|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1999 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1998 : ¥1,600,000 (Direct Cost : ¥1,600,000)
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.