Peter Stirling

DNA replication is a vulnerable time for genome stability maintenance. Endogenous add oncogenic stressors can challenge replication by fostering conflicts with transcription and stabilizing DNA:RNA hybrids called R-loops. The capacity for unscheduled R-loops to collapse stalled replication forks and induce genome instability has become apparent. In this work, I provide an overview of the causes and cellular responses to replication stress, detailing the importance of replication checkpoint signaling, replication fork remodelling, and the clinical relevance to cancer development. Recently progress has been made towards identifying R-loop and conflict regulators, however, our understanding of this regulation is incomplete. Building off past findings that utilized a genome-wide interaction screen in budding yeast lacking key R-loop degrading RNase H enzymes, I explored a network of potential R-loop tolerance genes. PCNA is a complex replisome component with over 200 binding partners and was identified as a hit alongside RAD18 and ATAD5 (Elg1 homolog), key factors required for PCNA ubiquitin regulation. Once ubiquitinated, PCNA catalyzes several downstream repair pathways. I validated the roles of these genes in R-loop biology, identifying an accumulation of DNA:RNA hybrids in several human cell lines after gene silencing and in conjugation-incompetent PCNA K164R mutants. In both RAD18- and ATAD5-deficient cells, I observed higher levels of replication stress, DNA damage, and transcription-replication conflicts, dependent on R-loops, transcription, and replication. To gain mechanistic insight into how RAD18 tolerates transcriptional stress, I utilized stressors aphidicolin and pladienolide B to stall replication and disrupt the R-loop landscape respectively. I found that RAD18 localization was dependent on these transcriptional stressors and R-loops. I explored the mechanistic connection between RAD18 and members of the Fanconi anemia (FA) pathway, showing DNA:RNA hybrid epistasis. RAD18 was important for recruitment of FANCD2, the key effector molecule with established roles in resolving R-loop-induced stress, to R-loop prone loci and common fragile sites. These findings expand upon a published connection between RAD18 and the FA pathway by establishing a mechanism involving RAD18 in opposing transcription-associated genome instability through FANCD2 recruitment. The result of my work establishes PCNA ubiquitination as a novel regulatory axis for the tolerance of transcriptional stress.

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RNA splicing mutants have been broadly implicated in genome stability, but mechanistic links are often unclear. Two predominant models have emerged: one involving changes in gene expression that perturb other genome maintenance factors and another in which genotoxic DNA:RNA hybrids, called R-loops, impair DNA replication. Recent efforts in whole genome sequencing have identified splicing factor mutations in several cancers, suggesting that splicing disruption may be a common mechanism involved in oncogenesis. To understand how splicing factor mutations contribute to genetic instability (GIN) in budding yeast, I selected strains with mutations in core snRNP complexes involved in establishing the splicing reaction and characterized GIN phenotypes to find that mitotic defects, and in some cases R-loop accumulation, are causes of GIN. I observed evidence of R-loop induced DNA damage in some cases, while all splicing mutants tested caused GIN through aberrant splicing of the TUB1 transcript, the protein product of which, α-tubulin, is critical in forming the mitotic spindle. GIN is exacerbated by loss of the spindle-assembly checkpoint protein Mad1, and moreover, removal of the intron from the α-tubulin gene TUB1 restores genome integrity. To gain functional insights to how HSH155 could influence GIN in the context of cancer progression, I studied five cancer-associated SF3B1 point mutations in the yeast ortholog HSH155. While the splicing activity in Hsh155 and SF3B1 were conserved, I did not observe measurable phenotypes in the yeast mutant strains. Thus, I used isogenic NALM6 human leukemia cell lines to investigate how a specific SF3B1 hotspot mutation, H662Q contributes to GIN. My data indicate that GIN occurs in two ways: 1) by induction of R-loop-mediated replication stress either directly or indirectly through suboptimal expression of an R-loop modulating factor, and 2) aberrant splicing of the multifunctional protein DYNLL1, which may potentially perturb double strand break repair pathway choice. The results of my study show how differing penetrance and selective effects on the transcriptome in yeast and human splicing factors contribute to GIN through R-loop accumulation and altered gene expression, adding to a growing body of evidence that splicing factors play a key role in genome maintenance across species.

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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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