VanLoock Margaret S, Alexandrov Alexander, Yu Xiong, Cozzarelli Nicholas R, Egelman Edward H
Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences, Box 800733, Charlottesville, VA 22908-0733, USA.
Curr Biol. 2002 Mar 19;12(6):472-6. doi: 10.1016/s0960-9822(02)00696-6.
Simian Virus 40 replication requires only one viral protein, the Large T antigen (T-ag), which acts as both an initiator of replication and as a replicative helicase (reviewed in ). We used electron microscopy to generate a three-dimensional reconstruction of the T-ag hexameric ring in the presence and absence of a synthetic replication fork to locate the T-ag domains, to examine structural changes in the T-ag hexamer associated with DNA binding, and to analyze the formation of double hexamers on and off DNA. We found that binding DNA to the T-ag hexamer induces large conformational changes in the N- and C-terminal domains of T-ag. Additionally, we observed a significant increase in density throughout the central channel of the hexameric ring upon DNA binding. We conclude that conformational changes in the T-ag hexamer are required to accommodate DNA and that the mode of DNA binding may be similar to that suggested for some other ring helicases. We also identified two conformations of T-ag double hexamers formed in the presence of forked DNA: with N-terminal hexamer-hexamer contacts, similar to those formed on origin DNA, or with C-terminal contacts, which are unlike any T-ag double hexamers reported previously.
猴病毒40（Simian Virus 40）的复制仅需要一种病毒蛋白，即大T抗原（T-ag），它既是复制起始因子，又是复制解旋酶（相关综述见 ）。我们利用电子显微镜对存在和不存在合成复制叉时的T-ag六聚体环进行三维重建，以定位T-ag结构域，检查与DNA结合相关的T-ag六聚体的结构变化，并分析DNA上和脱离DNA时双六聚体的形成。我们发现，将DNA与T-ag六聚体结合会在T-ag的N端和C端结构域诱导大的构象变化。此外，我们观察到DNA结合后六聚体环的整个中央通道密度显著增加。我们得出结论，T-ag六聚体的构象变化是容纳DNA所必需的，并且DNA结合模式可能与其他一些环状解旋酶的情况相似。我们还鉴定了在叉状DNA存在下形成的T-ag双六聚体的两种构象：一种是N端六聚体-六聚体接触，类似于在起始DNA上形成的接触；另一种是C端接触，这与之前报道的任何T-ag双六聚体都不同。
