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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

EB 病毒 (EBV) 的复制仅在 S 期发生一次，与宿主细胞周期相协调。 EBV 的主要复制起点 oriP 可以在病毒编码的 EBNA-1 蛋白存在的情况下支持质粒的自主复制。 oriP 依赖性复制是细胞周期调节的，取决于宿主因素，包括 ORC 和最有可能的 MCM，并支持质粒在细胞中的长期维持。因此，EBV可以作为一个优秀的模型复制子来探测细胞复制因子的作用并剖析真核复制复合物组装和激活的分子机制。我们首先在细胞水平上检查了oriP质粒的复制。它需要通过 M 期，并且似乎发生在 S 期晚期。与染色质蛋白形成的复合物中附加型质粒的大小在细胞周期中发生变化。我们还表明，oriP 复制需要 Cdc7 功能。接下来，我们尝试在 oriP 处组装 preRC（预复制复合体）。我们推测，组装功能性 preRC 需要适当的染色质结构，并建立了一种从哺乳动物细胞中快速分离染色质模板的方法。所开发的方法有两个特点。 1)将oriP质粒连接到SV40起点，并将复合复制子质粒引入表达大T抗原的细胞中，这允许从SV40起点高效瞬时质粒复制。这导致了质粒拷贝数的扩增，并允许回收足够量的模板 DNA 以用于进一步分析。 2) 将多拷贝的tetO序列（tetR蛋白的靶标）添加到质粒中，以便于通过tetR亲和珠回收模板染色质。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。我们已经过表达并纯化了 EBNA1、Cdt1 和 MCM 蛋白，现在正在纯化 Cdc6 和 ORC 复合物。我们相信，我们建立的系统对于真核染色体复制起点复制复合物的组装和激活的酶学表征非常有用。较少的