Mitochondrial transcription factor A promotes DNA strand cleavage at abasic sites

Abstract

In higher eukaryotic cells, mitochondria are essential subcellular organelles for energy production, cell signaling, and the biosynthesis of biomolecules. The mitochondrial DNA (mtDNA) genome is indispensable for mitochondrial function because it encodes protein subunits of the electron transport chain and a full set of transfer and ribosomal RNAs. MtDNA degradation has emerged as an essential quality control measure to maintain mtDNA and to cope with mtDNA damage resulting from endogenous and environmental factors. Among all types of DNA damage known, abasic (AP) sites, sourced from base excision repair and spontaneous base loss, are the most abundant endogenous DNA lesions in cells. In mitochondria, AP sites trigger rapid DNA loss; however, the mechanism and molecular factors involved in the process remain elusive. Herein, we demonstrate that the stability of AP sites is reduced dramatically upon binding to a major mtDNA packaging protein, mitochondrial transcription factor A (TFAM). The half-life of AP lesions within TFAM–DNA complexes is 2 to 3 orders of magnitude shorter than that in free DNA, depending on their position. The TFAM-catalyzed AP-DNA destabilization occurs with nonspecific DNA or mitochondrial light-strand promoter sequence, yielding DNA single-strand breaks and DNA–TFAM cross-links. TFAM–DNA cross-link intermediates prior to the strand scission were also observed upon treating AP-DNA with mitochondrial extracts of human cells. In situ trapping of the reaction intermediates (DNA–TFAM cross-links) revealed that the reaction proceeds via Schiff base chemistry facilitated by lysine residues. Collectively, our data suggest a novel role of TFAM in facilitating the turnover of abasic DNA.

Mitochondrial DNA degradation: A quality control measure for mitochondrial genome maintenance and stress response

“…APE1 is known to localize to mitochondria and particularly relevant to AP lesion repair. We found that TFAM competes actively with APE1-mediated DNA cleavage using mitochondrial extracts from HeLa and HEK-293 cells [162], suggesting that TFAM interacts strongly with AP lesions under physiological concentration ratios of TFAM to APE1, and TFAM could play a regulatory role in mtDNA repair, as suggested previously [161]. Because mtDNA degradation and repair may occur in parallel to alleviate abasic DNA damage [127], it is plausible that TFAM could act redundantly with APE1 to trigger mtDNA turnover or to regulate the partitioning between mtDNA degradation and repair.…”

“…Furthermore, TFAM has been proposed to play a role in regulating mtDNA repair [161], though the underlying mechanism remains to be clarified. Recently, my laboratory has identified a novel role for TFAM in facilitating the degradation of damaged mtDNA containing abasic (AP) sites [162], which are ubiquitous DNA lesions and an important intermediate during BER.…”

“…Considering the abundance of TFAM and its role as a histone-like protein in packaging mtDNA, it is inevitable that TFAM encounters the ubiquitous AP sites. Recently, my laboratory has shown that TFAM accelerates the degradation of AP lesion-containing DNA using biochemical assays and mitochondrial extracts from human cells [162]. Based on the proximity of the ε-NH 2 group of several lysine residues and the C1′-carbon of the nearby nucleotide residue in the TFAM-DNA crystal structures [38,160], we hypothesized that several lysine residues might react with AP sites nearby to form single-strand breaks ( Fig.…”

Mechanisms of replication and repair in mitochondrial DNA deletion formation

“…The selective depletion of damaged mtDNA lowers the overall population of mutated and/or deleted mtDNA molecules. For example, mtDNA molecules harboring abasic sites ( 77 , 78 ) or DNA double-stranded breaks (DSBs) ( 72 , 73 ) could be directly degraded rather than repaired. As mtDNA is under a strict copy-number control, selective depletion of damaged mtDNA molecules triggers replication of the undamaged mtDNA copies, reconstituting the mtDNA pool (reviewed in ( 79 )).…”

“…Among the proteins that negatively regulate BER, TFAM binds the damaged mtDNA in close proximity to an abasic site. By steric exclusion of BER proteins, such as glycosylases and the APE1 nuclease, TFAM inhibits BER activation and promotes the degradation of the targeted mtDNA molecule ( 77 , 78 ). In stark contrast with its established roles in promoting nuclear DNA repair, the PARP1 protein inhibits BER within mitochondria.…”