Myers R M, Tjian R
Proc Natl Acad Sci U S A. 1980 Nov;77(11):6491-5. doi: 10.1073/pnas.77.11.6491.
We have inserted a 311-base pair DNA fragment containing the simian virus 40 (SV40) origin of DNA replication, the early promoter, and the tumor (T) antigen binding sites into a bacterial plasmid and cloned it. This recombinant plasmid, pSV01, binds to a purified T antigen in vitro and replicates in monkey cells when supplied with large T antigen. A series of deletion mutations was generated in the origin sequences of pSV01 DNA by mutagenesis in vitro. The replication of these mutant DNAs in monkey cells was compared with their ability to bind to purified D2 protein. Mutant DNAs deficient in binding to D2 protein also exhibit reduced levels of replication in monkey cells. These findings provide biochemical evidence that the initiation of SV40 DNA synthesis may involve a direct interaction of T antigen with sequences at the origin of replication.
我们已将一段包含猿猴病毒40（SV40）DNA复制起点、早期启动子及肿瘤（T）抗原结合位点的311个碱基对的DNA片段插入到一个细菌质粒中并进行了克隆。这种重组质粒pSV01在体外能与纯化的T抗原结合，当提供大T抗原时可在猴细胞中复制。通过体外诱变在pSV01 DNA的起点序列中产生了一系列缺失突变。将这些突变DNA在猴细胞中的复制情况与其结合纯化D2蛋白的能力进行了比较。缺乏与D2蛋白结合能力的突变DNA在猴细胞中的复制水平也降低。这些发现提供了生化证据，表明SV40 DNA合成的起始可能涉及T抗原与复制起点序列的直接相互作用。
