Estudo de ß-glucanas extraídas dos fungos Geastrum saccatum e Polyporos dermoporus: características químicas e atividades biológicas

Fungal polysaccharides have received a great deal of attention due to itsbecause of their potential use in a wide rangegreat variety fromof industries. Some studies have demonstrated that polysaccharides extracted offrom basidiomycetes they have presented significant properties as anti-inflammatory, antimicrobial, antioxidant and anti-tumoral properties. In spite of thisDespite these potential properties, these mushrooms have not been insufficiently investigated, and the great number of antibiotics number produced forby these organisms suggests that they canmay be a new source of bioactives composites source. In tThe present work, reports onlated the chemical composition, potential antioxidant, antiinflammatory and citotoxycity of extracted polymers extracted offrom the fruits bodies of the fungiius Geastrum saccatum and Polyporus dermoporus, native mushrooms of the Atlantic forest inof the state of the Rio Grande do Norte, Brazil. The Cchemical analyses had revealed ademonstrated text of total sugar rates of 65% and 49%, and proteins of 7.0% for in extracts of G. saccatum and P. dermoporus extracts, respectively. The analyses ofNMR spectroscopy of RMN had demonstrated that these extracts are composites forof a complex involving β- glucans and- proteins complex. The inhibition of the formation of superoxide radicals formation was of 88.4% in G. saccatum and 83.3% in P. dermoporus, and 75 and 100% for inhibition of hydroxyls radicals inhibition. TopicalThe topic application of extracts the 10, 30 and 50 mg/kg extract in BALBc mice with cutaneous inflammation induced byfor croton oil demonstrated to inhibitedion of ear edema of ear and cells polimorfonuclears cells atin the inflamed siteplace, being this reply more effective in lower concentrations being more effective. The evaluation of the glucans of G. saccatum and P. dermoporus glucans under induced pleurisy for carrageenan-induced pleurisya of showed the antiinflammatory action of these composites., being analyzed tThe frame number in the pleural exudates and thedosage of nitric oxide dosage was also analyzed. The cytotoxic action of these polymers was analyzed throughthrough the mitochondrial function (MTT). The incubation of the glucans with mononuclear cells of the peripheral blood demonstrated that the extracted glucans extracted fromof G. saccatum havepossess a moderate cytotoxic action. These results suggest that these mushrooms possess polymers formed byfor a complex glucana-protein complex, with antiinflammatory and antioxidant actions

Design and synthesis of novel quinolones directed to the Plasmodium falciparum bc1 protein complex

Tese de Doutoramento, Química, Especialização em Química Orgânica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Convergent bioenergetic defects in Coenzyme Q10 depleted cells by pharmacological inhibition of coq2 enzyme (p-hydroxybenzoate polyprenyl transferase) and by genome editing technology targeting the encoding gene (COQ2)

Primary CoQ10 deficiency diseases encompass a heterogeneous spectrum of clinical phenotypes. Among these, defect or mutation on COQ2 gene, encoding a para-hydroxybenzoate polyprenyl transferase, have been associated with different diseases. Understanding the functional and metabolic impact of COQ2 mutation and the consequent CoQ10 deficiency is still a matter of debate. To date the aetiology of the neurological phenotypes correlated to CoQ10 deficiency does not present a clear genotype-phenotype association. In addition to the metabolic alterations due to Coenzyme Q depletion, the impairment of mitochondrial function, associated with the reduced CoQ level, could play a significant role in the metabolic flexibility of cancer. This study aimed to characterize the effect of varying degrees of CoQ10 deficiency and investigate the multifaceted aspect of CoQ10 depletion and its impact on cell metabolism. To induced CoQ10 depletion, different cell models were used, employing a chemical and genome editing approach. In T67 and MCF-7 CoQ10 depletion was achieved by a competitive inhibitor of the enzyme, 4-nitrobenzoate (4-NB), whereas in SH-SY5Y the COQ2 gene was edited via CRISPR-Cas9 cutting edge technology.

Whole Exome Sequencing widens the genetic landscape of Hereditary Optic Neuropathies

Hereditary optic neuropathies (HON) are a genetic cause of visual impairment characterized by degeneration of retinal ganglion cells. The majority of HON are caused by pathogenic variants in mtDNA genes and in gene OPA1. However, several other genes can cause optic atrophy and can only be identified by high throughput genetic analysis. Whole Exome Sequencing (WES) is becoming the primary choice in rare disease molecular diagnosis, being both cost effective and informative. We performed WES on a cohort of 106 cases, of which 74 isolated ON patients (ON) and 32 syndromic ON patients (sON). The total diagnostic yield amounts to 27%, slightly higher for syndromic ON (31%) than for isolated ON (26%). The majority of genes found are related to mitochondrial function and already reported for harbouring HON pathogenic variants: ACO2, AFG3L2, C19orf12, DNAJC30, FDXR, MECR, MTFMT, NDUFAF2, NDUFB11, NDUFV2, OPA1, PDSS1, SDHA, SSBP1, and WFS1. Among these OPA1, ACO2, and WFS1 were confirmed as the most relevant genetic causes of ON. Moreover, several genes were identified, especially in sON patients, with direct impairment of non-mitochondrial molecular pathways: from autophagy and ubiquitin system (LYST, SNF8, WDR45, UCHL1), to neural cells development and function (KIF1A, GFAP, EPHB2, CACNA1A, CACNA1F), but also vitamin metabolism (SLC52A2, BTD), cilia structure (USH2A), and nuclear pore shuttling (NUTF2). Functional validation on yeast model was performed for pathogenic variants detected in MECR, MTFMT, SDHA, and UCHL1 genes. For SDHA and UCHL1 also muscle biopsy and fibroblast cell lines from patients were analysed, pointing to possible pathogenic mechanisms that will be investigated in further studies. In conclusion, WES proved to be an efficient tool when applied to our ON cohort, for both common disease-genes identification and novel genes discovery. It is therefore recommended to consider WES in ON molecular diagnostic pipeline, as for other rare genetic diseases.

Defective protein folding and function in metabolic disorders: studies on the mitochondrial flavoenzyme ETF

Dissertation presented to obtain the PhD degree in Biochemistry at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

Postischemic recovery of heart metabolism and function: role of mitochondrial fatty acid transfer.

Postischemic recovery of contractile function is better in hearts from fasted rats than in hearts from fed rats. In this study, we examined whether feeding-induced inhibition of palmitate oxidation at the level of carnitine palmitoyl transferase I is involved in the mechanism underlying impaired recovery of contractile function. Hearts isolated from fasted or fed rats were submitted to no-flow ischemia followed by reperfusion with buffer containing 8 mM glucose and either 0.4 mM palmitate or 0.8 mM octanoate. During reperfusion, oxidation of palmitate was higher after fasting than after feeding, whereas oxidation of octanoate was not influenced by the nutritional state. In the presence of palmitate, recovery of left ventricular developed pressure was better in hearts from fasted rats. Substitution of octanoate for palmitate during reperfusion enhanced recovery of left ventricular developed pressure in hearts from fed rats. However, the chain length of the fatty acid did not influence diastolic contracture. The results suggest that nutritional variation of mitochondrial fatty acid transfer may influence postischemic recovery of contractile function.

The mitochondrial targeting function of randomly generated peptide sequences correlates with predicted helical amphiphilicity.

A pool of oligonucleotides encoding a start methionine and nine random amino acids was inserted at the 5'-end of the gene for the yeast cytochrome oxidase subunit IV lacking its own mitochondrial targeting sequence. Approximately one-quarter of the randomly generated sequences targeted subunit IV to its correct intramitochondrial location in vivo. Sequence analysis of 89 randomly generated sequences showed that their efficiencies as mitochondrial targeting signals correlated with the potential to fold into an amphiphilic alpha-helix. Functional targeting sequences were enriched in arginine and isoleucine residues but contained few aspartate, glutamate, and proline residues. Nonfunctional sequences predicted to have significant helical amphiphilicity often had at least one acidic or multiple helix-breaking residues that would be expected to interfere with targeting functioning. These results support the hypothesis that the signal for targeting a protein into the mitochondrial matrix is usually a positively charged amphiphilic helix.

Effect of aerobic exercise on skeletal muscle mitochondrial content and function in older adults

Regular aerobic exercise training, which is touted as a way to ameliorate metabolic diseases, increases aerobic capacity. Aerobic capacity usually declines with advanced age. The decline in aerobic capacity is typically associated by a decrease in the quality of skeletal muscle. At the molecular level, this decreased quality comes in part from perturbations in skeletal muscle mitochondria. Of particular is a decrease in the total amount of mitochondria that occupy the skeletal muscle volume. What is not well established is if this decrease in mitochondrial content is due to inactive lifestyle or the process of aging. Herein, the work of the thesis shows a clear connection between mitochondrial content and aerobic capacity. This indicates that active individuals with higher VChmax levels also contain higher volumes of mitochondria inside their muscle as opposed to sedentary counterparts who have lower levels of mitochondrial content. Upon taking these previously sedentary individuals and entering them into an aerobic exercise intervention, they are able to recover their mitochondrial content as well as function to similar levels of lifelong athletes of the same age. Furthermore, the results of this thesis show that mitochondrial content and function also correlate with exercise efficiency. If one is more efficient, he/she is able to expend less energy for a similar power output. Furthermore, individuals who increase in efficiency also increase in the ability to oxidize and utilize fat during pro-longed exercise. This increased reliance on fat after the intervention is associated with an increased amount of mitochondria, particularly in the intermyofibrillar region of skeletal muscle. Therefore, elderly adults who were once sedentary were able to recover mitochondrial content and function and are able to reap other health benefits from regular aerobic exercise training. Aging per se does not seem to be the culprit that will lead to metabolic diseases but rather it seems to be a lack of physical activity. -- Un entraînement sportif d'endurance, connu pour réduire le risque de développer des maladies métaboliques, augmente notre capacité aérobie. La capacité aérobie diminue généralement avec l'âge. Ce déclin est typiquement associé d'une diminution de la qualité du muscle squelettique. Au niveau moléculaire, cette diminution est due à des perturbations dans les mitochondries du muscle squelettique,, ce qui conduit à une diminution de la quantité totale des mitochondries présentes dans le muscle squelettique. Il n'a pas encore été établi si cette diminution de la teneur mitochondriale est due à un mode de vie sédentaire ou au processus du vieillissement. Ce travail de thèse montre un lien clair entre le contenu mitochondrial et la capacité aérobie. Il indique que des personnes âgées actives, avec des niveaux de V02max plus élevés, possèdent également un volume plus élevé de mitochondries dans leurs muscles en comparaison à leurs homologues sédentaires. En prenant des individus sédentaires et leur faisant pratiquer une activité physique aérobie, il est possible d'accroître leur contenu de même que leur fonction mitochondriale à des niveaux similaires à ceux d'athlètes du même âge ayant pratiqué une activité physique tout au long de leur vie. De plus, les résultats de ce travail démontrent que le contenu et la fonction mitochondriale sont en corrélation avec l'efficiscience lors d'exercice physique. En agumentant l'effiscience, les personnes sont alors capables de dépenser moins d'énergie pour une puissance d'exercice similaire. Donc, un volume mitochondrial accru dans le muscle squelettique, associé à une fonction mitochondriale améliorée, est associté à une augmentation de l'effiscience. En outre, les personnes qui augmentent leur effiscience, augmentent aussi leur capacité à oxyder les graisses durant l'exercice prolongé. Une augmentation du recours au graisses après l'intervention est associée à une quantité accrue de mitochondries, en particulier dans la région inter-myofibrillaire du muscle squelettique. Par conséquent, les personnes âgées autrefois sédentaires sont en mesure de récupérer leur contenu et leur fonction mitochondriale ainsi que d'autres avantages pour la santé grâce à un entraînement aérobie régulier. Le vieillissement en soi ne semble donc pas être le coupable conduisant aux maladies métaboliques qui semblent plutôt être lié à un manque d'activité physique.

Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role

Plants and some other organisms including protists possess a complex branched respiratory network in their mitochondria. Some pathways of this network are not energy-conserving and allow sites of energy conservation to be bypassed, leading to a decrease of the energy yield in the cells. It is a challenge to understand the regulation of the partitioning of electrons between the various energy-dissipating and -conserving pathways. This review is focused on the oxidase side of the respiratory chain that presents a cyanide-resistant energy-dissipating alternative oxidase (AOX) besides the cytochrome pathway. The known structural properties of AOX are described including transmembrane topology, dimerization, and active sites. Regulation of the alternative oxidase activity is presented in detail because of its complexity. The alternative oxidase activity is dependent on substrate availability: total ubiquinone concentration and its redox state in the membrane and O2 concentration in the cell. The alternative oxidase activity can be long-term regulated (gene expression) or short-term (post-translational modification, allosteric activation) regulated. Electron distribution (partitioning) between the alternative and cytochrome pathways during steady-state respiration is a crucial measurement to quantitatively analyze the effects of the various levels of regulation of the alternative oxidase. Three approaches are described with their specific domain of application and limitations: kinetic approach, oxygen isotope differential discrimination, and ADP/O method (thermokinetic approach). Lastly, the role of the alternative oxidase in non-thermogenic tissues is discussed in relation to the energy metabolism balance of the cell (supply in reducing equivalents/demand in energy and carbon) and with harmful reactive oxygen species formation.

Studies of OPA1 pathogenic mechanisms in Dominant Optic Atrophy and novel protein function in mitochondrial DNA stability

The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for eukaryotic cell physiology. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. These organelles are organized in an extended network that dynamically fuses and divides. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of “mitochondria-shaping” proteins. It is becoming clear that defects in mitochondrial dynamics can impair mitochondrial respiration, morphology and motility, leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in vivo in humans. Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1 is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is anchored or associated on the inner mitochondrial membrane facing the intermembrane space. Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b have been described (Delettre et al., 2001). These variants greatly vary among diverse organs and the presence of specific isoforms has been associated with various mitochondrial functions. The different spliced exons encode domains included in the amino-terminal region and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in maintenance of the mitochondrial membrane potential and in the fusion of the network. Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to vertebrates (Olichon et al., 2007). While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause accumulation of multiple deletions in skeletal muscle. The syndrome associated to these mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy, progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati- Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA “breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009). In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane. Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately associated to the maintenance of the mitochondrial genome, as their silencing leads to mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids distribution in the mitochondrial network. In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide embedded in the inner membrane by a second transmembrane domain, that seems to be crucial for mitochondrial genome maintenance and does correspond to the second transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and specifically interacts with two major components of the mitochondrial nucleoids: the polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1- exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a process that is required for the initiation of mtDNA replication and for the distribution of nucleoids along the network. These data provide new crucial insights in understanding the mechanism involved in maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also an important role. Noticeably, the effect on mtDNA is different depending on the specific OPA1 isoforms down-regulated, suggesting the involvement of two different combined mechanisms. Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have been described to date, including substitutions, deletions or insertions. Some mutations are predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense nucleotide substitutions result in aminoacidic changes which affect conserved positions of the OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA patients are poorly understood. Phosphorus MR spectroscopy in patients with the c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1 R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008). In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency (DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and compared with control fibroblasts. Although both types of DOA fibroblasts maintained a similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense substitutions. Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing response to oligomycin in DOA-H cell lines only. This finding clearly supports the hypothesis that these mutations cause a significant alteration in the respiratory chain function, which can be unmasked only when the operation of the ATP synthase is prevented. Noticeably, oligomycin-induced depolarization in these cells was almost completely prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability transition pore (PTP). This results is very important because it suggests for the first time that the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue has not yet been addressed in the present study, several are the mechanisms that have been proposed to lead to PTP deregulation, including in particular increased reactive oxygen species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP opening is currently under investigation. Identification of the mechanisms leading to altered threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but also provide a strategy for therapeutic intervention.

Mitochondrial translation factors of Trypanosoma brucei: elongation factor-Tu has a unique subdomain that is essential for its function

Mitochondrial translation in the parasitic protozoan Trypanosoma brucei relies on imported eukaryotic-type tRNAs as well as on bacterial-type ribosomes that have the shortest known rRNAs. Here we have identified the mitochondrial translation elongation factors EF-Tu, EF-Ts, EF-G1 and release factor RF1 of trypanosomatids and show that their ablation impairs growth and oxidative phosphorylation. In vivo labelling experiments and a SILAC-based analysis of the global proteomic changes induced by EF-Tu RNAi directly link EF-Tu to mitochondrial translation. Moreover, EF-Tu RNAi reveals downregulation of many nuclear encoded subunits of cytochrome oxidase as well as of components of the bc1-complex, whereas most cytosolic ribosomal proteins were upregulated. Interestingly, T. brucei EF-Tu has a 30-amino-acid-long, highly charged subdomain, which is unique to trypanosomatids. A combination of RNAi and complementation experiments shows that this subdomain is essential for EF-Tu function, but that it can be replaced by a similar sequence found in eukaryotic EF-1a, the cytosolic counterpart of EF-Tu. A recent cryo-electron microscopy study revealed that trypanosomatid mitochondrial ribosomes have a unique intersubunit space that likely harbours the EF-Tu binding site. These findings suggest that the trypanosomatid-specific EF-Tu subdomain serves as an adaption for binding to these unusual mitochondrial ribosomes.

Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^

Mechanisms of Human Mitochondrial DNA Maintenance: The Determining Role of Primary Sequence and Length over Function

Although the regulation of mitochondrial DNA (mtDNA) copy number is performed by nuclear-coded factors, very little is known about the mechanisms controlling this process. We attempted to introduce nonhuman ape mtDNA into human cells harboring either no mtDNA or mutated mtDNAs (partial deletion and tRNA gene point mutation). Unexpectedly, only cells containing no mtDNA could be repopulated with nonhuman ape mtDNA. Cells containing a defective human mtDNA did not incorporate or maintain ape mtDNA and therefore died under selection for oxidative phosphorylation function. On the other hand, foreign human mtDNA was readily incorporated and maintained in these cells. The suicidal preference for self-mtDNA showed that functional parameters associated with oxidative phosphorylation are less relevant to mtDNA maintenance and copy number control than recognition of mtDNA self-determinants. Non–self-mtDNA could not be maintained into cells with mtDNA even if no selection for oxidative phosphorylation was applied. The repopulation kinetics of several mtDNA forms after severe depletion by ethidium bromide treatment showed that replication and maintenance of mtDNA in human cells are highly dependent on molecular features, because partially deleted mtDNA molecules repopulated cells significantly faster than full-length mtDNA. Taken together, our results suggest that mtDNA copy number may be controlled by competition for limiting levels of trans-acting factors that recognize primarily mtDNA molecular features. In agreement with this hypothesis, marked variations in mtDNA levels did not affect the transcription of nuclear-coded factors involved in mtDNA replication.

Mitochondrial DNA ligase function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (Δdnl4). The Escherichia coli EcoRI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant EcoRI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and Δdnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage.