Publication Date
2025
Document Type
Thesis
Committee Members
Yong-jie Xu, M.D., Ph.D. (Advisor); Michael Kemp, Ph.D. (Committee Member); Ravi P. Sahu, Ph.D. (Committee Member)
Degree Name
Master of Science (MS)
Abstract
To maintain genome stability, which is essential for cell proliferation and survival, cells rely on cell cycle checkpoint pathways to deal with perturbed DNA replication or replication stress. The fission yeast Schizosaccharomyces pombe serves as a powerful model organism for studying checkpoint mechanisms because its fundamental cellular processes are highly conserved in higher eukaryotes. During a genetic screening for mutants affecting genome integrity, we identified a novel mutant called C3 that exhibits a unique phenotype. Unlike wild-type cells, where phosphorylation of the checkpoint mediator Mrc1 is typically observed only in response to exogenously induced replication stress, such as the treatment with hydroxyurea (HU), which depletes cellulat dNTPs, or the DNA-damaging agent, methyl methane sulfonate (MMS). The C3 mutant shows significant Mrc1 phosphorylation under physiological conditions without the treatment with a replication stress inducer, leading to the activation of the replication checkpoint. This suggests that a persistent endogenous replication stress occurs inside the C3 mutant cell. The characterization of the C3 mutant by drug sensitivity assays revealed that the mutant is highly sensitive to HU, but not MMS, highlighting a stress that mainly occurs during the S phase. This HU-sensitive phenotype remains after three backcrosses with wild-type strains, which rules out the possibility of multiple mutations in the genome. To further investigate the molecular mechanisms, we examined the phosphorylation of Mrc1 and Chk1in the replication checkpoint and the DNA damage checkpoint pathways to identify any potential defects in checkpoint signaling. The results showed that Mrc1 was phosphorylated even in the absence of iv HU treatment, which confirms the preliminary data. Consistent with MMS resistance, Chk1 phosphorylation was not much affected in the C3 mutant after MMS treatment . To identify the mutated gene in C3, we transformed the mutant with defined libraries consisting of the genes involved in DNA replication initiation and fork protection. However, none of these genes could rescue the mutant, suggesting that these genes are unlikely to be mutated in C3. Thus, ongoing efforts are focused on identifying the causative gene and elucidating the underlying molecular mechanism in endogenous replication stress.
Page Count
45
Department or Program
Department of Pharmacology and Toxicology
Year Degree Awarded
2025
Copyright
Copyright 2025, all rights reserved. My ETD will be available under the "Fair Use" terms of copyright law.
