Date of Award

1-1-2025

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Deyu Li

Abstract

DNA is constantly under attack - from both internal cellular processes and external environmental sources. This ongoing stress creates a wide range of damage that can compromise genomic stability. Some stubborn and damaging lesions are etheno DNA adducts and interstrand cross-links (ICLs), which pose serious mutagenic and cytotoxic threats. While classic repair pathways like base excision repair (BER), nucleotide excision repair (NER), and direct reversal mechanisms have been well studied, recent findings suggest that the AlkB family of enzymes may have a broader role than previously thought - especially when it comes to handling oxidative and alkylative DNA damage, including etheno adducts and cross-links.

This dissertation dives into the discovery of new types of ICLs, examines the repair dynamics of etheno adducts, and explores how various AlkB homologs help mitigate these forms of DNA damage.

A key part of this work focuses on identifying and characterizing novel ICLs that form through etheno adduct chemistry. Using high-resolution mass spectrometry (HRMS) and LC-MS, we confirmed the formation of 3,N4-ethenocytosine (εC)-derived ICLs, which appeared in significantly higher yields than previously reported etheno cross-links. By using carefully designed oligonucleotide sequences, we also discovered that εC-ICL formation is sequence-dependent, which could help explain why certain genomic regions are more prone to mutation.

In addition, we uncovered that 3,N4 etheno-5-methylcytosine (ε5mC) is capable of forming stable ICLs with cytosine - a finding that broadens the known chemistry of etheno adducts. We also identified a high-yield εA-ICL, with formation rates much higher than those of previously described etheno adenine cross-links. This suggests εA damage may have a larger role in genome instability than we realized. To push the envelope further, we explored the possibility that small molecules could drive cross-link formation involving εA and εC. This opens intriguing questions about how such pathways might contribute to persistent DNA lesions or even therapeutic applications.

Beyond these discoveries, the project also expands what we know about FTO, an AlkB family enzyme typically known for its role in RNA modification. Through a combination of biochemical assays and kinetic studies, we identified several new substrates for FTO beyond ,N6-methyladenosine (m6A), showing that its role in nucleic acid modulation may be more diverse than explored. These findings offer fresh insight into how the AlkB family helps safeguard the genome.

To support this work, we successfully expressed and purified a full suite of AlkB proteins - AlkB, ALKBH2, ALKBH3, ALKBH5, FTO, and others - setting the stage for in-depth biochemical and structural studies. With these tools in hand, we explored each enzyme’s substrate preferences, kinetics, and potential for repairing complex DNA and RNA modifications.

Altogether, this dissertation pushes the boundaries of what we know about DNA repair. By uncovering new types of DNA cross-links, mapping enzyme-substrate relationships, and highlighting unexplored repair pathways, it adds to the growing understanding of how cells respond to DNA damage. These insights have potential implications for cancer biology, epigenetic regulation, and the future design of therapies targeting DNA repair systems.

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Available for download on Thursday, May 27, 2027

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