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’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

Escherichia coli in Europe: An Overview

Escherichia coli remains one of the most frequent causes of several common bacterial infections in humans and animals. E. coli is the prominent cause of enteritis, urinary tract infection, septicaemia and other clinical infections, such as neonatal meningitis. E. coli is also prominently associated with diarrhoea in pet and farm animals. The therapeutic treatment of E. coli infections is threatened by the emergence of antimicrobial resistance. The prevalence of multidrug-resistant E. coli strains is increasing worldwide principally due to the spread of mobile genetic elements, such as plasmids. The rise of multidrug-resistant strains of E. coli also occurs in Europe. Therefore, the spread of resistance in E. coli is an increasing public health concern in European countries. This paper summarizes the current status of E. coli strains clinically relevant in European countries. Furthermore, therapeutic interventions and strategies to prevent and control infections are presented and discussed. The article also provides an overview of the current knowledge concerning promising alternative therapies against E. coli diseases

A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication

The unavailability of tractable reverse genetic analysis approaches represents an obstacle to a better understanding of mitochondrial DNA replication. Here, we used CRISPR-Cas9 mediated gene editing to establish the conditional viability of knockouts in the key proteins involved in mtDNA replication. This observation prompted us to develop a set of tools for reverse genetic analysis in situ, which we called the GeneSwap approach. The technique was validated by identifying 730 amino acid (aa) substitutions in the mature human TFAM that are conditionally permissive for mtDNA replication. We established that HMG domains of TFAM are functionally independent, which opens opportunities for engineering chimeric TFAMs with customized properties for studies on mtDNA replication, mitochondrial transcription, and respiratory chain function. Finally, we present evidence that the HMG2 domain plays the leading role in TFAM species-specificity, thus indicating a potential pathway for TFAM-mtDNA evolutionary co-adaptations

Presequence-independent mitochondrial import of LigA-myc is inefficient.

<p>A. Genotyping cell lines transduced with either MTS-LigA-myc or with LigA-myc; B. mtDNA content in the parental 4B6 cells, and in cells in which mtDNA replication is supported by either MTS-LigA-myc (+MTS) or LigA-myc (No MTS); C. Whole cell and mitochondrial fractions from cells that express either MTS-LigA-myc or LigA-myc were subjected to western blotting with myc-tag antibodies, or with antibodies against reference proteins TOM70 (resides in the mitochondrial outer membrane) or MnSOD (resides in the mitochondrial matrix).</p

ChVlig can support mtDNA replication at reduced copy number.

<p>A, 4B6 cells were sequentially transduced with retroviruses encoding ChVLigAnd Cre recombinase, and Lig3 excision was tested in 13 resulting clones. B, resulting clones have variable mtDNA copy number as compared to an arbitrarily chosen clone#1. C, Clones #5, #6, and #13 maintained >90% reduced mtDNA copy number upon 3-week propagation in the selective medium. D, mtDNA copy number in the 4B6 cells transduced with ChVlig, two clones (#3 and #14), which were derived from the original ChVlig-transduced clone by Lig3 excision, and in the clone#14 after transduction with either WT or catalytically inactive mLig3.</p

The effect of LigA on mtDNA copy number in 4B6 cells.

<p>A, 4B6 cells were transduced with a retrovirus encoding LigA without MTS, and mtDNA copy number was determined in four resulting subclones. B, Subclone #11 was transduced with a retrovirus encoding Cre recombinase, and genomic DNA from ten clones was PCR-analyzed for the presence of unexcised Lig3 allele (Lig3), excised Lig3 allele (ΔLig3), ρ⁰ phenotype (mtDNA and nDNA primers), and for the presence of LigA (LigA). Primer sets and primer sequences are listed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152705#pone.0152705.s005" target="_blank">S1 Table</a>.</p