Steven J. Berberich (Committee Member), Kozak J. Ashot (Committee Member), Michael Leffak (Committee Member), Robert W. Putnam (Committee Member), Yong-jie Xu (Advisor)
Doctor of Philosophy (PhD)
Hydroxyurea (HU, also known as hydroxycarbamide) is a well known ribonucleotide reductase (RNR) inhibitor that depletes cellular deoxyribonucleoside triphosphates (dNTPs, the building blocks of DNA). Depleted dNTP pools inside the proliferating cells stalls ongoing DNA replication forks, leading to the activation of the well-conserved DNA replication checkpoint (also known as intra-S phase checkpoint) pathway. One of the major functions of the checkpoint pathway is to protect ongoing forks from collapsing. Stalled forks, if not protected by the checkpoint pathway, lead to DNA damage and ultimately cell death. Thus it is believed that DNA damage resulting from collapsed forks is the underlying cause of cell death induced by HU, although HU may kill the cells by other mechanisms. This dissertation focuses on defining a novel cell killing mechanism of HU in the fission yeast Schizosaccharomyces pombe. A mutation has been identified in hem13+ encoding coproporphyrinogen III oxidase, an essential enzyme involved in the heme biosynthetic pathway that significantly sensitizes the cells to HU. We found that the HU sensitivity of the hem13 mutant is a consequence of increased production of reactive oxygen species caused by HU treatment, not the DNA damage generated by fork collapsing. This newly identified cell killing mechanism of HU in hem13 mutant has great implications in antifungal therapy. The first part of this dissertation focuses on the discovery of the novel hem13 mutant in S. pombe using genetic screening. The second part rules out the possibility of a checkpoint defect in the screened mutant. The third part describes oxidative stress as the novel cell killing mechanism of HU in hem13 mutant. In the last part, we show the strong synergistic cell killing effect of sampangine (SMP), an inhibitor of heme synthesis and HU in S. pombe, S. cerevisiae and the pathogenic fungus Candida albicans. Taken together, this dissertation defines a novel cell killing mechanism of HU in S. pombe and the synergistic effect of HU and SMP in suppressing cell growth of wild type S. pombe, S. cerevisiae and pathogenic fungus C. albicans.
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