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 r epeated sequences (FR) within the Epstein-Barr virus latent origin of replication oriP, both in a small plasmid and in the intact Epstein-Ba rr virus genome, Each of the 20 repeated sequences within the FR conta ins a binding site for Epstein-Barr nuclear antigen 1 (EBNA-1), the on ly viral protein required for latent replication. We showed that the E BNA-1 protein enhances the accumulation of paused replication forks at the FR. In this study, we have investigated a series of truncated EBN A-1 proteins to determine the portion of the EBNA-1 protein that is re sponsible 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 vario us deletions to assess the extent of fork pausing at the FR. We conclu de that a portion of the DNA binding domain is important for fork paus ing, We also present evidence indicating that phosphorylation of the E BNA-1 protein or EBNA-1-truncated derivatives is not essential for pau sing, To investigate the mechanism of EBNA-1-mediated pausing of repli cation 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 Escheri chia coil dnaB helicases in an orientation-independent manner.