Publication Date
2010
Document Type
Thesis
Committee Members
Steven Berberich (Committee Member), Andrew Hsu (Other), Michael Leffak (Committee Member), Yong-jie Xu (Advisor)
Degree Name
Master of Science (MS)
Abstract
The initiation of DNA replication is a highly conserved process in all eukaryotes. However, the underlying mechanism is not well understood. Genetic studies in the fission yeast S. pombe have contributed greatly to and will continue to provide insights to our understanding of this important biological process.
In the first chapter, we have used a complementary method to test three recently identified human replication proteins DUE-B, Ticrr/Treslin, and GEMC1 as the candidate functional homologue of Sld3 in S. pombe. Sld3 is an essential replication initiation protein discovered in yeasts. Since no apparent sequence similarity can be found, its homologue in higher eukaryotes remains to be uncovered. In fact, among all yeast replication proteins, Sld3 is the only protein whose functional homologue has not been found in metazoan. Our preliminary results showed that all three human replication proteins failed to complement the function of Sld3 in fission yeast. Unlike DUE-B, whose expression does not perturb the cell cycle progression in fission yeast, overexpression of Ticrr and GEMC1 in S. pombe can suppress the cell growth.
In the second chapter, we have developed a screening strategy in S. pombe to discover new gene(s) that may function in the initiation of DNA replication. A yeast strain has been made in which the promoter of Chk1, the effector kinase of the DNA damage checkpoint in fission yeast, was replaced with a thiamine-repressive promoter. The Chk1 expression in this strain can be completely shut off by adding thiamine in the culture medium. It is known that defects in the replication initiation require the checkpoint function for cell survival. A mutation in the gene involved in the initiation of DNA replication is expected to be sensitive to the depletion of the Chk1 function. In a small-scale screening, we obtained 15 so-called Kds (Chk1 dependent survival) mutants. Characterization of one the mutants Kds15 in the preliminary study identified a structure-destructive mutation in Psf2, a subunit of the essential GINS complex required for the initiation of DNA replication. This result validates the strategy and shows that the screening can be productive.
Page Count
88
Department or Program
Department of Biochemistry and Molecular Biology
Year Degree Awarded
2010
Copyright
Copyright 2010, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.
