In higher eukaryotes, mitochondria are essential organelles for energy production, biosynthesis, and signaling pathways. One unique feature of these organelles is that mitochondria have their own genome, mitochondrial DNA (mtDNA). The circular mtDNA molecules encode thirteen proteins involved in the oxidative phosphorylation (OXPHOS) process and a set of transfer and ribosomal RNAs. Therefore, the maintenance of mtDNA integrity is critical for normal cellular activities. Compared to the genomic DNA, mitochondrial DNA (mtDNA) are more susceptible to damage due to its multi-copy nature and the lack of certain repair pathways. To cope with the difficult-to-repair lesions, or counteract excessive DNA damage, damaged mtDNA molecules will undergo degradation. Recently, mtDNA degradation has emerged as a key mechanism of mtDNA maintenance. Nonetheless, the mechanism of mtDNA degradation remains poorly understudied. Our group reported that mitochondrial transcription factor A (TFAM) facilitates the mtDNA strand cleavage activity at the abasic (AP) sites, a ubiquitous DNA lesion and common DNA repair intermediate. We demonstrated that the process occurs via Schiff base chemistry facilitated by a number of lysine residues. Considering that TFAM is rich in glutamate residues and its common role as a general base in enzyme catalysis, the objective aims to uncover the role of glutamate in TFAM facilitated AP-DNA degradation. When TFAM crosslink with AP-DNA, through Schiff base chemistry which will produce uncleaved intact DNA-protein complex (DPCunc), followed by β-elimination process which will generate the cleaved DPC (DPCcl), and finally the single strand breaks (SSB). We discovered a decreasing DPCunc content, and an elevated DPCunc /DPCcl ratio when using glutamate substituted TFAM variants, suggesting an impeded β-elimination step without the aid from glutamate. This result strongly supports our conclusion that glutamate residues play a catalytic role in the TFAM facilitated AP-DNA strand cleavage process.

Uncovering the role of glutamate in mitochondrial transcription factor a facilitated AP-DNA cleavage reaction

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