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Study on the initiation mechanism of higher eukaryotic DNA replication.

高等真核生物DNA复制启动机制的研究。

基本信息

批准号：
09680669
负责人：
SHIRAKATA Masaki
金额：
$ 1.92万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680669/
关键词：
DNA replication Epstein-Barr virus oriP replication licensing signal transduction TRAF cell cycle regulation

项目摘要

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.

EB病毒oriP的DNA复制由病毒复制因子EBNA 1和细胞因子介导。由于复制在每个细胞周期中只发生一次，我们使用oriP作为模型复制起点来研究DNA复制起始的分子机制。首先，我们已经确定了二分体对称（DS）元素的oriP作为一个最小的oriP元素，是必要的和足够的DNA复制。DS元件仅包含四个EBNA 1结合位点。这表明DNA复制是通过EBNA 1将细胞复制因子募集到最小oriP上或附近而启动的。我们还证明了oriP质粒需要一个细胞周期窗口，包括早期G1期的复制在下一个S期。这表明，DS依赖的DNA复制oriP需要复制许可。与此一致，MCM 2在G1晚期与oriP微型染色体结合，但在G2/M期不结合，这种结合需要质粒中的DS元件。EBNA 1和MCM 5在DS元件上的相互作用也被提出。从这些结果，我们认为，细胞许可机制控制复制从oriP。我们还发现EBV编码的潜伏膜蛋白1（LMP 1）的信号转导抑制oriP。LMP 1中的TRAF结合基序PxQxT对于启动导致oriP抑制的信号级联是必需的且足够的，oriP抑制的信号级联不同于导致NF-κB、JNK、JAK-stat和MAPK活化的信号级联。TRAF 6在TRAF 5的帮助下的过量表达也诱导了在LMP 1不存在的情况下对oriP的抑制。因此，来自LMP 1和细胞膜受体的TRAF介导信号调节oriP作为DNA复制起点的功能。