Title

DNA polymerase epsilon is important for the initiation of DNA replication in Schizosaccharomyces pombe

Date of Award

2002

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Gennaro D'Urso, Committee Chair

Abstract

DNA polymerase epsilon (Pol epsilon) is one of the three eukaryotic DNA polymerases that are essential for chromosomal DNA replication. Pol epsilon purified from both S. cerevisiae and human contains at least four subunits. In S. pombe, the composition of the holoenzyme is unknown. The large catalytic subunit of S. pombe Pol epsilon is a 256-kD polypeptide, encoded by the essential cdc20 + gene. The amino-terminal catalytic domains (approximately half of the polypeptide) are dispensable for cell viability in S. pombe. The carboxyl-terminal non-catalytic domain is both essential and sufficient for cell viability and chromosomal DNA replication. We propose that the C terminus of Pol epsilon is important for the initiation of DNA replication by facilitating the assembly of a replicative/initiation complex. However, S. pombe cells lacking the N-terminal catalytic domains of Pol epsilon are delayed in both S and G2 phase progression, sensitive to DNA damaging agents and are dependent on the intact DNA damage checkpoint for viability. These observations suggest that the N terminus of Pol epsilon, though dispensable for viability, normally does participate in chromosomal DNA replication and perhaps DNA repair. We also cloned the second largest regulatory subunit of Pol epsilon in S. pombe, named dpb2+. Using a chromatin immunoprecipitation (ChIP) assay, we demonstrate that both Dpb2p and the C terminus of Pol epsilon bind to the origins of DNA replication early during S phase, similar to Mcm6p, a component of the pre-replicative complex. Our data support the hypothesis that the essential function of Pol epsilon during chromosomal DNA replication is the assembly of an initiation complex. Finally, multi-copy suppressor screening of a Pol epsilon mutant, cdc20-M10, identified a gene sds23+. The sds23+ gene was previously implicated in the regulation of ubiquitin-dependent proteolysis pathway. Through a yeast two-hybrid screen using Sds23p as the bait, we identified a protein Mts2p that specifically interact with Sds23p. Mts2p is a conserved ATPase of the 19S regulatory complex of the 26S proteasome. We suggest that the overproduction of Sds23p up-regulates the ubiquitin-dependent proteolysis, likely through its interaction with the proteasome. The mechanism by which overproduction of Sds23p suppresses the cdc20 mutant will be speculated.

Keywords

Biology, Molecular; Biology, Genetics; Chemistry, Biochemistry

Link to Full Text

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