DNA-protein cross-links (DPCs) are deleterious DNA lesions containing a covalent bond between DNA and a protein. DPCs can be induced by ionizing radiation, anticancer agents, and endogenous chemicals. DPCs are strong blocks to DNA replication and transcription and can lead to genomic instability and pathogenesis. Mitochondrial DNA (mtDNA)encodes 13 protein subunits in the oxidative phosphorylation system, 22 tRNA, and 2rRNAs. Considering mitochondria lack nucleotide excision repair pathways, which are key to the removal of DPC in nuclear DNA, DPC may accumulate in mtDNA and pose threats to mtDNA integrity. Unfortunately, the lack of robust methods to measure mitochondrial DPC has hindered the confirmation of the presence of such lesions in mtDNA and clarification of their biological consequence.<br/><br/>Herein, we developed an ELISA-based method for detecting DNA-protein-crosslinks in mitochondria from mammalian cell cultures. We use mitochondria transcription factor A (TFAM)-induced DPCs as a model system. TFAM is one of the most abundant proteins in mitochondrial nucleoid. Previously, we demonstrated that TFAM processed abasic (AP) sites via Schiff base chemistry and that reaction intermediate DPCs can be trapped <i>in vitro</i>. In our ELISA-based method, we optimized the procedure using synthetic TFAM-DNA cross-links. We show that the classic phenol/chloroform extraction failed to remove free TFAM and that commercial solid-phase extraction kits exhibit different recovery and efficiency. We also examined the effect of surface treatment of microplates, coating solutions, and HRP substrates on the specificity and sensitivity of the ELISA-based method. Our method uses commercially available materials and can distinguish between TFAM in TFAM-DNA cross-links and non-covalently associated TFAM. Our method confirms the presence of TFAM-DNA cross-links in human cells and has the potential to be applied to the detection of other forms of DPC in mitochondria.

An ELISA-based method for detecting DNA-protein-crosslinks in mitochondria from human cell cultures

Fall 2022 abstracts, posters, and presentations will launch in mid-September.

ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.