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Nucleic Acids Research 2004 32(16):4945-4953; doi:10.1093/nar/gkh811
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Published online 23 September 2004

Nucleic Acids Research, Vol. 32 No. 16 © Oxford University Press 2004; all rights reserved

Identification and cloning of two putative subunits of DNA polymerase epsilon in fission yeast

Maria-Grazia Spiga and Gennaro D'Urso*

Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, PO Box 016129, Miami, FL 33101-1019, USA

* To whom correspondence should be addressed. Tel: +1 305 243 3105; Fax: +1 305 243 3064; Email: gdurso{at}miami.edu

Received May 11, 2004; Revised July 20, 2004; Accepted August 18, 2004

DNA polymerase epsilon (Pol {epsilon}) is a multi-subunit enzyme required for the initiation of chromosomal DNA replication. Here, we report the cloning of two fission yeast genes, called dpb3+ and dpb4+ that encode proteins homologous to the two smallest subunits of Pol {epsilon}. Although Dpb4 is not required for cell viability, {Delta}dpb4 mutants are synthetically lethal with mutations in four genes required for DNA replication initiation, cdc20+ (encoding DNA Pol {epsilon}), cut5+ (homologous to DPB11/TopBP1), sna41+ (homologous to CDC45) and cdc21+ (encoding Mcm4, a component of the pre-replicative complex). In contrast to Dpb4, Dpb3 is essential for cell cycle progression. A glutathione S-transferase pull-down assay indicates that Dpb3 physically interacts with both Dpb2 and Dpb4, suggesting that Dpb3 associates with other members of the Pol {epsilon} complex. Depletion of Dpb3 leads to an accumulation of cells in S phase consistent with Dpb3 having a role in DNA replication. In addition, many of the cells have a bi-nucleate or multinucleate phenotype, indicating that cell separation is also inhibited. Finally, we have examined in vivo localization of green fluorescent protein (GFP)-tagged Dpb3 and Dpb4 and found that both proteins are localized to the nucleus consistent with their proposed role in DNA replication. However, in the absence of Dpb3, GFP-Dpb4 appears to be more dispersed throughout the cell, suggesting that Dpb3 may be important in establishing or maintaining normal localization of Dpb4.


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