这些结果表明，一种新型动物细胞DNA聚合酶，即DNA聚合酶δ，参与了体外复制性DNA合成的延伸阶段。
