| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1999: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1998: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1997: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
DNA replication from oriP of Epstein-Barr virus is mediated by the virus replication factor EBNA1 and cellular factors. Since the replication occurs only once in each cell cycle, we used oriP as a model replication origin to investigate the molecular machinery for the initiation of DNA replication. First, we have identified the dyad symmetry (DS) element of oriP as a minimal oriP element that is necessary and sufficient for DNA replication. The DS element contains only the four EBNA1-binding sites. This suggests that DNA replication is initiated by recruitment of cellular replication factors onto or near the minimal oriP by EBNA1. We also demonstrate that the oriP plasmid required a cell cycle window including early G1 phase for the replication in next S phase. This suggests that the DS-dependent DNA replication from oriP requires the replication licensing. Consistent with this, MCM2 associates with the oriP minichromosome at late G1 but not at G2/M, and this association requires the DS-element in the plasmid. Interaction of EBNA1 and MCM5 on the DS element is also suggested. From these results, we suggest that the cellular licensing mechanism controls the replication from oriP. We also find that the signal transduction form the EBV-encoded latent membrane protein 1 (LMP1) suppresses oriP. The TRAF-binding motif in LMP1, PxQxT, is essential and sufficient to initiate the signal cascade leading to the suppression of oriP, which is distinct from signal cascades leading to activation of NF-κB, JNK, JAK-stat and MAPKs. Excess expression of TRAF6 with a help of TRAF5 also induces suppression of oriP in the absence of LMP1. Thus, the TRAF-mediating signals from LMP1 and a cellular membrane receptor regulate the oriP's function as a DNA replication origin.
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