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

eb病毒（EBV）的复制只在S期发生一次，与宿主细胞周期协调。EBV的主要复制起源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。orp依赖性复制是细胞周期调控的，依赖于宿主因子，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可以作为一个很好的模型复制子，用于探索细胞复制因子的作用，以及剖析真核生物复制复合体的组装和激活的分子机制。我们首先在细胞水平上研究了oriP质粒的复制。它需要通过M期，似乎发生在S期后期。在染色质蛋白复合体中，表观质粒的大小在细胞周期中发生变化。我们还证明了Cdc7功能是oriP复制所必需的。接下来，我们尝试在oriP上组装prec（预复制复合体）。我们推测，正确的染色质结构将需要组装一个功能性的preRC，并建立了一种方法快速分离染色质模板从哺乳动物细胞。所开发的方法有两个特点。1)将oriP质粒与SV40源连接，将复合复制子质粒导入表达大t抗原的细胞中，实现了SV40源质粒的高效瞬时复制。这导致质粒拷贝数的扩增，并允许恢复足够数量的模板DNA进行进一步分析。2)在质粒中加入多份tetO序列（tetR蛋白的靶点），通过tetR亲和珠促进模板染色质的恢复。这些修饰使我们能够快速分离出足够数量的oriP质粒染色质。我们现在正在对分离的染色质模板进行表征，以确定复合体中存在的复制蛋白。我们还计划使用EBNA1蛋白和纯化的preRC组分在染色质模板上重建preRC。我们已经过表达和纯化了EBNA1、Cdt1和MCM蛋白，现在正在纯化Cdc6和ORC复合物。我们相信，我们建立的系统将对真核生物染色体复制起点上复制复合体的组装和激活的酶学表征非常有用。少

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