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-EBNA1复制系统在持续感染的细胞中维持EB病毒基因组的机制。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，准确分析这种起始蛋白对于了解Ori P复制系统是非常重要的。在这方面，我们一直在对哺乳动物细胞中的DNA复制起始蛋白进行功能分析，重点是Horc、hCDC6和HMCM蛋白。目前，DNA复制的细胞周期调控模型如下。一个可能的六聚体哺乳动物ORC与核基质上的染色质相互作用。CDC6也与基质有关。通过ORC/CDC6，MCM异六聚体复合体主要被加载到与基质无关的染色质区域。结合的MCM可能通过磷酸化被激活。激活的MCM在DNA复制中发挥着未知但必不可少的作用。一旦解离的MCM的重新加载，以及随后的重新复制，可能会被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相互作用而参与Ori P复制系统。使用各种技术，我们已经解决了这种可能性。然而，到目前为止，我们还没有发现它们之间存在显著的互动。考虑到我们的数据以及最近的发现，EBNA1不一定是Ori P复制所必需的，Ori P的复制可能依赖于细胞复制起始蛋白，如ORC、CDC6和MCM。在这方面，研究这些蛋白是否与体内EBV基因组相关，如果是，与它们相关的基因组区域以及这种相关性是如何调节的，这将是重要的。我们现在正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.