Author ORCID Identifier
0000-0002-0917-8265
Date of Graduation
5-2022
Document Type
Dissertation (PhD)
Program Affiliation
Microbiology and Molecular Genetics
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Nayun Kim, PhD
Committee Member
Ambro van Hoof, PhD
Committee Member
Anne-Marie Krachler, Ph.D.
Committee Member
Margarida Albuquerque Almeida Santos, Ph.D.
Committee Member
Grzegorz Ira, Ph.D.
Abstract
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 Saccharomyces cerevisiae. 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 S. cerevisiae. 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 in vitro, 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 TOP1-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 in vitro 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 in vivo. 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.
Keywords
G-quadruplex, Topoisomerase 1