Title

Studies on the two smallest subunits of DNA polymerase epsilon, and a DNA replication origin binding protein in fission yeast

Date of Award

2004

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Gennaro D'Urso, Committee Chair

Abstract

Replication of chromosomal DNA is initiated at multiple origin sites called ARS elements. Before DNA synthesis can begin a number of proteins are recruited to these origins and sequentially assembled to form a pre-replicative complex. It is generally believed that, following activation by cyclin dependent kinases, DNA polymerases are loaded and DNA synthesis begins.The DNA polymerase epsilon complex (Pol epsilon) is one of three DNA polymerases required for eukaryotic chromosomal DNA replication and consists of four subunits that are evolutionary conserved. Interestingly, all the domains required for DNA synthesis activity, which reside in the N-terminal half of the large catalytic subunit, are dispensable for cell viability in yeast, indicating that Pol epsilon might be involved in other cellular processes. To investigate the function(s) of the DNA polymerase epsilon complex, I have identified and cloned the two smallest subunits of Pol epsilon in fission yeast, called Dpb3 and Dpb4. My results show that Dpb3 is essential for viability. Cells deleted for dpb3 display an elongated cdc phenotype distinctive of cell cycle defective mutants. However, analysis of their DNA content by FACS analysis indicate that cells arrest in G2 with a 2C DNA content and that a small number of these cells display 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. On the other hand, Dpb4 is not essential; however, synthetic lethality with a number of known temperature sensitive initiation mutants suggests that Pol epsilon and its associated subunits participates in the early events of DNA replication and might be part of pre-replication and/or initiation complexes present at the origins. Both Dpb3 and Dpb4 localize to the nucleus; however, in the absence of Dpb3, Dpb4 is dispersed in the cytoplasm suggesting that Dpb3 is essential for the proper localization of Dpb4 and perhaps might be necessary for the entire Pol epsilon complex stability.Additionally, I investigated the DNA replication role of the origin binding protein, Abp1. This protein had been originally purified based on its ARS binding activity and we had later found a yeast-two hybrid interaction with the essential replication protein Cdc23. We have identified and cloned two fission yeast homologues of Abp1, called Abp3 and Cbh. Genetic analysis of double and triple mutants together with FACS analysis of triple mutant cells depleted of Abp1 show that these three proteins display a moderate S-phase defect.

Keywords

Biology, Molecular

Link to Full Text

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