Stillman B, Gerard R D, Guggenheimer R A, Gluzman Y
EMBO J. 1985 Nov;4(11):2933-9. doi: 10.1002/j.1460-2075.1985.tb04026.x.
A cell-free system for replication of SV40 DNA was used to assess the effect of mutations altering either the SV40 origin of DNA replication or the virus-encoded large tumor (T) antigen. Plasmid DNAs containing various portions of the SV40 genome that surround the origin of DNA replication support efficient DNA synthesis in vitro and in vivo. Deletion of DNA sequences adjacent to the binding sites for T antigen either reduce or prevent DNA synthesis. This analysis shows that sequences that had been previously defined by studies in vivo to constitute the minimal core origin sequences are also necessary for DNA synthesis in vitro. Five mutant T antigens containing amino acid substitutions that affect SV40 replication have been purified and their in vitro properties compared with the purified wild-type protein. One protein is completely defective in the ATPase activity of T antigen, but still binds to the origin sequences. Three altered proteins are defective in their ability to bind to origin DNA, but retain ATPase activity. Finally, one of the altered T antigens binds to origin sequences and contains ATPase activity and thus appears like wild-type for these functions. All five proteins fail to support SV40 DNA replication in vitro. Interestingly, in mixing experiments, all five proteins efficiently compete with the wild-type protein and reduce the amount of DNA replication. These data suggest that an additional function of T antigen other than origin binding or ATPase activity, is required for initiation of DNA replication.
利用一种用于SV40 DNA复制的无细胞系统，来评估改变SV40 DNA复制起点或病毒编码的大T抗原的突变的影响。含有围绕DNA复制起点的SV40基因组各部分的质粒DNA，在体外和体内均支持高效的DNA合成。删除与T抗原结合位点相邻的DNA序列会减少或阻止DNA合成。该分析表明，先前在体内研究中确定为构成最小核心起点序列的序列，对于体外DNA合成也是必需的。已纯化出五种含有影响SV40复制的氨基酸取代的突变T抗原，并将它们的体外特性与纯化的野生型蛋白进行了比较。一种蛋白在T抗原的ATP酶活性方面完全有缺陷，但仍能与起点序列结合。三种改变的蛋白在与起点DNA结合的能力方面有缺陷，但保留了ATP酶活性。最后，一种改变的T抗原与起点序列结合并具有ATP酶活性，因此在这些功能方面看起来像野生型。所有这五种蛋白在体外均无法支持SV40 DNA复制。有趣的是，在混合实验中，所有这五种蛋白都能与野生型蛋白有效竞争，并减少DNA复制量。这些数据表明，DNA复制起始需要T抗原除起点结合或ATP酶活性之外的其他功能。
