Frédérique Weber-Lotfi

DNA repair in organelles: Pathways, organization, regulation, relevance in disease and aging.

Both endogenous processes and exogenous physical and chemical sources generate deoxyribonucleic acid (DNA) damage in the nucleus and organelles of living cells. To prevent deleterious effects, damage is balanced by repair pathways. DNA repair was first documented for the nuclear compartment but evidence was subsequently extended to the organelles. Mitochondria and chloroplasts possess their own repair processes. These share a number of factors with the nucleus but also rely on original mechanisms. Base excision repair remains the best characterized. Repair is organized with the other DNA metabolism pathways in the organelle membrane-associated nucleoids. DNA repair in mitochondria is a regulated, stress-responsive process. Organelle genomes do not encode DNA repair enzymes and translocation of nuclear-encoded repair proteins from the cytosol seems to be a major control mechanism. Finally, changes in the fidelity and efficiency of mitochondrial DNA repair are likely to be involved in DNA damage accumulation, disease and aging. The present review successively addresses these different issues.

Mitochondrial Targeting and Membrane Anchoring of a Viral Replicase in Plant and Yeast Cells

ABSTRACT Replication of the Carnation Italian ringspot virus genomic RNA in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection. ORF1 in the viral genome encodes a 36-kDa protein, while ORF2 codes for the 95-kDa replicase by readthrough of the ORF1 stop codon. We have shown previously that the N-terminal part of ORF1 contains the information leading to vesiculation of mitochondria and that the 36-kDa protein localizes to mitochondria. Using infection, in vivo expression of green fluorescent protein fusions in plant and yeast cells, and in vitro mitochondrial integration assays, we demonstrate here that both the 36-kDa protein and the complete replicase are targeted to mitochondria and anchor to the outer membrane with the N terminus and C terminus on the cytosolic side. Analysis of deletion mutants indicated that the anchor sequence is likely to correspond approximately to amino acids 84 to 196, containing two transmembrane domains. No evidence for a matrix-targeting presequence was found, and the data suggest that membrane insertion of the viral proteins is mediated by an import receptor-independent signal-anchor mechanism relying on the two transmembrane segments and multiple recognition signals present in the N-terminal part of ORF1.

The plant mitochondrial genome: Dynamics and maintenance

Plant mitochondria have a complex and peculiar genetic system. They have the largest genomes, as compared to organelles from other eukaryotic organisms. These can expand tremendously in some species, reaching the megabase range. Nevertheless, whichever the size, the gene content remains modest and restricted to a few polypeptides required for the biogenesis of the oxidative phosphorylation chain complexes, ribosomal proteins, transfer RNAs and ribosomal RNAs. The presence of autonomous plasmids of essentially unknown function further enhances the level of complexity. The physical organization of the plant mitochondrial DNA includes a set of sub-genomic forms resulting from homologous recombination between repeats, with a mixture of linear, circular and branched structures. This material is compacted into membrane-bound nucleoids, which are the inheritance units but also the centers of genome maintenance and expression. Recombination appears to be an essential characteristic of plant mitochondrial genetic processes, both in shaping and maintaining the genome. Under nuclear surveillance, recombination is also the basis for the generation of new mitotypes and is involved in the evolution of the mitochondrial DNA. In line with, or as a consequence of its complex physical organization, replication of the plant mitochondrial DNA is likely to occur through multiple mechanisms, potentially involving recombination processes. We give here a synthetic view of these aspects.

Developing a genetic approach to investigate the mechanism of mitochondrial competence for DNA import

Mitochondrial gene products are essential for the viability of eukaryote obligate aerobes. Consequently, mutations of the mitochondrial genome cause severe diseases in man and generate traits widely used in plant breeding. Pathogenic mutations can often be identified but direct genetic rescue remains impossible because mitochondrial transformation is still to be achieved in higher eukaryotes. Along this line, it has been shown that isolated plant and mammalian mitochondria are naturally competent for importing linear DNA. However, it has proven difficult to understand how such large polyanions cross the mitochondrial membranes. The genetic tractability of Saccharomyces cerevisae could be a powerful tool to unravel this molecular mechanism. Here we show that isolated S. cerevisiae mitochondria can import linear DNA in a process sharing similar characteristics to plant and mammalian mitochondria. Based on biochemical data, translocation through the outer membrane is believed to be mediated by voltage-dependent anion channel (VDAC) isoforms in higher eukaryotes. Both confirming this hypothesis and validating the yeast model, we illustrate that mitochondria from S. cerevisiae strains deleted for the VDAC-1 or VDAC-2 gene are severely compromised in DNA import. The prospect is now open to screen further mutant yeast strains to identify the elusive inner membrane DNA transporter.

DNA delivery to mitochondria: sequence specificity and energy enhancement.

ABSTRACTPurposeMitochondria are competent for DNA uptake in vitro, a mechanism which may support delivery of therapeutic DNA to complement organelle DNA mutations. We document here key aspects of the DNA import process, so as to further lay the ground for mitochondrial transfection in intact cells.MethodsWe developed DNA import assays with isolated mitochondria from different organisms, using DNA substrates of various sequences and sizes. Further import experiments investigated the possible role of ATP and protein phosphorylation in the uptake process. The fate of adenine nucleotides and the formation of phosphorylated proteins were analyzed.ResultsWe demonstrate that the efficiency of mitochondrial uptake depends on the sequence of the DNA to be translocated. The process becomes sequence-selective for large DNA substrates. Assays run with a natural mitochondrial plasmid identified sequence elements which promote organellar uptake. ATP enhances DNA import and allows tight integration of the exogenous DNA into mitochondrial nucleoids. ATP hydrolysis has to occur during the DNA uptake process and might trigger phosphorylation of co-factors.ConclusionsOur data contribute critical information to optimize DNA delivery into mitochondria and open the prospect of targeting whole mitochondrial genomes or complex constructs into mammalian organelles in vitro and in vivo.

Targeting nucleic acids into mitochondria: Progress and prospects

Given the essential functions of these organelles in cell homeostasis, their involvement in incurable diseases and their potential in biotechnological applications, genetic transformation of mitochondria has been a long pursued goal that has only been reached in a couple of unicellular organisms. The challenge led scientists to explore a wealth of different strategies for mitochondrial delivery of DNA or RNA in living cells. These are the subject of the present review. Targeting DNA into the organelles currently shows promise but remarkably a number of alternative approaches based on RNA trafficking were also established and will bring as well major contributions.

Mitochondrial transfection for studying organellar DNA repair, genome maintenance and aging

Maintenance of the mitochondrial genome is a major challenge for cells, particularly as they begin to age. Although it is established that organelles possess regular DNA repair pathways, many aspects of these complex processes and of their regulation remain to be investigated. Mitochondrial transfection of isolated organelles and in whole cells with customized DNA synthesized to contain defined lesions has wide prospects for deciphering repair mechanisms in a physiological context. We document here the strategies currently developed to transfer DNA of interest into mitochondria. Methodologies with isolated mitochondria claim to exploit the protein import pathway or the natural competence of the organelles, to permeate the membranes or to use conjugal transfer from bacteria. Besides biolistics, which remains restricted to yeast and Chlamydomonas reinhardtii, nanocarriers or fusion proteins have been explored as methods to target custom DNA into mitochondria in intact cells. In further approaches, whole mitochondria have been transferred into recipient cells. Repair failure or error-prone repair leads to mutations which potentially could be rescued by allotopic expression of proteins. The relevance of the different approaches for the analysis of mitochondrial DNA repair mechanisms and of aging is discussed.

Nucleic acid import into mitochondria: New insights into the translocation pathways

Mitochondria have retained indispensable but limited genetic information and they import both proteins and nucleic acids from the cytosol. RNA import is essential for gene expression and regulation, whereas competence for DNA uptake is likely to contribute to organellar genome dynamics and evolution. Contrary to protein import mechanisms, the way nucleic acids cross the mitochondrial membranes remains poorly understood. Using proteomic, genetic and biochemical approaches with both plant and yeast organelles, we develop here a model for DNA uptake into mitochondria. The first step includes the voltage-dependent anion channel and an outer membrane-located precursor fraction of a protein normally located in the inner membrane. To proceed, the DNA is then potentially recruited in the intermembrane space by an accessible subunit of one of the respiratory chain complexes. Final translocation through the inner membrane remains the most versatile but points to the components considered to make the mitochondrial permeability transition pore. Depending on the size, DNA and RNA cooperate or compete for mitochondrial uptake, which shows that they share import mechanisms. On the other hand, our results imply the existence of more than one route for nucleic acid translocation into mitochondria.

Biochemical and molecular studies on declining and decline-resistant spruce in the north-east of France.

In declining forests of the Vosges mountains (northeast of France), we previously observed that the yellowing of spruce (Picea abies L. cv. Karsten) needles was associated with impairment of the free radical scavenging capacity of the cells and coincided with chronic exposure to ozone. Chloroplasts of yellow needles were characterized by an abnormal accumulation of photosystem II (PSII) D1-protein in the thylakoids. Further experiments carried out on declining and decline-resistant individual spruce trees characterized in previous studies showed that needle yellowing was associated with impairment of the overall anti-oxidative defense in both the cytosol and the chloroplasts. Both enzymic (peroxidases) and non-enzymic (carotenoids) oxidant scavengers were shown to be affected in the declining spruce. PSII D1-protein accumulation seemed to result from a stabilization of the polypeptide, which led us to hypothesize that oxidative processes might interfere with the specific degradation of this protein in declining spruce, with destructive consequences for the photosystems.

DNA import into mitochondria

In recent decades, it has become evident that the condition for normal functioning of mitochondria in higher eukaryotes is the presence of membrane transport systems of macromolecules (proteins and nucleic acids). Natural competence of the mitochondria in plants, animals, and yeasts to actively uptake DNA may be directly related to horizontal gene transfer into these organelles occurring at much higher rate compared to the nuclear and chloroplast genomes. However, in contrast with import of proteins and tRNAs, little is known about the biological role and molecular mechanism underlying import of DNA into eukaryotic mitochondria. In this review, we discuss current state of investigations in this area, particularly specificity of DNA import into mitochondria and its features in plants, animals, and yeasts; a tentative mechanism of DNA import across the mitochondrial outer and inner membranes; experimental data evidencing several existing, but not yet fully understood mechanisms of DNA transfer into mitochondria. Currently available data regarding transport of informational macromolecules (DNA, RNA, and proteins) into the mitochondria do not rule out that the mechanism of protein and tRNA import as well as tRNA and DNA import into the mitochondria may partially overlap.