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.
Ermakova
O V
OV
Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461, USA.
Frappier
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L
Schildkraut
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CL
D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002474:Cell-Free System; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D053488:DnaB Helicases; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism*; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D046911:Macromolecular Substances; D013329:Structure-Activity Relationship; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming; D001426:Bacterial Proteins; D004279:DNA, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; D046911:Macromolecular Substances; D004265:DNA Helicases; D053488:DnaB Helicases