Doctor of Philosophy (PhD)
Eukaryotic organisms implement conserved surveillance machinery to sense and respond to DNA damage. Fundamental to the repair process is coordinated regulation of repair genes and initiation of cell cycle arrest protocols. Failure to preserve these checkpoints results in accumulation of mutated DNA and aberrant cell phenotypes that are characteristic of human disease. The yeast, Saccharomyces cerevisiae, utilizes the MEC1 checkpoint pathway to regulate the DNA damage response. This study addressed two overarching themes of transcriptional and post-translational regulation within the MEC1 pathway. We first applied our understanding of the MEC1 DNA damage transcriptional response to develop advanced luciferase whole cell biosensors that could detect a broad range of carcinogens using the promoter sequence of the MEC1 DNA repair gene, HUG1. The enhanced whole cell yeast biosensor exhibited improved sensitivity and dynamic range when compared to fluorescent-based biosensors while reducing reporter read-out processing time through a one-step, in vivo measurement regime. Previous global transcription studies performed in our lab identified a dose-dependent biphasic response of MEC1 repair genes to alkylating agents. The origin of this unique profile, however, remained unknown. Using a GFP promoter-reporter construct placed under MEC1 pathway genes, we found that the biphasic response persists through the MEC1 pathway, and that neither reactive oxygen species accumulation nor pro-apoptotic genes contributed to the expression profile. Cell cycle analysis revealed that cells immediately enter a senescent state after experiencing high alkylating concentrations which we proposed was the root cause of the MEC1 pathway gene repression. The role of a functionally uncharacterized MEC1 DNA repair protein, HUG1, in the DNA damage response was also explored. Using overexpression phenotype and subcellular localization assays, we demonstrated that HUG1 is a negative regulator of the MEC1 pathway and that its co-localization with the positive MEC1 effector, Rnr2p, was likely the source of its regulation. Protein affinity assays confirmed the Hug1p-Rnr2p interaction while mutagenesis analysis probed domains within Hug1p to determine regions necessary to its inhibitory action. Finally, we discovered that Hug1p also interacts with human ribonucleotide reductase homologs, p53R2 and hRRM2, demonstrating that Hug1p uses a conserved interaction motif for its inhibition.
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Ainsworth, William Barrett, "Analyses of the MEC1 DNA Damage Pathway in Saccharomyces cerevisiae" (2015). LSU Doctoral Dissertations. 1185.