Dean F B, Dodson M, Echols H, Hurwitz J
Memorial Sloan-Kettering Cancer Center, Program in Molecular Biology, New York, NY 10021.
Proc Natl Acad Sci U S A. 1987 Dec;84(24):8981-5. doi: 10.1073/pnas.84.24.8981.
The large tumor antigen (T antigen) specified by simian virus 40 (SV40) is required for viral DNA replication. To carry out its function, T antigen binds to duplex DNA at the origin of replication (oriSV40) and exerts a helicase activity that unwinds the two DNA strands. Previous work has defined two binding sites for T antigen near oriSV40, designated sites I and II; site II is within the 64-base-pair core sequence absolutely required for viral DNA replication. We have used electron microscopy and gel electrophoresis to characterize the interaction of T antigen with the origin region. We have found that effective binding to site II under conditions that support DNA replication requires ATP or a nonhydrolyzable analog. In the absence of ATP, T antigen binds mainly to site I; in the presence of ATP, both sites I and II are occupied, and binding is markedly increased. The ATP-dependent reaction generates a complex multimeric structure for T antigen. We conclude that T antigen forms an ATP-dependent nucleoprotein structure at oriSV40. We suggest that this nucleoprotein complex provides for the precise initiation of SV40 DNA replication.
猴病毒40（SV40）所编码的大肿瘤抗原（T抗原）是病毒DNA复制所必需的。为了执行其功能，T抗原在复制起点（oriSV40）处与双链DNA结合，并发挥解旋酶活性解开两条DNA链。先前的研究确定了oriSV40附近T抗原的两个结合位点，分别称为位点I和位点II；位点II位于病毒DNA复制绝对必需的64个碱基对的核心序列内。我们利用电子显微镜和凝胶电泳来表征T抗原与起始区域的相互作用。我们发现，在支持DNA复制的条件下，与位点II的有效结合需要ATP或不可水解的类似物。在没有ATP的情况下，T抗原主要与位点I结合；在有ATP的情况下，位点I和位点II都被占据，且结合显著增加。ATP依赖的反应为T抗原生成了一种复杂的多聚体结构。我们得出结论，T抗原在oriSV40处形成了一种ATP依赖的核蛋白结构。我们认为这种核蛋白复合物为SV40 DNA复制的精确起始提供了条件。
