ROLES OF THE PIF1 HELICASE AND THE G-QUADRUPLEX STABILIZING DRUG PHENDC3 IN THE MAINTENACE OF GENOMIC INTEGRITY IN S. CEREVISIAE

Abstract

ABSTRACT:

In this thesis, I describe several approaches to gaining insight into the functions of S. cerevisiae Pif1 (ScPif1) and G-quadruplex structures. ScPif1 is the founding member of the Pif1 family of DNA helicases and has multiple roles in maintaining genomic stability.

My thesis consists of three different parts each designed to obtain a better understanding of how ScPif1 performs its various functions.

First, to identify genes that work in parallel to ScPIF1, I performed a synthetic genetic analysis using the mitochondrially proficient and nuclear deficient mutant, pif1-m2. The synthetic genetic analysis was performed by mating pif1-m2 to the entire prototrophic deletion collection, which consists of 4,783 single non-essential gene deletions, thus creating double mutants containing pif1-m2 and the deletion of one other non-essential gene. Strains that failed to form a double mutant haploid or that grew more slowly than either haploid contain a gene that is synthetically lethal or synthetically sick with pif1-m2. Twelve genes are synthetic lethal with pif1-m2, including the gene UMP1, which is required for the proper assembly of the proteasome. Three genes were synthetic sick with pif-1m2, including DEF1, a gene that, like PIF1, plays a role in telomere maintenance and replication fork progression.

Second, to understand how cells tolerate G-quadruplexes, I performed several analyses using the G-quadruplex stabilizing drug, PhenDC3. I first tested the entire prototrophic deletion collection for growth on phenDC3, identifying genes whose deletions resulted in an increased or decreased sensitivity to PhenDC3. Several genes whose deletions resulted in increased sensitivity to PhenDC3 are involved in autophagy and the maintenance of mitochondria. I then performed the PhenDC3 sensitivity test on double mutants containing both the pif1-m2 mutant and the deletion of one other non-essential gene, which identified several genes related to mitochondria as being important for growth in PhenDC3 in the pif1-m2 strain, including UTH1, which is a gene involved in the autophagy of the mitochondria (mitophagy). I also performed microarrays comparing the transcriptome of wild-type cells to each of the following: pif1-m2 cells, pif1-m2 cells treated with PhenDC3 and wild type cells treated with PhenDC3. Transcriptional changes in pif1-m2 cells and in wild type cells treated with PhenDC3 support a novel model of autophagy induction via endoplasmic reticulum stress induction and initiation of the unfolded protein response. Future work is needed to identify a link between G-quadruplex forming motifs and differential gene expression in pif1-m2 cells and wild type cells treated with G-quadruplex stabilizers.

Thirdly, I investigated the role of ScPif1 and PhenDC3 in autophagy. I discovered that the pif1-m2 strain has an elevated level of autophagy. The data from my synthetic genetic screen shows that pif1-m2 is synthetically lethal with ump1Δ, a key gene required for proper formation of the proteasome. Data from the microarray performed here demonstrated a downregulation of genes involved in endoplasmic-reticulum maintenance and an upregulation in genes involved in the unfolded protein response. Together, this suggests a model where the depletion of nuclear ScPif1 causes endoplasmic reticulum stress, which leads to an unfolded protein response that overwhelms the proteasome, requiring autophagy induction to tolerate the excess unfolded proteins. In the absence of a functional proteasome, as is seen with ump1∆ cells, the proposed increase in unfolded proteins could be toxic to the cell, explaining the synthetic lethal relationship between pif1-m2 and UMP1. Similarly, PhenDC3 treatment of wild type cells was found to induce autophagy, downregulate genes involved in endoplasmic reticulum maintenance and upregulate genes involved in the unfolded protein response.

Taken together, this work presents a novel relationship between the S. cerevisiae DNA helicase ScPif1, proteasome maintenance, protein folding and autophagy. Future studies will be needed to elucidate the relationship between ScPif1 induced transcriptional changes; PhenDC3 induced changes and the regulation of autophagy and protein folding.