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

分析 EB 病毒 EBNA-1 蛋白与参与宿主 DNA 复制起始步骤的细胞蛋白之间的相互作用

基本信息

批准号：
10670296
负责人：
FUJITA Masatoshi
金额：
$ 1.92万
依托单位：
Aichi Cancer Center
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

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.

该项目的目的是了解ORI P-EBNA1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。现在建议，参与宿主DNA复制的涉及的细胞蛋白在ORI P复制系统的调节中也起着作用。因此，精确分析这种起始蛋白以理解Ori P复制系统显然很重要。在这方面，我们一直在哺乳动物细胞中的DNA复制起始蛋白进行功能分析，重点是HORC，HCDC6和HMCM蛋白。 DNA复制的细胞周期调节的当前模型如下。推定的六聚体哺乳动物兽人在核基质上与染色质相互作用。 Cdc6也与矩阵有关。兽人/cdc6加载了MCM杂己聚集络合物，主要是与矩阵无关的染色质区域。结合的MCM可以通过磷酸化激活。激活的MCM在DNA复制中起未知但至关重要的作用。 CDK激酶可能会抑制一次分解的MCM并随后的重新加载，直到下一个G1相。一个有趣的可能性是，这些起始蛋白可以通过与EBNA-1的相互作用参与ORI P复制系统。使用各种技术，我们已经解决了这种可能性。但是，到目前为止，我们还找不到它们之间的显着相互作用。考虑到我们的数据以及最近的发现，即EBNA1不一定需要进行ORI P复制，可能是ORI P复制取决于细胞复制起始蛋白，例如ORC，CDC6和MCM。在这方面，重要的是研究这些蛋白是否与体内EBV基因组相关，如果是哪个基因组区域与它们相关的基因组区域以及如何调节关联。我们现在使用染色质免疫沉淀测定法解决了这些点。