Müller F, Seo Y S, Hurwitz J
Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Institute, New York, New York 10021.
J Biol Chem. 1994 Jun 24;269(25):17086-94.
The in vitro replication of DNA containing the bovine papillomavirus (BPV-1) origin has been carried out with cell-free extracts from mouse FM3A and human HeLa cells. DNA synthesis required the E1 protein, the minimal origin of replication (nucleotides 7911-22 of the BPV-1 genome), and, at low levels of FM3A extract, the addition of the human single-stranded DNA-binding protein (also called RP-A or RF-A). The E2 protein was not absolutely required, but could stimulate DNA synthesis at low levels of E1. DNA synthesis was also reconstituted using purified proteins from HeLa cells. These protein factors included human single-stranded DNA-binding protein, topoisomerase I, and DNA polymerase (pol) alpha-primase complex. At low concentrations of pol alpha-primase complex, the formation of high molecular weight products was dependent on the addition of DNA polymerase delta holoenzyme containing proliferating cell nuclear antigen and activator 1, also called RF-C. We have overexpressed and isolated the E1 protein from bacteria. This protein also supported BPV DNA synthesis, both in crude extracts and with purified proteins suggesting that E1 phosphorylation is not required for BPV DNA replication in vitro.
利用来自小鼠FM3A细胞和人HeLa细胞的无细胞提取物，对含有牛乳头瘤病毒（BPV - 1）复制起点的DNA进行了体外复制。DNA合成需要E1蛋白、最小复制起点（BPV - 1基因组的核苷酸7911 - 22），并且在低水平的FM3A提取物中，还需要添加人单链DNA结合蛋白（也称为RP - A或RF - A）。E2蛋白不是绝对必需的，但在低水平的E1时可刺激DNA合成。也使用来自HeLa细胞的纯化蛋白重建了DNA合成。这些蛋白因子包括人单链DNA结合蛋白、拓扑异构酶I和DNA聚合酶（pol）α - 引发酶复合物。在低浓度的polα - 引发酶复合物时，高分子量产物的形成依赖于添加含有增殖细胞核抗原和激活因子1（也称为RF - C）的DNA聚合酶δ全酶。我们已经在细菌中过表达并分离了E1蛋白。该蛋白在粗提取物和纯化蛋白中均支持BPV DNA合成，这表明体外BPV DNA复制不需要E1磷酸化。
