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

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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.