UC Riverside

Mitochondria are important subcellular compartments, crucial for energy production, metabolism, and cell signaling. Mitochondrial dysfunction is known to cause nearly 200 mitochondrial disorders and has been associated with aging and a variety of human diseases. Mitochondrial DNA (mtDNA) encodes 37 genes, including 13 proteins and a set of tRNA and rRNA. mtDNA is constantly threatened by chemical and physical assaults. Because mitochondria have limited DNA repair pathways, mtDNA damage accumulates and occurs at a higher level compared to nuclear DNA. Abasic (AP) sites are abundant DNA lesions that can be generated from various pathways, including base excision repair (BER). AP sites are highly reactive and can form secondary DNA adducts, DNA-interstrand cross-links (ICLs), and DNA-protein cross-links (DPCs). My dissertation project exploits the chemistry of AP sites and develops methods to explore biological processes pertinent to AP sites. First, I developed a mass spectrometry-based method to identify the cross-linked amino acid residues in DNA-protein cross-links. I designed DNA substrates with ribonucleotides (rNMPs), which provide chemical-labile sites for DNA strand cleavage reactions and produce structurally defined DPCs. Also, I developed a program (AP_CrosslinkFinder) to accelerate data analysis. The method was applied to identify the cross-linking amino acid residues in DPCs derived from mitochondrial transcription factor A (TFAM). Second, I developed a method to prepare model ICLs using rNMP-containing DNA with a nucleotide analog 2-aminopurine. The alkaline lability of rNMP enables the generation of strand breaks at specific sites. AP sites react with 2-aminopurine with high yield and high rate. This method provides a simple and straightforward tool for investigating the impact of ICLs during the repair process. Third, I investigated the DNA terminal structures generated in TFAM-catalyzed AP-DNA strand cleavage. Quantification of reaction rates in the presence of biological amines and thiols demonstrates that GSH competes with TFAM for AP site strand breaks, suggesting a possible strategy to limit the formation of DPC and control the strand break terminus in cells. Removal of DNA terminal modifications by relevant DNA repair enzymes was also evaluated. Together, results from my dissertation provide insights into the complexity of AP site chemistry with important biological implications.