Cell-Free Reconstitution of Site-Specific Assembly of Human Pre-Replicative Complex

EB病毒的基因组DNA在其潜伏的附加体状态下，在EBNA 1（一种病毒编码的核蛋白）存在下每个细胞周期复制一次。病毒附加体的潜伏复制依赖于人类复制前复合体（pre-replicative complex，pre-RC），包括Orc（origin-recognition complex）、Cdc 6、Cdt 1和Mcm（minichromosome maintenance complex）。我打算使用含有oriP的质粒DNA作为模型复制子，在体外重建EB病毒oriP上的复制起始。oriP上的pre-RC形成依赖于EBNA 1与DSE（二分体对称元件）的结合，DSE是oriP上的一个必需顺式元件。端粒保护因子TRF 2最近被确定为招募DSE兽人的另一个重要因素。我已经在人293 T细胞中过表达重组EBNA 1、TRF 2、Orc 1和Cdc 6，然后制备用于无细胞前RC组装的核提取物。发现人Orc 2是Orc的核心亚基之一，在重组EBNA 1存在下，与固定化的oriP以及短DSE片段结合，增加了TRF 2和Orc 1。cdc 6表现出几乎相关的行为，只有在他们的存在。当DSE缺失时，即使重组EBNA 1、TRF 2和Orc 1增加父性，Orc 2也不与oriP结合。另一方面，内源性Cdt 1与oriP的关联仅需要重组EBNA 1，而不需要增加TRF 2或Orc 1。此外，令人惊讶的是，Cdt 1仅在重组EBNA 1存在时才能与缺乏DSE的oriP结合。目前，我正在为重组Cdt 1和不同细胞周期阶段的核提取物制备过表达器，以在oriP模板DNA上完全重建前RC。

Genomic DNA of Epstein-Barr (EB) virus, in its latent episomal state, replicates once per cell cycle in the presence of EBNA1, a virus-encoded nuclear protein. The latent replication of viral episome is believed to depend on human pre-replicative complex (pre-RC), including Orc (origin-recognition complex), Cdc6, Cdt1 and Mcm (minichromosome maintenance complex). I intend to reconstitute initiation of replication on oriP of EB virus in vitro using oriP-containing plasmid DNA as a model replicon. Pre-RC formation on oriP is dependent on EBNA1 binding to DSE (dyad symmetry element), an essential cis-element on oriP. A telomere protection factor, TRF2, was recently identified as another essential factor for recruiting Orc on DSE. I have overexpressed recombinant EBNA1, TRF2, Orc1, and Cdc6 in human 293T cells, and then prepared nuclear extracts for cell-free pre-RC assembly. I found that human Orc2, one of core subunit of Orc, was associated with immobilized oriP as well as short DSE fragments in the presence of recombinant EBNA1, increased TRF2 and Orc1. Cdc6 exhibited almost correlated behavior only in their presence. Orc2 did not bind to oriP when DSE was deleted even if recombinant EBNA1, TRF2 and Orc1 increased father. On the other hand, association of endogeneous Cdt1 with oriP required only recombinant EBNA1, but not increased TRF2 or Orc1. Moreover, it was somehow surprising that Cdt1 could bind to oriP lacking DSE only when recombinant EBNA1 was present. At present, I am making overexpreser for recombinant Cdt1 and nuclear extracts of various cell cycle stages for complete reconstitution of pre-RC on oriP template DNA.