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

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

From the ^‡ Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461 and the
^§ Institute for Molecular Biology and Biotechnology, Cancer Research Group, McMaster University, Hamilton, Ontario L8N 3Z5, Canada

↵^∥To whom correspondence should be addressed: Dept. of Cell Biology (CH 416), Albert Einstein College of Medicine, 1300 Morris Park Ave., New York, NY 10461. Tel.: 718-430-2097; Fax: 718-430-8574.

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.

Footnotes

↵^¶ A Research Scientist of the NCIC.
↵* This work was supported by National Institutes of Health Grant RO1 GM45751 and Cancer Center Core Grant NIH/NCI P30-CA13330 (to C. L. S.) and by a grant from the National Cancer Institute of Canada (NCIC) (to L. F.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.