Enzymatic analyses of eukaryotic chromosomal DNA replication using the Epstein Barr Virus-derived oriP replicon

使用 Epstein Barr 病毒衍生的 oriP 复制子对真核染色体 DNA 复制进行酶促分析

基本信息

批准号：
14208079
负责人：
MASAI Hisao
金额：
$ 34.61万
依托单位：
Tokyo Metropolitan Organization for Medical Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

Replication of Epstein Barr Viruses (EBV) occurs only once during S phase in coordination with the host cell cycle. The predominant replication origin of EBV, oriP, can support autonomous replication of a plasmid in the presence of virus-encoded EBNA-1 protein. oriP-dependent replication is cell cycle-regulated and depends on the host factors including ORC and most likely MCM, and supports a long-term maintenance of the plasmid in cells. Thus, EBV may serve as an excellent model replicon to probe the roles of cellular replication factors and to dissect the molecular mechanisms of the assembly and activation of eukaryotic replication complexes.We first examined replication of the oriP plasmid on a cellular level. It required the passage through M phase and appears to occur during late S phase. The size of the episomal plasmid in a complex with chromatin proteins changes during cell cycle. We have also shown that Cdc7 function is required for oriP replication. We next attempted to assemb … More le the preRC (prereplicative complex) at the oriP. We speculated that proper chromatin structures would be required for assembly of a functional preRC and established a method for rapid isolation of the chromatin template form mammalian cells. The developed method has two features. 1) The oriP plasmid was linked to SV40 origin and the composite replicon plasmid was introduced into cells expressing the large T-antigen, which permitted highly efficient transient plasmid replication from the SV40 origin. This led to amplification of the plasmid copy number and allowed recovery of a sufficient amount of the template DNA for further analyses. 2) A multiple copies of the tetO sequence (the target of tetR protein) were added to the plasmid to facilitate the recovery of the template chromatin through tetR affinity beads. These modifications enabled us to rapidly isolate a sufficient quantity of the oriP plasmid chromatin.We are now in the process of characterizing the isolated chromatin template to determine the replication proteins present in the complex. We also plan to reconstitute the preRC on the chromatin template using EBNA1 protein and the purified preRC components. We have already overexpressed and purified EBNA1, Cdt1 and MCM proteins, and are now purifying The Cdc6 and ORC complexes. We believe that the system we have established will be very useful for enzymatic characterization of the assembly and activation of the replication complexes at eukaryotic chromosomal replication origins. Less

爱泼斯坦巴尔病毒（EBV）的复制仅在S期与宿主细胞周期的协调过程中发生一次。 EBV的主要复制起源，Orip可以支持在病毒编码的EBNA-1蛋白存在下质粒的自主复制。 Orip依赖性复制是细胞周期调节的，并取决于包括ORC和最有可能MCM在内的宿主因子，并支持细胞中质粒的长期维持。这是EBV可以作为探测细胞复制因子的作用并剖析组装的分子机制和真核复制复制复合物的分子机制的出色模型复制品。我们首先检查了细胞水平上的Orip质粒的复制。它需要通过M相通过，并且似乎在S后期发生。在细胞周期期间，具有染色质蛋白的复合物中的伴有质粒的大小。我们还表明，CDC7功能是Orip复制所必需的。接下来，我们试图组装……更多的是在Orip上的Prerc（prequality Complex）。我们推测，组装功能性PRERC需要适当的染色质结构，并建立了一种快速分离染色质模板形成哺乳动物细胞的方法。开发的方法具有两个功能。 1）将Orip质粒与SV40起源有关，并将复合复制质粒引入表达大型T抗原的细胞中，这导致了质粒拷贝数的扩增，并允许恢复足够量的模板DNA以进行进一步分析。 2）将TETO序列的多个副本（TER蛋白的靶标）添加到质粒中，以促进通过TETR亲和珠的模板染色质回收。这些修饰使我们能够快速分离足够数量的Orip质粒染色质。我们现在正在表征分离的染色质模板以确定复合物中存在的复制蛋白。我们还计划使用EBNA1蛋白和纯化的PRERC成分在染色质模板上重建PRERC。我们已经过表达和纯化的EBNA1，CDT1和MCM蛋白，现在正在纯化CDC6和ORC复合物。我们认为，我们已经建立的系统对于在真核染色体复制起源处的组装和激活的酶促表征非常有用。较少的