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A more recent version of this article appeared on August 1, 2003
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Submitted on February 18, 2003
Revised on April 9, 2003
Accepted on April 11, 2003
1 Department of Biochemistry and Molecular Biology, P.O. Box 016129, University of Miami School of Medicine, Miami, FL 33101-6129 (present address: Department of Genome Sciences, University of Washington, Box 357730, Seattle WA 98195)
2 Department of Biochemistry and Molecular Biology, P.O. Box 016129, University of Miami School of Medicine, Miami, FL 33101-6129
* Corresponding author. E-mail address: gdurso{at}miami.edu.
Genetic evidence suggests that DNA polymerase epsilon (Pol 
) has a non-catalytic essential role during the early stages of DNA replication initiation. Here we report the cloning and characterization of the second largest subunit of Pol ?in fission yeast, called Dpb2. We demonstrate that Dpb2 is essential for cell viability, and that a temperaturesensitive mutant of dpb2 arrests with a 1C DNA content, suggesting that Dpb2 is required for initiation of DNA replication. Using a chromatin immunoprecipitation assay (ChIP), we show that Dpb2, binds preferentially to origin DNA at the beginning of S phase. We also show that the C-terminus of Pol ?associates with origin DNA at the same time as Dpb2. We conclude that Dpb2 is an essential protein required for an early step in DNA replication. We propose that the primary function of Dpb2 is to facilitate assembly of the replicative complex at the start of S phase. These conclusions are based on the novel cell cycle arrest phenotype of the dpb2 mutant, on the previously uncharacterized binding of Dpb2 to replication origins, and on the observation that the essential function of Pol ?is not dependent on its DNA synthesis activity.
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