UBC Theses and Dissertations
Genome instability in multiple myeloma-associated DIS3 exonuclease domain mutants Milbury, Karissa Lynn
Chromosome instability (CIN) is characterized by an increased rate of the unequal distribution of DNA between daughter cells. Such changes in chromosome structure or number can occur due to both mitotic defects leading to aneuploidy and DNA damage-induced chromosome rearrangements. Previous large-scale screens for CIN genes in the model organism Saccharomyces cerevisiae identified DIS3, which encodes a catalytic component of the core RNA exosome complex, as a novel CIN gene. Mutations in human DIS3 have been identified in roughly 11% of multiple myeloma (MM) cases. I sought to recapitulate MM-associated point mutations at conserved sites in yeast cells, in order to understand the mechanism of emergent CIN in MM. I have found that MM-associated DIS3 exonuclease mutations do increase the frequency of CIN. A temperature sensitive DIS3 mutant accumulates DNA:RNA hybrids, however analysis of DNA damage foci by microscopy revealed no increase in double-strand breaks in any of the tested strains. Yeast DIS3 exonuclease mutants experience growth retardation, temperature sensitivity, and an altered cell cycle. Microarray analysis of one MM mutant has additionally demonstrated downregulation of cell cycle components, consistent with the potential for mitotic defects, in addition of upregulation of a host of metabolic pathways. Further, genetic interaction profiling by synthetic genetic array indicates MM-associated DIS3 mutations synthetically interact with rRNA processing proteins, as well as a host of mitotic regulators and metabolic pathways, particularly those involved in spindle and kinetochore function. Further, I verify that DIS3 mutants have a functional spindle assembly checkpoint, and are in fact resistant to microtubule poisons. Finally, I discover that the fitness defects induced by these mutations can be abrogated through culturing on media containing only a non-fermentable carbon source, suggesting that growth on poor carbon sources may also rescue CIN. Together, these results demonstrate extensive phenotypic consequences of MM-associated point mutations in DIS3, and support a model for CIN in DIS3 mutants involving defects in mitotic progression.
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