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

Abstract

In higher eukaryotic cells, mitochondria are essential organelles for energy production, metabolism, and signaling. Mitochondrial DNA (mtDNA) encodes 13 protein subunits for oxidative phosphorylation and a set of tRNAs and rRNAs. mtDNA damage, sourced from endogenous chemicals and environmental factors, contributes to mitochondrial genomic instability, which has been associated with various mitochondrial diseases. DNA–protein cross-links (DPCs) are deleterious DNA lesions that threaten genomic integrity. Although much has been learned about the formation and repair of DPCs in the nucleus, little is known about DPCs in mitochondria. Here, we present in vitro and in cellulo data to demonstrate the formation of DPCs between a prevalent abasic (AP) DNA lesion and a DNA-packaging protein, mitochondrial transcription factor A (TFAM). TFAM cleaves AP-DNA and forms DPCs and single-strand breaks (SSB). Lys residues of TFAM are critical for the formation of TFAM-DPC and a reactive 3′-phospho-α,β-unsaturated aldehyde (3′pUA) residue on SSB. The 3′pUA residue reacts with two Cys of TFAM and contributes to the stable TFAM-DPC formation. Glutathione reacts with 3′pUA and competes with TFAM-DPC formation, corroborating our cellular experiments showing the accumulation of TFAM-DPCs under limiting glutathione. Our data point to the involvement of TFAM in AP-DNA turnover and fill a knowledge gap regarding the protein factors in processing damaged mtDNA.

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

“…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.…”

“…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. AP sites are one of the most abundant DNA lesions in mtDNA and nDNA, present at a level of 1 AP lesion/10 5 –10 6 nt …”

“…Key to the AP lyase activity of TFAM is its high Lys content (15% of the amino acid residues in the mature TFAM). A proximal Lys activates an AP lesion for β-elimination via Schiff-base chemistry to form single-strand breaks (SSBs). , A body of literature in the DNA repair field has shown that for DNA glycosylases, the β-elimination step can be catalyzed by carboxylate-containing Glu or Asp residues (e.g., T4 endonuclease V), primary or secondary amines (e.g., NEIL1, also reviewed in ref ). Similarly, pol β uses E71 to perform a water-assisted H-abstraction at the C2′ position to achieve its dRp-lyase activity .…”

“…Intriguingly, more than half of these Glu residues in TFAM are present in KE clusters (Figure B). Considering that multiple Lys residues can be involved in Schiff base formation with AP sites, ,, a proximal Glu would be able to facilitate β-elimination by the deprotonating the 2′ carbon atom of the sugar ring, or serving as a Schiff base counterion, or both. Yet, the potential contribution of the highly abundantly Glu residues to the AP-lyase activity of TFAM has not been explored.…”

“…Herein, we clarify the role of a Glu residue (E187) in TFAM-mediated AP-DNA cleavage using wet-lab experiments and computer simulations. E187 is chosen given its proximity to the hotspot K186 in TFAM-facilitated AP-lyase reactions. ,, We demonstrate that E187 facilitates β-elimination through detailed kinetic analysis and molecular dynamics (MD) simulations. Our kinetic assays and simulations show that the TFAM E187A variant has a reduced rate of β-elimination.…”