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

Epstein巴尔病毒（EBV）的复制在与宿主细胞周期协调的S期期间仅发生一次。EBV的主要复制起点oriP可以在病毒编码的EBNA-1蛋白存在下支持质粒的自主复制。oriP依赖性复制是细胞周期调节的，并且取决于宿主因子，包括ORC和最可能的MCM，并且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，以探测细胞复制因子的作用，并剖析真核复制复合物的组装和激活的分子机制。它需要通过M期，似乎发生在S期晚期。在细胞周期中，与染色质蛋白复合的附加型质粒的大小发生变化。我们还表明，Cdc 7功能是oriP复制所需的。我们接下来试图组装 ...更多信息 也就是oriP上的preRC（prereplicative complex）。我们推测，正确的染色质结构将需要一个功能性preRC的组装，并建立了一种方法，用于快速分离的染色质模板哺乳动物细胞。开发的方法有两个特点。1)将oriP质粒连接到SV 40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SV 40起点进行高效瞬时质粒复制。这导致质粒拷贝数的扩增，并允许回收足够量的模板DNA用于进一步分析。2)将tetO序列（tetR蛋白的靶标）的多个拷贝添加到质粒中，以促进通过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EBNA 1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EBNA 1、Cdt 1和MCM蛋白，现在正在纯化Cdc 6和ORC复合物。我们相信，我们已经建立的系统将是非常有用的酶的组装和激活的复制复合物在真核染色体复制起点的表征。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