An Enzyme-Linked Immunosorbent Assay for the Detection of Mitochondrial DNA–Protein Cross-Links from Mammalian Cells

Abstract

DNA–Protein cross-links (DPCs) are cytotoxic DNA lesions with a protein covalently bound to the DNA. Although much has been learned about the formation, repair, and biological consequences of DPCs in the nucleus, little is known regarding mitochondrial DPCs. This is due in part to the lack of robust and specific methods to measure mitochondrial DPCs. Herein, we reported an enzyme-linked immunosorbent assay (ELISA)-based method for detecting mitochondrial DPCs formed between DNA and mitochondrial transcription factor A (TFAM) in cultured human cells. To optimize the purification and detection workflow, we prepared model TFAM-DPCs via Schiff base chemistry using recombinant human TFAM and a DNA substrate containing an abasic (AP) lesion. We optimized the isolation of TFAM-DPCs using commercial silica gel-based columns to achieve a high recovery yield for DPCs. We evaluated the microplate, DNA-coating solution, and HRP substrate for specific and sensitive detection of TFAM-DPCs. Additionally, we optimized the mtDNA isolation procedure to eliminate almost all nuclear DNA contaminants. For proof of concept, we detected the different levels of TFAM-DPCs in mtDNA from HEK293 cells under different biological conditions. The method is based on commercially available materials and can be amended to detect other types of DPCs in mitochondria.

DNA–protein cross-links between abasic DNA damage and mitochondrial transcription factor A (TFAM)

“…The accumulation of TFAM-DPCs in HeLa cells under limited GSH (∼70% relative to the control) and an elevated AP lesion level, but not the induction of AP sites alone, suggests a role of GSH in regulating the TFAM-DPC formation. Interestingly, in HEK293 cells, we observed that an elevation of mitochondrial AP site level is sufficient to cause a statistically significant increase of TFAM-DPCs ( 25 ). This can be explained by the overall lower level of total cellular glutathione (GSH and GSSG) in HEK293 cells compared to HeLa cells ( 51 ).…”

“…To quantify TFAM-DPCs in HeLa cells, we devised an ELISA-based method to measure TFAM-DPCs ( 25 ). ELISA was chosen to measure the total amount of TFAM covalently attached to mtDNA regardless of the cross-linking chemistry.…”

“…The development and validation our ELISA-based method for detecting TFAM-DPCs were described previously ( 25 ). Briefly, isolated mitochondria were resuspended in 50 μl of ice-cold 1× PBS, which contained 60 μg of RNase A (NEB, T3018L).…”

Key Amino Acid Residues of Mitochondrial Transcription Factor A Synergize with Abasic (AP) Site Dynamics To Facilitate AP-Lyase Reactions

“…63 The high abundance of Lys and Glu residues in TFAM supports the role of TFAM in facilitating strand scission at ubiquitous AP sites. Although the observed rate of AP-DNA cleavage is much slower than other DNA repair enzymes, the abundance of TFAM in nucleoids and our detection of elevated TFAM-DPCs upon inducing mitochondrial AP sites in HeLa and HEK293 cells 21,22 argue the involvement TFAM in processing AP sites in mitochondria. The strand scission at AP sites converts AP lesions to more deleterious "roadblocks" in the forms of DPCs and SSBs to various mtDNA transactions, which could potentially serve as signals for recruiting additional factors (e.g., DNA nucleases) or for purifying selection against damaged mtDNA molecules.…”

“…Pol γ and MGME1 process mtDNA containing induced double-strand breaks into shorter fragments in mammalian cells and mouse models. − FEN1 has been implicated in promoting mtDNA fragment release into cytoplasm on the basis of reduced cytosolic mtDNA fragments upon silencing FEN1 . In addition, mitochondrial transcription factor A (TFAM) has emerged as a new player in mtDNA turnover. − As a key mtDNA-packaging protein, TFAM organizes mtDNA into DNA-protein complexes known as nucleoids . In addition, TFAM is an essential factor in mtDNA transcription activation. , Recently, TFAM has been shown to cleave DNA molecules containing abasic (AP) sites in vitro and in cells, , arguing its role in damaged mtDNA turnover.…”