Abstract
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, Δ
dpb4
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.
