Date of Award
2025
Degree Type
Dissertation
Degree Name
Doctor of Philosophy in Pharmaceutical Sciences
Specialization
Medicinal Chemistry
Department
Biomedical and Pharmaceutical Sciences
First Advisor
Deyu Li
Abstract
Modifications of DNA and RNA, in particular methylations - such as N6-methyladenine (m6A), 5-methylcytosine (m5C), 1-methyladenine (m1A), and 3-methylcytosine (m3C) are crucial for gene regulation, genome integrity, and cellular adaptation. This dissertation investigates the role of the ALKB family of alpha-ketoglutarate (αKG) and iron-dependent dioxygenases in modulating these modifications, with a focus on how their activity is influenced by pH and metabolic factors, underscoring the connection between environmental conditions and epigenetic and genetic stability.
Chapter 1 provides a comprehensive literature review on the epigenetic and genetic regulation of DNA and RNA modifications, with a particular focus on methylation and its demethylation by the ALKB family of enzymes. This chapter discusses the mechanisms and biological significance of key modifications, including 5-methylcytosine (m5C) and N6-methyladenosine (m6A), detailing their roles in gene expression, genomic stability, and disease pathology. The structural and functional characteristics of the ALKB family are explored in depth, highlighting their substrate specificity and repair mechanisms. Additionally, the chapter examines how environmental and metabolic factors, such as pH and oncometabolites, influence ALKB-mediated demethylation, establishing a foundation for the experimental investigations presented in subsequent chapters.
Chapter 2 addresses the pH sensitivity of ALKB, ALKBH2, and ALKBH3, characterizing how even subtle pH changes can impact their efficiency in repairing methylated DNA substrates. These findings reveal that the enzymatic activity of ALKB family members is closely tuned to pH, influencing DNA stability across varying pH environments.
Chapter 3 investigates the role pH plays in the demethylation of the epigenetic marker m6A by its eraser enzymes, FTO (also known as ALKBH9) and ALKBH5, in a specific context often called the “motif” or “consensus” sequence. This chapter highlights how pH variations affect m6A levels, thereby modulating gene expression and potentially contributing to disease pathways when dysregulated.
Chapter 4 turns to the effects oncometabolites impart on AlkB family mediated DNA demethylation. Here, the investigation focuses on the inhibitory effects of Krebs Cycle metabolites implicated in the progression of cancer, hence “oncometabolites”. In addition, the kinetics of αKG are investigated as a pathway leading to investigating, kinetically, the roles these oncometabolites play.
Collectively, this dissertation presents a comprehensive view of AlkB family enzymes as critical sensors and mediators of genetic and epigenetic modifications. By elucidating how pH sensitivity and the inhibitory effects of oncometabolites govern demethylation processes, these findings suggest new avenues for therapeutic strategies potentially targeting epigenetic, genetic, and/or metabolic imbalances in disease.
Recommended Citation
Mylie, Quentin, "THE EFFECTS OF PH AND ONCOMETABOLITES ON DNA REPAIR MEDIATED BY ALKB FAMILY ENZYMES" (2025). Open Access Dissertations. Paper 4480.
https://digitalcommons.uri.edu/oa_diss/4480