Publication Date
2025
Document Type
Thesis
Committee Members
Yong-Jie Xu, M.D., Ph.D. (Advisor); Michael G. Kemp, Ph.D. (Committee Member); Ravi P. Sahu, Ph.D. (Committee Member)
Degree Name
Master of Science (MS)
Abstract
Ribonucleotide reductase (RNR) is a key enzyme for the biosynthesis of dNTPs for DNA replication and repair. It is composed of a large subunit, Cdc22, and a small subunit, Suc22, in the fission yeast S. pombe. We have recently reported a suc22-S239F mutation in the small subunit of RNR, causing replication stress and hypersensitivity to hydroxyurea (HU), an inhibitor of RNR. Interestingly, this mutation does not sensitize the cell to acute treatment with HU, which mainly generates replication stress, suggesting that the mechanism of HU sensitivity in the suc22-S239F mutant remains to be fully understood. To better understand the cell-killing mechanism of HU in the suc22-S239F mutant, suppressor mutations that restore HU resistance in the suc22-S239F mutant were screened by random mutagenesis of the genome. The screened mutants were validated by spot assays, DNA sequencing to identify the mutations and the mutated genes, and genomic integrations of the identified mutations. We found that while some of the suppressor mutants carry secondary mutations in suc22-S239F, the other suppressor mutants do not have secondary suc22 mutations, suggesting that unknown mutations remain to be identified in the genome. Genomic DNAs have been purified from several such mutants for iv genome sequencing. It is anticipated that more mutations will be identified. Although the research is still ongoing, our preliminary data suggest that the suppressor mutations can restore dNTP balance or bypass the checkpoint defects. These results may provide insights into RNR regulation, the interplay between RNR regulation and checkpoint signaling, and a potential therapeutic strategy to increase replication stress for cancer treatment.
Page Count
74
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.
