Molecular mechanisms of mammalian mitochondrial DNA replication and transcription

Abstract

Mitochondria are cytoplasmic organelles in eukaryotic cells, mainly devoted to the synthesis of ATP, through the Oxydative Phospshorylation System (OXPHOS). The organelle has its own genome, the mitochondrial DNA (mtDNA) that encodes for all the RNA components of the mitochondrial translation apparatus (two rRNAs and 22 tRNAs) and for 13 subunits of the OXPHOS complexes. Many trans-acting factors involved in the replication and expression of the mtDNA have been identified, but the molecular mechanisms regulating these processes are still poorly understood.

In our work, we have reconstituted the minimal mammalian mtDNA replisome in vitro. Together, the mitochondrial DNA polymerase (POLgamma) and the mitochondrial DNA helicase TWINKLE form a processive replication machinery, which can use doublestranded DNA (dsDNA) as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. The addition of the mitochondrial ssDNA-binding protein (mtSSB) stimulates the reaction, generating DNA products of about 16 kb, the size of the mammalian mtDNA molecule. These findings provide biochemical evidence for TWINKLE being the helicase at the mtDNA replication fork and define the minimal mitochondrial replisome.

Previous studies had shown that the human mitochondrial transcription termination factor (mTERF) governs site-specific termination of the mitochondrial transcription process. We used bioinformatic analysis and could demonstrate that mTERF is a member of a protein family (the MTERF family) shared among metazoans and plants. Interestingly, we identified three novel MTERF genes in vertebrates (MTERF2 - 4), which all encode proteins with predicted mitochondrial localization.

To analyze the function of one of these MTERF proteins, MTERF3, we used a combination of mouse genetics and biochemistry. We could demonstrate that MTERF3 is a negative regulator of mtDNA transcription initiation. The MTERF3 gene is essential because homozygous knockout mouse embryos die in midgestation. Heart-specific disruption of MTERF3 gene impairs mtDNA transcription and causes severe respiratory chain deficiency. Biochemical evidences show that MTERF3 binds the mtDNA promoter region and depletion of MTERF3 increases transcription initiation on both mtDNA strands. MTERF3 was the first described mitochondrial protein that specifically repressed mammalian mtDNA transcription initiation in vivo.

A biochemical approach has been used to characterize another member of the MTERF family, MTERF2. We have confirmed the mitochondrial localization of the protein and the position of the cleavage site of its mitochondrial targeting peptide was identified. MTERF2 is a monomer in isolation and it binds mtDNA in a non sequence-specific manner. In vivo quantification experiments show that MTERF2 is relatively abundant, with one monomer present per ∼ 265 bp of mtDNA. Using formaldehyde cross-linking we demonstrated that MTERF2 is present in nucleoids, and therefore located in close proximity to mtDNA. The functional role of MTERF2 is still unknown.