DNA POLYMERASE EPSILON DPB3 IS ESSENTIAL FOR CELL VIABILITY IN FISSION YEAST

M. Grazia Spigaand Gennaro D’Urso
Department of Biochemistry and Molecular Biology,
University of Miami School of Medicine, Miami Fl 33101

Introduction.  Eukaryotic chromosomal DNA replication requires the activity of at least three different DNA polymerases, alpha (Pol a), delta (Pol d) and epsilon (Pol e).  Pol a is thought to be responsible for primer synthesis whilst Pol d and e are involved in strand elongation.  However, recently it has been shown that in human cells Pol e may only be required for DNA synthesis in late S-phase (1).  Nevertheless, studies in both budding and fission yeast demonstrate that Pol e is required for chromosomal DNA replication (2,3).  Our results in fission yeast suggest that Pol e may have a specific role in the initiation step (4,5,6). 

DNA polymerase e consists of four subunits that are evolutionary conserved.  All the domains required for DNA synthesis activity reside in the N-terminal half of the large catalytic subunit, called Cdc20 in fission yeast.  We have previously shown that the N-terminal half of Pol e is dispensable for cell viability, suggesting that Pol e provides additional function(s) to the DNA replication complex (5).  We have also reported that the second largest subunit of Pol e, called Dpb2, is required early in S phase and binds directly to origin-associated chromatin providing further support that Pol e is required for initiation (6).  Here we report the cloning of the two remaining subunits of Pol e in fission yeast, called Dpb3 and Dpb4.

Methods.  We have used one step gene replacement to replace either dpb3+ or dpb4+ with the ura4+ gene marker.  To examine the role of the essential dpb3+ gene, we have used standard recombinant DNA techniques and constructed a Ddpb3 strain containing one copy of the dpb3+ gene under the control of a thiamine repressible promoter. Nuclei were counted after DAPI staining. To analyze synthetic-lethal phenotypes, we created double mutants between Ddpb4 strain and other replication mutants and analyzed their phenotypes at different temperatures. To study localization of each protein, the genes were endogenously tagged at the C-terminus with GFP. To analyze physical interactions between the different Pol e subunits, we used a GST pull down assay on proteins tagged with either GST- or Myc- followed by western blotting.

Results and Discussion.  Although Dpb4 is not required for cell viability, dpb4 deletion mutants are synthetically lethal with mutations in cdc20, which encodes the catalytic subunit of Pol e, cut5, sna41, and cdc21, proteins required for DNA replication initiation.  In contrast to Dpb4, Dpb3 is essential for cell cycle progression.  GST pull down assays indicate that Dpb3 physically interacts with Dpb2, suggesting that Dpb3 is a component of the Pol e complex.  Interestingly, a high percentage of fission yeast cells depleted for Dpb3 arrest with a bi-nucleate or multi-nucleate phenotype indicating that in addition to its putative role in DNA replication, Dpb3 may have additional functions during the later stages of cytokinesis.  Finally, we have examined the in vivo localization of GFP tagged Dpb3 and Dpb4 and found that both proteins are present in the nucleus consistent with a role for these proteins in DNA replication.  In the absence of Dpb3, GFP-Dpb4 is absent from the nucleus suggesting that Dpb3 may be important to establish or maintain normal localization of Dpb4.  

Acknowledgments.
M.G. Spiga is supported by an American Heart Association pre-doctoral fellowship.  G. D’Urso is supported by the National Institute of Health (NCI grant IR01-CA-099034)

References.

1. Fuss J, Linn S.  J Biol Chem. 2002 Mar 8;277(10):8658-66.
2. Aparicio OM, Weinstein DM, Bell SP.  Cell. 1997 Oct 3;91(1):59-69.
3. Masumoto H, Sugino A, Araki H.  Mol Cell Biol. 2000 Apr;20(8):2809-17.
4. D'Urso G, Nurse P. Proc Natl Acad Sci USA. 1997 Nov11; 94(23):12491-6.
5. Feng W, D'Urso G.  Mol Cell Biol. 2001 Jul;21(14):4495-504.
6. Feng W, Rodriguez-Menocal L, Tolun G, D'Urso G.  Mol Biol Cell. 2003 Aug;14(8):3427-36.