UC Riverside

Guanine quadruplex (G4) structures are nonconical DNA conformations that play crucial roles in various cellular processes, including DNA replication and transcription regulation. G4 structures are frequently found in promoter regions of oncogenes, which render G4 structures promising therapeutic targets. However, the mechanisms through which G4 structures regulate biological functions remain underexplored. This dissertation focuses on the development of novel quantitative proteomic methods to identify putative G4-binding proteins, to characterize novel G4-binding proteins, to explore cellular modifiers of G4 stability, and to discover of novel biomarkers in breast cancer cells. In chapter 2, we employed an affinity-based quantitative proteomics analysis to identify novel G4-binding proteins. By utilizing three different biotinylated G4-forming oligonucleotides as probes, I achieved a comprehensive analysis of proteins interacting with various G4 conformations, which led to the discovery of over 30 G4-binding proteins. Building upon the pull-down experiments, I introduced, in Chapter 3, the use of photo-crosslinking G4 probes to capture G4-binding proteins. This approach enabled the capture of weak and transient interactions, where harsh washing conditions eliminate protein-protein interactions during the pull-down process. By Using this method, I identified 99 putative G4-binding proteins, and I also characterized one of these proteins, HELLS as a novel G4 helicase. In Chapter 4, I demonstrated, for the first time, that G4 DNA structures can undergo phase separation. By utilizing immunofluorescence microscopy and ChIP-seq analysis, I observed that phase separation modulates the stabilities of G4 structures in vitro and in cells. This discovery provides new insights into factors that modulate the formation and stabilities of G4 structures in cells. In Chapter 5, I identified novel small GTPases as biomarkers of radioresistance in breast cancer cells through a multiple-reaction monitoring-based targeted proteomics analysis. The study revealed ARFRP1 as a novel radioresistance biomarker in breast cancer cells, where its downregulation promotes radioresistance. In conclusion, this dissertation presents novel proteomic approaches for the identification of G4-binding proteins, understanding G4 stability modulation, and the discovery of potential biomarkers in breast cancer cells. These findings enhance our understanding of G4 biology and offer new avenues for therapeutic interventions and biomarker-driven cancer research.