Journal Article

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

Maria-Grazia Spiga and Gennaro D'Urso

in Nucleic Acids Research

Volume 32, issue 16, pages 4945-4953
Published in print September 2004 | ISSN: 0305-1048
Published online September 2004 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/gkh811
Identification and cloning of two putative subunits of DNA polymerase epsilon in fission yeast

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DNA polymerase epsilon (Pol ϵ) 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 ϵ. Although Dpb4 is not required for cell viability, Δdpb4 mutants are synthetically lethal with mutations in four genes required for DNA replication initiation, cdc20+ (encoding DNA Pol ϵ), 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 ϵ 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.

Journal Article.  5960 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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