Role of the EBNA-1 Protein in Pausing of Replication Forks in the Epstein-Barr Virus Genome

Olga V Ermakova,Lori Frappier,Carl L Schildkraut

doi:10.1074/jbc.271.51.33009

Abstract

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.

Highlights

The Epstein-Barr virus latent origin of replication, oriP, was originally identified as a genetic element that conferred the ability to replicate autonomously upon small plasmids in EBV1-transformed cells [1]
There are several similarities between pausing of replication forks at the family of repeats (FR) and replication pause sites that have been observed in prokaryotes
One difference between the termination pause sites in prokaryotes and the FR of the EBV site is that the pause sites in prokaryotes are polar and relatively efficient, whereas Epstein-Barr nuclear antigen 1 (EBNA-1)-mediated pausing at the FR occurs in both directions and is less efficient

Summary

Introduction

The Epstein-Barr virus latent origin of replication, oriP, was originally identified as a genetic element that conferred the ability to replicate autonomously upon small plasmids in EBV1-transformed cells [1]. Since EBNA-1 binds to multiple sites within the FR, we have begun to investigate its role in the pausing of replication forks. Toward this aim, we have used an in vitro replication system in which replication of plasmids containing the FR is initiated from the SV40 origin in the presence of T antigen and a soluble HeLa cell extract. The substitution of three tandem copies of the DS region (12 EBNA-1 binding sites) for the FR results in pausing of replication forks, suggesting that multiple EBNA-1 binding sites are important irrespective of spacer DNA sequences [18]. We show that the full-length EBNA-1 protein enhances the

Methods

Results

Conclusion