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

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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.

Original languageEnglish (US)
Pages (from-to)33009-33017
Number of pages9
JournalJournal of Biological Chemistry
Volume271
Issue number51
DOIs
StatePublished - 1996

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Nuclear Antigens
Human Herpesvirus 4
Viruses
Genes
Genome
Proteins
DnaB Helicases
Polyomavirus Transforming Antigens
Phosphorylation
Replication Origin
Agar Gel Electrophoresis
DNA sequences
DNA
Electrophoresis, Gel, Two-Dimensional
Viral Proteins
Nuclear Proteins
Electrophoresis
Sepharose
Escherichia coli
Plasmids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Role of the EBNA-1 protein in pausing of replication forks in the Epstein-Barr virus genome. / Ermakova, Olga V.; Frappier, Lori; Schildkraut, Carl L.

In: Journal of Biological Chemistry, Vol. 271, No. 51, 1996, p. 33009-33017.

Research output: Contribution to journalArticle

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