Role of the EBNA-1 protein in pausing of replication forks in the Epstein-Barr virus genome

We have previously shown that replication forks stall at a family of repeated sequences (FR) within the Epstein-Barr virus latent origin of replication oriP, both in a small plasmid and in the intact Epstein-Barr virus genome. Each of the 20 repeated sequences within the FR contains a binding site for Epstein-Barr nuclear antigen 1 (EBNA-1), the only viral protein required for latent replication. We showed that the EBNA-1 protein enhances the accumulation of paused replication forks at the FR. In this study, we have investigated a series of truncated EBNA-1 proteins to determine the portion of the EBNA-1 protein that is responsible for pausing of forks at the FR. Two-dimensional agarose gel electrophoresis was performed on the products of in vitro replication reactions in the presence of full-length EBNA-1 or proteins with various deletions to assess the extent of fork pausing at the FR. We conclude that a portion of the DNA binding domain is important for fork pausing. We also present evidence indicating that phosphorylation of the EBNA-1 protein or EBNA-1-truncated derivatives is not essential for pausing. To investigate the mechanism of EBNA-1-mediated pausing of replication forks, we asked whether EBNA-1 could inhibit the DNA unwinding activity of replicative helicases. We found that EBNA-1, when bound to the FR, inhibits DNA unwinding in vitro by SV40 T antigen and Escherichia coli dnaB helicases in an orientation-independent manner.