Analysis of the interaction between EB viral EBNA-1 protein and cellular proteins involved in the initiation step of host DNA replication

本项目的目的是了解Ori P-EBNA 1复制系统的机制，该系统在持续感染的细胞中维持EB病毒基因组。目前认为，参与宿主DNA复制起始的细胞蛋白也在Ori P复制系统的调节中发挥作用。因此，精确分析这种起始蛋白对于理解Ori P复制系统显然是重要的。在这方面，我们一直在进行哺乳动物细胞中DNA复制起始蛋白的功能分析，重点是hORC，hCDC 6和hMCM蛋白。目前DNA复制的细胞周期调控模型如下。一个推定的六聚体哺乳动物ORC与核基质上的染色质相互作用。CDC 6也与基质相关。MCM异六聚体复合物通过ORC/CDC 6主要装载到与基质不相关的染色质区域上。结合的MCMs可能通过磷酸化被激活。激活的MCM在DNA复制中起着未知但重要的作用。一旦解离的MCM的增殖，以及随后的再复制，可以被CDK激酶抑制，直到下一个G1期。一个有趣的可能性是，这些起始蛋白可能通过与EBNA-1相互作用参与Ori P复制系统。我们使用各种技术来解决这种可能性。然而，到目前为止，我们还没有发现它们之间有显著的相互作用。考虑到我们的数据以及最近的发现，即EBNA 1不一定是Ori P复制所必需的，可能是Ori P复制依赖于细胞复制起始蛋白，如ORC、CDC 6和MCM。在这方面，重要的是研究这些蛋白质是否与体内EBV基因组相关，如果是这样，什么基因组区域与它们相关，以及如何调节这种关联。我们现在使用染色质免疫沉淀分析来解决这些问题。

The purpose of this project is to understand the mechanism of Ori P-EBNA1 replication system, which maintains EB viral genome in persistently infected cells. It is now suggested that cellular proteins involved in the initiation of host DNA replication also play roles in regulation of Ori P replication system. Therefore, it is clearly important to precisely analyze such the initiation protein for understanding Ori P replication system. In this regard, we have been performing functional analysis of DNA replication initiation proteins in mammalian cells, focusing on hORC, hCDC6 and hMCM proteins. Current model for the cell cycle regulation of DNA replication is as follows. A putative hexameric mammalian ORC interacts with chromatin on nuclear matrix. CDC6 is also associated with the matrix. MCM heterohexameric complexes are loaded, by ORC/CDC6, mainly onto chromatin regions not associated with the matrix. The bound MCMs might be activated through phosphorylation. The activated MCM plays an unknown but essential role in DNA replication. Reloading of once dissociated MCM, and subsequent rereplication, may be suppressed by CDK kinase until next G1 phase. An interesting possibility is that these initiation proteins may participate in Ori P replication system via interaction with EBNA-1. Using various techniques, we have addressed this possibility. However, so far we can not find significant interaction between them. Considering our data together with recent findings that EBNA1 is not necessarily required for Ori P replication, it could be that Ori P replication is dependent on the cellular replication initiation proteins such as ORC, CDC6 and MCM. In this regard, it will be important to investigate whether these proteins are associated with EBV genome in vivo and if so what genome region is associated with them and how the association is regulated. We are now addressing these points using chromatin immunoprecipitation assay.