Murakami Y, Hurwitz J
Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
J Biol Chem. 1993 May 25;268(15):11018-27.
The ATP-dependent binding of simian virus 40 (SV40) large tumor antigen (T antigen) to the SV40 origin of replication is an essential step in the initiation of SV40 DNA synthesis. Previous studies indicated that the ATP-dependent complex consists of a double hexamer of T antigen at the origin. The binding reaction and the subsequent unwinding of the duplex DNA from the origin were examined using a gel mobility shift assay. T antigen bound to the core origin cooperatively in the presence of ATP. In the presence of human single-stranded DNA-binding protein (HSSB), T antigen, complexed to the core origin, started the unwinding of duplex DNA. At low concentrations of T antigen and in the presence of ATP, DNA polymerase alpha (pol alpha) stimulated the binding of T antigen to the core origin, while HSSB did not. This stimulation resulted in an increase in the subsequent unwinding reaction in the presence of HSSB. Primase alone did not affect the binding reaction and was not required for the stimulation by pol alpha. The stimulation required the hydrolysis of ATP and the AT tract domain of the core origin. Kinetic studies showed that while pol alpha stimulated the binding of T antigen to the core origin, it did not stabilize the complex. Pol alpha also stimulated the formation of the ATP-dependent T antigen-site I complex, a region that also contains an AT-rich sequence. These observations imply a regulatory role for pol alpha in the initiation of SV40 DNA replication.
猿猴病毒40（SV40）大T抗原（T抗原）与SV40复制起点的ATP依赖性结合是启动SV40 DNA合成的关键步骤。先前的研究表明，该ATP依赖性复合物由位于复制起点的T抗原双六聚体组成。使用凝胶迁移率变动分析检测了结合反应以及随后双链DNA从复制起点的解旋情况。在ATP存在下，T抗原与核心复制起点协同结合。在人单链DNA结合蛋白（HSSB）存在的情况下，与核心复制起点结合的T抗原开始双链DNA的解旋。在低浓度T抗原且存在ATP的情况下，DNA聚合酶α（polα）刺激T抗原与核心复制起点的结合，而HSSB则无此作用。这种刺激导致在HSSB存在时随后的解旋反应增强。单独的引发酶不影响结合反应，也不是polα刺激所必需的。这种刺激需要ATP水解以及核心复制起点的富含AT区域。动力学研究表明，虽然polα刺激T抗原与核心复制起点的结合，但它并不稳定复合物。Polα还刺激了ATP依赖性T抗原-位点I复合物的形成，该区域也包含富含AT的序列。这些观察结果暗示polα在SV40 DNA复制起始过程中具有调节作用。
