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.

Mitochondrial DNA (mtDNA) is a circular DNA molecule existing in multiple copies in mitochondria. mtDNA contains a higher level of chemical-induced DNA lesions than nuclear DNA due to the accumulation of lipophilic and charged chemicals in the mitochondria and the lack of certain DNA repair pathways. mtDNA lesions can alter mitochondrial function, such as the reduction of mitochondrial respiration, mitochondrial membrane potential and an elevation of mitochondrial reactive oxygen species (ROS). In response to mtDNA damage, DNA repair, mtDNA degradation and mitochondrial dynamics are the major pathways to eliminate mtDNA damage. The relative contributions of mtDNA repair, degradation, and mitochondrial dynamics in response to different types of mtDNA damage remains an outstanding question. To address this question, characterizing these pathways in mtDNA damage cell models was conducted in this study. First, inducible mitochondrial targeting uracil DNA glycosylase 1 variant (UNG1-Y147A) transduced HeLa and HEK293 cells with APE1 siRNA transfection were used as mitochondrial abasic site (AP) lesion models. The mtDNA copy numbers were significantly reduced in the UNG1-Y147A-overexpressed HeLa and HEK293 cells. However, PCR-blocking lesions on mtDNA were not increased in the UNG1-Y147A- overexpressed or APE1-knockdown cells. DNA repair and mitochondria dynamics-related genes were not significantly altered. mtDNA degradation was the main response in the mtDNA AP lesion models. Second, mitochondrial targeting chemical mt-Ox was used to generate mtDNA oxidative damage in HeLa and HEK293 cells. Different from UNG1-Y147A, mt-Ox increased PCR-blocking lesions on mtDNA without reducing mtDNA copy number. mt-Ox also induced the expressions of DNA repair and mitochondria dynamics-related genes in HeLa cells. DNA repair and mitochondria dynamics were more significant in response to mtDNA oxidative damage. The difference in response might be due to the types and amounts of lesions. The mechanisms of damage response activation and decision need further investigation. In conclusion, this study provides solid evidence that cells had different damage responses in the mtDNA AP lesions and oxidative damage models. The evidence provides insights into different cellular responses to different types of mtDNA lesions and guides future research for illustrating the mtDNA damage induced adverse outcome pathways.

This study enhances our understanding of mitochondrial transcription factor A (TFAM) and its pivotal roles in maintaining mitochondrial DNA (mtDNA) integrity through a series of investigations. First, we focused on Glutamic acid 187 (E187) in the β-elimination reaction, which is an essential step in mtDNA repair. Using kinetic analyses and comprehensive computer simulations, we demonstrated how E187 accelerates the reaction rates quantitatively, providing critical insights into the molecular mechanisms that facilitate DNA repair within mitochondria.Next, our study has demonstrated the intrinsic 5’dRp lyase activity of TFAM, observed through in vitro experiments. The activity rate is comparable with that of polymerase β, implying TFAM's vital role in the mitochondrial Base Excision Repair (BER) pathway. Such functionality is particularly significant since it indicates TFAM can reduce the accumulation of toxic DNA repair intermediates. These intermediates if unrepaired, could lead to mitochondrial dysfunction, thus highlighting the broader implications of TFAM in maintaining cellular health and preventing disease progression. Finally, we are working on developing an innovative mass spectrometry method to detect TFAM-DNA-protein complexes (DPCs) in cells. This novel method aims to provide structural insights into the ways TFAM interacts with damaged mtDNA and allow us to detect other protein candidates that are binding with AP-mtDNA under physiological condition. The development of this technique is an important step towards understanding the complex dynamics of mtDNA-protein interactions within mitochondria. Taken together, the above projects help with our understanding of TFAM's molecular interactions and regulatory roles and broaden our knowledge of mtDNA stability. By elucidating the mechanisms that protect mtDNA integrity, our research contributes to the broader scientific understanding of mitochondrial function and its impact on cellular health.

Mitochondrial DNA (mtDNA) is required to maintain the function of the oxidative phosphorylation pathway required for bulk ATP synthesis. Mitochondrial dysfunction results from loss-of-function (mtDNA) maintenance proteins, and results in human pathologies such as cancer, cardiovascular, inflammatory, and neurological disorders. In this project, we report findings related to enzymes involved in mtDNA repair, replication, and degradation to gather better understanding of their function, interactions, and roles.First, we aimed to establish kinetic details of mitochondrial genome maintenance nuclease MGME1 to explain its previously reported bidirectional exonuclease activity and possible role in mtDNA replication. We found that in addition to preferential binding to a 5'-phosphate end, MGME1 prefers to degrade in the 5' to 3' direction, supporting its previous reports that it is a core participant in the mitochondrial replisome, where it aids mitochondrial polymerase γ, which only carries 3' to 5' exonuclease function. Next, we investigated the modulation of base-excision repair (BER) enzymes by TFAM. In mitochondria, mtDNA is often subject to exogenous and endogenous agents which can introduce DNA. We studied the effects of TFAM-DNA compaction on three glycosylases related to mitochondrial BER: uracil deglycosylase (UNG1), alkyladenine deglycosylase (AAG), and 8-oxoguanine deglycosylase (OGG1). We found that only UNG1 activity is stimulated by TFAM. Inclusion of AP endonuclease 1 (APE1), the enzyme that follows after the glycosylase excision in BER, reveals that TFAM also aided in generating strand-breaks in addition to simulation, overall illustrating a multi-layer regulation of mtDNA repair. Finally, we probed the modulation of mtDNA degradation nucleases MGME1 and flap endonuclease 1 FEN1 by mtDNA replication enzymes TFAM and mitochondrial single-stranded binding protein (mtSSB). MGME1 and FEN1 are reported to aid in degradation and release of oxidized mtDNA but the mechanism for degradation is unclear. Here, we report preliminary findings that may explain certain fragmentation patterns of products released as well as the tight control between discarding and maintaining mtDNA. Together these findings provide critical information from a biochemical perspective that improves our current understanding of mtDNA maintenance. In efforts to demystify enzymes involved in key steps in repair, replication, and degradation, we have found details that contribute to the broader scientific knowledge of mitochondrial genome maintenance, which by extension aids comprehension of mitochondrial and cellular health and function.