Investigation of Abasic Site-Induced Transcription Factor A-DNA Cross-Links
Boyd, Riley M.
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Damage to mitochondrial DNA plays a significant role in aging, as well as in various diseases. With the electron transport chain in close proximity to mitochondrial DNA, significant oxidative damage is regularly incurred. During repair of such damage, apurinic/apyrimidinic, or abasic, site formation is relatively common. Reactive abasic sites in nuclear DNA are known to form covalent bonds with proteins and their complementary strand of DNA, however such reactions have not been studied within mitochondrial DNA. This study explores the possibility of cross-linking between abasic site-containing DNA and an abundant mitochondrial protein that extensively interacts with mitochondrial DNA, mitochondrial transcription factor A. This work demonstrates the successful expression and isolation of mitochondrial transcription factor A through multiple purification steps including his-tag affinity purification, cation exchange chromatography, and size exclusion chromatography using fast protein liquid chromatography. Following analysis, it was determined that the isolated protein was not only free of major contaminants, but also showed to be fully active. The results of this study purified mitochondrial transcription factor A and abasic site containing DNA duplex, while also demonstrating DNA-protein cross-linking with mitochondrial transcription factor A and abasic site-containing DNA under reducing conditions. Further work is to explore cross-linking in environments more similar to those in vivo and with mitochondrial transcription factor A mutants in order to identify which residues may be important to this process.