Gillette T G, Borowiec J A
Department of Biochemistry and Kaplan Comprehensive Cancer Center, New York University Medical Center, New York, New York 10016, USA.
J Virol. 1998 Jul;72(7):5735-44. doi: 10.1128/JVI.72.7.5735-5744.1998.
The modulation of DNA replication by transcription factors was examined by using bovine papillomavirus type 1 (BPV). BPV replication in vivo requires two viral proteins: E1, an origin-binding protein, and E2, a transcriptional transactivator. In the origin, E1 interacts with a central region flanked by two binding sites for E2 (BS11 and BS12), of which only BS12 has been reported to be essential for replication in vivo. Using chemical interference and electrophoretic mobility shift assays, we found that the binding of E2 to each site stimulates the formation of distinct E1-origin complexes. A high-mobility C1 complex is formed by using critical E2 contacts to BS12 and E1 contacts to the dyad symmetry element. In contrast, interaction of E2 with the BS11 element on the other origin flank promotes the formation of the lower-mobility C3 complex. C3 is a novel species that resembles C2, a previously identified complex that is replication active and formed by E1 alone. The binding of E1 greatly differs in the C1 and C3 complexes, with E1 in the C1 complex limited to the origin dyad symmetry region and E1 in the C3 complex encompassing the region from the proximal edge of BS11 through the distal edge of BS12. We found that the presence of both E2-binding sites is necessary for wild-type replication activity in vivo, as well as for maximal production of the C3 complex. These results show that in the normal viral context, BS11 and BS12 play separate but synergetic roles in the initiation of viral DNA replication that are dependent on their location within the origin. Our data suggest a model in which the binding of E2 to each site sequentially stimulates the formation of distinct E1-origin complexes, leading to the replication-competent complex.
利用1型牛乳头瘤病毒（BPV）研究了转录因子对DNA复制的调控作用。BPV在体内的复制需要两种病毒蛋白：E1，一种起源结合蛋白；E2，一种转录反式激活因子。在病毒起源区域，E1与一个中央区域相互作用，该区域两侧有两个E2结合位点（BS11和BS12），据报道其中只有BS12对体内复制至关重要。通过化学干扰和电泳迁移率变动分析，我们发现E2与每个位点的结合会刺激形成不同的E1-起源复合物。通过E2与BS12的关键接触以及E1与二元对称元件的接触形成高迁移率的C1复合物。相比之下，E2与另一个起源侧翼上的BS11元件相互作用会促进低迁移率的C3复合物的形成。C3是一种新的复合物，类似于C2，C2是先前鉴定的具有复制活性且仅由E1形成的复合物。E1在C1和C3复合物中的结合有很大差异，C1复合物中的E1局限于起源二元对称区域，而C3复合物中的E1涵盖从BS11近端边缘到BS12远端边缘的区域。我们发现，两个E2结合位点的存在对于体内野生型复制活性以及C3复合物的最大产量都是必需的。这些结果表明，在正常病毒环境中，BS11和BS12在病毒DNA复制起始过程中发挥着各自但协同的作用，这取决于它们在起源区域内的位置。我们的数据提出了一个模型，其中E2与每个位点的结合依次刺激形成不同的E1-起源复合物，从而导致具有复制能力的复合物形成。
