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Vol. 14, Issue 8, 3427-3436, August 2003

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Schizosacchromyces pombe Dpb2 Binds to Origin DNA Early in S Phase and Is Required for Chromosomal DNA Replication

Wenyi Feng ^*, Luis Rodriguez-Menocal, Gökhan Tolun, and Gennaro D'Urso 

Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101-6129

Submitted February 18, 2003; Revised April 9, 2003; Accepted April 9, 2003
Monitoring Editor: Mark Solomon

Genetic evidence suggests that DNA polymerase epsilon (Pol ) has a noncatalytic essential role during the early stages of DNA replication initiation. Herein, 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 temperature-sensitive mutant of dpb2 arrests with a 1C DNA content, suggesting that Dpb2 is required for initiation of DNA replication. Using a chromatin immunoprecipitation assay, 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.

Abbreviations used: ChIP, chromatin immunoprecipitation; Pol, DNA polymerase; PCR, polymerase chain reaction.

^* Present address: Department of Genome Sciences, University of Washington, Box 357730, Seattle, WA 98195.

Corresponding author. E-mail address: gdurso{at}miami.edu.

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