Replication of simian virus 40 origin-containing DNA in vitro with purified proteins.
Replication of simian virus 40 origin-containing DNA in vitro with purified proteins.
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DOI：
10.1073/pnas.84.7.1834
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发表时间：
1987-04
影响因子：
11.1
通讯作者：
C. Wobbe;L. Weissbach;J. Borowiec;F. Dean;Y. Murakami;P. Bullock;J. Hurwitz
中科院分区：
文献类型：
--
作者：
C. Wobbe;L. Weissbach;J. Borowiec;F. Dean;Y. Murakami;P. Bullock;J. Hurwitz
猴病毒40（SV 40）DNA复制依赖于SV 40复制起点和SV 40大肿瘤（T）抗原，已经用从HeLa细胞分离的纯化蛋白组分在体外重建。除SV 40 T抗原外，这些组分还包括DNA聚合酶α-引发酶复合物、拓扑异构酶I和含有单链DNA结合蛋白的组分。后者的蛋白质，沉淀在5.1 S的甘油梯度和copurifies与两个主要的蛋白质种类的72和76 kDa的，被单独分离的能力，以支持SV 40 DNA复制。纯化的系统保留了物种特异性DNA聚合酶α-引发酶的要求，以前观察到的粗馏分，从HeLa细胞的复合物支持SV 40复制，而从小牛胸腺和小鼠细胞没有。含有多瘤病毒复制起点的DNA在含有多瘤病毒T抗原、含有HeLa单链DNA结合蛋白的级分和来自小鼠但不是HeLa细胞的DNA聚合酶α-引发酶复合物的系统中复制。虽然粗级分产生闭合环状双链DNA，但用纯化的系统未检测到。然而，向纯化的系统中加入粗级分产生封闭的环状单体产物。
Simian virus 40 (SV40) DNA replication dependent on the SV40 origin of replication and the SV40 large tumor (T) antigen has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, these components included the DNA polymerase alpha-primase complex, topoisomerase I, and a fraction that contained a single-stranded DNA binding protein. The latter protein, which sediments at 5.1 S on glycerol gradients and copurifies with two major protein species of 72 and 76 kDa, was isolated solely by its ability to support SV40 DNA replication. The purified system retained the species-specific DNA polymerase alpha-primase requirement previously observed with crude fractions; the complex from HeLa cells supported SV40 replication, whereas that from calf thymus and mouse cells did not. DNA containing the polyomavirus origin of replication was replicated in a system containing polyomavirus T antigen, the HeLa single-stranded DNA binding protein-containing fraction, and DNA polymerase alpha-primase complex from mouse, but not HeLa, cells. While crude fractions yielded closed circular duplex DNA, none was detected with the purified system. Nevertheless, the addition of a crude fraction to the purified system yielded closed circular monomer products.