Melendy T, Stillman B
Cold Spring Harbor Laboratory, New York 11724.
J Biol Chem. 1991 Jan 25;266(3):1942-9.
DNA replication from the SV40 origin can be reconstituted in vitro using purified SV40 large T antigen, cellular topoisomerases I and II, replication factor A (RF-A), proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), and a phosphocellulose fraction (IIA) made from human cell extracts (S100). Fraction IIA contains all DNA polymerase activity required for replication in vitro in addition to other factors. A newly identified factor has been purified from fraction IIA. This factor is required for complete reconstitution of SV40 DNA replication and co-purifies with a PCNA-stimulated DNA polymerase activity. This DNA polymerase activity is sensitive to aphidicolin, but is not inhibited by butylanilinodeoxyadenosine triphosphate or by monoclonal antibodies which block synthesis by DNA polymerase alpha. The polymerase activity is synergistically stimulated by the combination of RF-A, PCNA, and RF-C in an ATP-dependent manner. Purified calf thymus polymerase delta can fully replace the purified factor in DNA replication assays. We conclude that this factor, required for reconstitution of SV40 DNA replication in vitro, corresponds to human DNA polymerase delta.
利用纯化的猿猴病毒40（SV40）大T抗原、细胞拓扑异构酶I和II、复制因子A（RF-A）、增殖细胞核抗原（PCNA）、复制因子C（RF-C）以及从人细胞提取物（S100）制备的磷酸纤维素级分（IIA），可在体外重建源自SV40起始点的DNA复制。级分IIA除了其他因子外，还包含体外复制所需的所有DNA聚合酶活性。一种新鉴定的因子已从级分IIA中纯化出来。该因子是完全重建SV40 DNA复制所必需的，并且与PCNA刺激的DNA聚合酶活性共纯化。这种DNA聚合酶活性对阿非迪霉素敏感，但不受丁基苯胺脱氧三磷酸腺苷或阻断DNA聚合酶α合成的单克隆抗体的抑制。RF-A、PCNA和RF-C的组合以ATP依赖的方式协同刺激该聚合酶活性。在DNA复制测定中，纯化的小牛胸腺聚合酶δ可以完全替代纯化的因子。我们得出结论，这种体外重建SV40 DNA复制所需的因子相当于人DNA聚合酶δ。
