Browsing by Subject "DNA damage response"
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Item Conserved and Unconventional Responses to DNA Damage in Tetrahymena(2012-07-16) Sandoval Oporto, PamelaHere the ciliate protozoa Tetrahymena thermophila was used as a model system to study the DNA damage response. Tetrahymena enclose nuclear dimorphism, a polyploid somatic macronucleus (MAC), which is transcriptionally active and maintains vegetative growth, and a diploid germline micronucleus (MIC) responsible for the transmition of genetic information during conjugation. Previous studies have identified Tif1p, a novel protein involved in the regulation of rDNA replication in Tetrahymena. TIF1 hypomorphic strains acquire spontaneous DNA damage during vegetative cell cycle and are hypersensitive to DNA damaging agents. TIF1-deficient strains acquire DNA damage in both nuclear compartments, suggesting a global role of Tif1p in the maintenance of genomic stability. In my dissertation research, I studied the role of Tif1p during the cell cycle progression. To this end, I generated tagged-Tif1p strains, which revealed that the subcellular localization of Tif1p is dynamic throughout the cell cycle. However, the addition of epitope tag to this protein generated phenotypes analogous to ones observed in a TIF1-deficient strain. This suggested that the addition of epitope tag to Tif1p severely affects the properties of Tif1p and hence the overall integrity of the cell. To overcome these limitations, a peptide antibody specific to Tif1p was generated to study the endogenous protein. This work revealed that the abundance of Tif1p protein is not cell cycle regulated and that Tif1p is absent in starved cells. Furthermore, the specific binding of TIf1p to rDNA minichromosome was studied during vegetative cell cycles. Chromatin immunoprecipitation studies revealed that the specific binding of Tif1p extends beyond the cis-acting determinant of replication present at the rDNA origin and promoter. This suggests that coding regions may be targeted for the binding of Tif1p to previously uncharacterized sequences, and that Tif1p preferentially localizes on the rDNA minichromosome. I also studied the induction of DNA damage response, demonstrating that Tetrahymena activates a checkpoint response mediated by an ATR-like pathway. Studies with a hypomorphic TIF1 strain revealed that Tif1p mediates proper activation of the DNA damage response. Further characterization of the response to genotoxic agents showed that Tetrahymena is able to activate a G1/S and intra-S phase DNA damage response. The results presented here suggest that a caffeine-dependent checkpoint activator protein modulates the response to DNA damage. In addition, a subunit of the replicative helicase, Mcm6p, is directly affected by the induction of DNA damage. This suggests that Tetrahymena uses a novel mechanism to halt the progression of DNA replication forks during genotoxic stress through degradation of Mcm6p.Item The tumor-promoting functions of Ataxia-telangiectasia mutated (ATM) in cancer cells(2015-05) Chen, Wei-Ta, Ph. D.; Miller, Kyle M.; Van den berg, CarlaAtaxia-telangiectasia mutated (ATM) protein kinase regulates the DNA damage response (DDR) and is associated with cancer suppression by protecting cells from DNA double-strand breaks (DSBs). However, how ATM functions outside of DSB signaling is less clearly understood. Here, we report a new cancer-promoting role for ATM in stimulating cell migration and invasion independently of DSB signaling or induction. We used two highly metastatic human breast cancer cell lines to corroborate that ATM is required for cell migration and invasion. Microarray analysis of cells depleted for ATM identified interleukin-8 (IL-8) as a target since the exogenous addition of IL-8 rescued migration and invasion defects in ATM-deficient cells. Finally, ATM depletion in human cancer cells reduced lung metastasis in a mouse xenograft model. These findings shed light on tumor-promoting functions of ATM. Therefore, in addition to its canonical roles in tumor suppression, ATM promotes tumor progression as well.