Investigation of Genomic Instability Induced By G-Quadruplexes In The Absence of Functional to poisomerase 1 In Yeast

<p>Topoisomerase 1 (Top1) is an enzyme that removes transcriptionally generated negative supercoils by binding and nicking DNA. Since transcription of guanine-rich DNA leads to the formation of G-quadruplex (G4) structures, Top1’s function likely suppresses G4-formation. In support of this, Top1 significantly reduces co-transcriptional G4 DNA-associated genomic instability at a model G4-motif in <em>Saccharomyces cerevisiae</em>. However, whether Top1 suppresses G4-formation on a genome-wide scale in yeast remains unexplored. Therefore, I aimed to uncover if deletion of Top1 enhances genome-wide G4-formation in <em>S. cerevisiae</em>. As an approach to quantify global G4-formation, I expressed the G4-specific antibody BG4 from a yeast vector to perform chromatin immunoprecipitation next generation sequencing (ChIP-seq) and immunofluorescence experiments. While the G4-antibody’s function was verified <em>in vitro</em>, ChIP and immunofluorescence experiments failed, possibly due to localization of BG4 to the cytoplasm rather than the nucleus of yeast cells. Thus, future attempts at enumerating G4s in <em>TOP1</em>-deletion yeast cells should include the usage of expressed BG4 fused to a nuclear localization signal sequence or purified BG4 protein. Top1 mutants arise in cancer cells treated with the Top1-targeting anticancer drug camptothecin (CPT). Here, I show that the impact on G4-induced recombination in yeast depends on the type of CPT-resistant Top1 mutant expressed. While expression of a Top1 mutant defective in duplex DNA binding results in G4-recombination levels equivalent to cells completely lacking Top1, expression of cleavage-defective Top1 mutants has an even greater impact on G4-mediated instability. I also find that Top1 cleavage-defective mutants bind G4s <em>in vitro</em> and that the SPRTN homolog Wss1 involved in DNA/protein crosslink resolution partly suppresses G4-induced recombination in yeast cells expressing Top1 cleavage-defective mutants. Collectively, these data suggest that Top1 cleavage-defective mutants induce instability at guanine-rich DNA through G4-stabilization <em>in vivo</em>. Further, I uncovered that another G4-binding protein, Nsr1 or yeast nucleolin, contributes to G4-instability in yeast cells expressing Top1 mutants and provide additional evidence indicating that Top1 cleavage-defective mutants and Nsr1 interact when bound to G4s to form a potential replication block. Bioinformatic data revealed that cancer genomes harboring Top1 mutants predicted to be functionally defective exhibit enriched mutagenesis at G4-motifs. Yeast genetic datum showing that Top1 cleavage-defective mutants and Nsr1 have a synergistic effect on G4-instability through cooperative G4-binding taken together with the result of bioinformatic analyses suggest that CPT-resistance conferring Top1 mutants could induce mutagenesis at G4-motifs in cancer cells and complicate patient treatment. Since loss of Top1 function increases G4-instability, identifying additional protein factors that suppress or instigate G4-mediated DNA damage in the absence of functional Top1 is an attractive future direction of this work.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2022

Authors dc:creator

• Berroyer, Alexandra
• <p>0000-0002-0917-8265</p>

Contributors dc:contributor

• Nayun Kim, PhD
• Ambro van Hoof, PhD
• Anne-Marie Krachler, Ph.D.

• G-quadruplex
• Topoisomerase 1
• Biology
• Medicine and Health Sciences
• Microbiology

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/1165

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-2218