Oxidative stress induces degradation of mitochondrial DNA

Mitochondrial DNA (mtDNA) is located in close proximity of the respiratory chains, which are the main cellular source of reactive oxygen species (ROS). ROS can induce oxidative base lesions in mtDNA and are believed to be an important cause of the mtDNA mutations, which accumulate with aging and in diseased states. However, recent studies indicate that cumulative levels of base substitutions in mtDNA can be very low even in old individuals. Considering the reduced complement of DNA repair pathways available in mitochondria and higher susceptibility of mtDNA to oxidative damage than nDNA, it is presently unclear how mitochondria manage to maintain the integrity of their genetic information in the face of the permanent exposure to ROS. Here we show that oxidative stress can lead to the degradation of mtDNA and that strand breaks and abasic sites prevail over mutagenic base lesions in ROS-damaged mtDNA. Furthermore, we found that inhibition of base excision repair enhanced mtDNA degradation in response to both oxidative and alkylating damage. These observations suggest a novel mechanism for the protection of mtDNA against oxidative insults whereby a higher incidence of lesions to the sugar–phosphate backbone induces degradation of damaged mtDNA and prevents the accumulation of mutagenic base lesions

TFAM in mtDNA Homeostasis: Open Questions

Transcription factor A, mitochondrial (TFAM) is a key player in mitochondrial DNA (mtDNA) transcription and replication [...

The mitochondrial genome sequence of the BS-C-1 cell line is at odds with the reported derivation from Cercopithecus aethiops

The BS-C-1 cell line widely used in virological research was reportedly derived from the African green monkey Cercopithecus (Chlorocebus) aethiops. We used Sanger sequencing to determine the full nucleotide sequence of mtDNA in BS-C-1 cells. The mitochondrial genome in this cell line is 16,456 bp long and has an organization typical of that in other mammalian genomes with 37 genes, including 2 rRNAs, 22 tRNAs, and 13 polypeptide-encoding genes. Surprisingly, this genome is most closely related to that of Chlorocebus pygerythrus, which does not agree with the reported derivation of this cell line from C. aethiops. Another unexpected observation is the presence of mtDNA length heteroplasmy within the MT-ND6 gene, with the variant containing a stretch of 8 Cs encoding a functional gene, whereas the variant containing 9 Cs encodes a frameshifted gene. It is unclear whether the latter variant is nonfunctional or whether it is corrected by programmed translational ribosomal −1 frameshifting. The availability of the full mtDNA sequence for the BS-C-1 cell line should increase its utility by enabling studies on mtDNA transcription and replication

TFAM’s Contributions to mtDNA Replication and OXPHOS Biogenesis Are Genetically Separable

The ability of animal orthologs of human mitochondrial transcription factor A (hTFAM) to support the replication of human mitochondrial DNA (hmtDNA) does not follow a simple pattern of phylogenetic closeness or sequence similarity. In particular, TFAM from chickens (Gallus gallus, chTFAM), unlike TFAM from the “living fossil” fish coelacanth (Latimeria chalumnae), cannot support hmtDNA replication. Here, we implemented the recently developed GeneSwap approach for reverse genetic analysis of chTFAM to obtain insights into this apparent contradiction. By implementing limited “humanization” of chTFAM focused either on amino acid residues that make DNA contacts, or the ones with significant variances in side chains, we isolated two variants, Ch13 and Ch22. The former has a low mtDNA copy number (mtCN) but robust respiration. The converse is true of Ch22. Ch13 and Ch22 complement each other’s deficiencies. Opposite directionalities of changes in mtCN and respiration were also observed in cells expressing frog TFAM. This led us to conclude that TFAM’s contributions to mtDNA replication and respiratory chain biogenesis are genetically separable. We also present evidence that TFAM residues that make DNA contacts play the leading role in mtDNA replication. Finally, we present evidence for a novel mode of regulation of the respiratory chain biogenesis by regulating the supply of rRNA subunits

35 Years of TFAM Research: Old Protein, New Puzzles

Transcription Factor A Mitochondrial (TFAM), through its contributions to mtDNA maintenance and expression, is essential for cellular bioenergetics and, therefore, for the very survival of cells. Thirty-five years of research on TFAM structure and function generated a considerable body of experimental evidence, some of which remains to be fully reconciled. Recent advancements allowed an unprecedented glimpse into the structure of TFAM complexed with promoter DNA and TFAM within the open promoter complexes. These novel insights, however, raise new questions about the function of this remarkable protein. In our review, we compile the available literature on TFAM structure and function and provide some critical analysis of the available data

Complete replacement of basic amino acid residues with cysteines in Rickettsia prowazekii ATP/ADP translocase

AbstractThe ATP/ADP translocase (Tlc) of Rickettsia prowazekii is a basic protein with isoelectric point (pI)=9.84. It is conceivable, therefore, that basic residues in this protein are involved in electrostatic interactions with negatively charged substrates. We tested this hypothesis by individually mutating all basic residues in Tlc to Cys. Unexpectedly, mutations of only 20 out of 51 basic residues resulted in greater than 80% inhibition of transport activity. Moreover, 12 of 51Cys-substitution mutants exhibited higher than wild-type (WT) activity. At least in one case this up-effect was additive and the double mutant Lys422Cys Lys427Cys transported ATP five-fold better than WT protein. Since in these two single mutants and in the corresponding double mutant Km's were similar to that of WT protein, we conclude that Tlc may have evolved a mechanism that limits the transporter's exchange rate and that at least these two basic residues play a key role in that mechanism.Based on the alignment of 16 Tlc homologs, the loss of activity in the mutants poorly correlates with charge conservation within the Tlc family. Also, despite the presence of three positively charged and one negatively charged intramembrane residues, we have failed to identify potential charge pairs (salt bridges) by either charge reversal or charge neutralization approaches