Mip1p is the highly processive monomeric mitochondrial DNA polymerase in Saccharomyces cerevisiae. Despite differences in enzyme structure, substrate topology, and possible nucleoid interactions, Mip1p continues to be used as a model for human mitochondrial DNA polymerase (POLG) variants associated with various human mitochondrial diseases. Structurally, Mip1p functions as a monomer, whereas, the POLG holoenzyme contains a catalytic subunit (POLGA) complexed with a dimeric form of an accessory subunit (POLGB) which functions by loading the enzyme onto mitochondrial DNA and enhancing processivity. However, Mip1p does contain a 279-residue carboxyl-terminal extension (CTE) absent in the structure of POLG. The function of the CTE has not yet been determined although studies of truncation variants identify 74 N-terminal residues are essential for Mip1p wild-type activity. Furthermore, regions encompassing Mip1p residues N1033 – E1038 and Y1039 – A1049 are suggested to function in mitochondrial DNA maintenance and fidelity, respectively. This study has developed a mutagenic strategy to systematically replace the residues in the mitochondrial DNA maintenance region with glycine in order to identify residues critical for Mip1p function. Using in vivo respiratory competence and erythromycin resistance assays accompanied by an in vitro non-radioactive DNA polymerase assay, this study has identified two key residues, E1036 and D1037 that may function in the exonuclease-polymerase coupling mechanism of Mip1p.

Mip1p is the highly processive monomeric mitochondrial DNA polymerase in Saccharomyces cerevisiae. Despite differences in enzyme structure, substrate topology, and possible nucleoid interactions, Mip1p continues to be used as a model for human mitochondrial DNA polymerase (POLG) variants associated with various human mitochondrial diseases. Structurally, Mip1p functions as a monomer, whereas, the POLG holoenzyme contains a catalytic subunit (POLGA) complexed with a dimeric form of an accessory subunit (POLGB) which functions by loading the enzyme onto mitochondrial DNA and enhancing processivity. However, Mip1p does contain a 279-residue carboxyl-terminal extension (CTE) absent in the structure of POLG. The function of the CTE has not yet been determined although studies of truncation variants identify 74 N-terminal residues are essential for Mip1p wild-type activity. Furthermore, regions encompassing Mip1p residues N1033 – E1038 and Y1039 – A1049 are suggested to function in mitochondrial DNA maintenance and fidelity, respectively. This study has developed a mutagenic strategy to systematically replace the residues in the mitochondrial DNA maintenance region with glycine in order to identify residues critical for Mip1p function. Using in vivo respiratory competence and erythromycin resistance assays accompanied by an in vitro non-radioactive DNA polymerase assay, this study has identified two key residues, E1036 and D1037 that may function in the exonuclease-polymerase coupling mechanism of Mip1p.

L’instabilité d’ADN mitochondrial (ADNmt) peut être quantitative avec la déplétion de l’ADNmt ou qualitative avec des délétions de l’ADNmt. Ces anomalies sont une des causes les plus commmunes des maladies mitochondriales. Un des gènes qui contrôle la stabilité et le maintien de l’ADNmt est POLG. Ce gène code pour la polymerase gamma mitochondriale. Chez l’homme, les mutations dans le gène POLG sont liées aux maladies mitochondriales telle que; l’insuffisance hépatique, le syndrome d’Alpers, le PEO ou Progressive External Ophtalmoplegia, la neuropathie sensorielle et l’ataxie. Des mutations dans le gène POLG sont aussi associées au syndrome de Parkinson. Aujourd’hui, il n’existe aucune thérapie pour ces maladies. Compte tenu de la conservation évolutive de la fonction mitochondriale de la levure à l’homme, nous avons utilisé deux organismes modèles, Saccharomyces cerevisiae et Caenorhabditis elegans, pour identifier des molecules chimiques capables de compenser l’instabilité de l’ADNmt liée à des mutations du gène POLG dans des fibroblastes d’un patient. Nous avons trouvé trois molécules candidates potentielles: MRS2, MRS3 et MRS4, à partir d’un criblage primaire chez la levure, en utilisant une chimiothèque d’environ 2000 molécules chimiques. MRS3 est la molécule candidate la plus efficace pour la stabilization d’ADNmt chez des mutants POLG de la levure, du champignon filamenteux, du nématode et sur des fibroblastes de patients. MRS3, ou clofilium tosylate (CLO), est un agent antiarrhytmique, médicament pour soigner les troubles du rythme cardiaque. Dans cette étude, nous avons aussi montré que deux autres antiarrhythmiques appartenant à la même classe que CLO avaient un effet positive chez un mutant POLG de C. elegans. En utilisant une approche de chemogénomique chez la levure, nous avons identifié Fis1, un acteur de la fission mitochondriale qui pourrait être impliqué dans la mode d’action de CLO. Fis1 est requise pour la viabilité cellulaire en concentration légèrement toxique de CLO et nécesaire pour la stabilization de l’ADNmt par CLO. L’ensemble de ces résultats ont montré que CLO pourrait être la première molécule chimique qui stimule la réplication de l’ADNmt et qui pourrait être développée pour le traitement des maladies liées à des mutations dans le gène POLG. Ces résultats ont aussi permis de mettre en évidence une nouvelle connexion entre replication de l’ADNmt et la fission mitochondriale. / The instability of mitochondrial DNA (mtDNA) in form of mtDNA depletion (quantitative instability) or large deletion (qualitative instability) is one of the most common cause of mitochondrial diseases.. One of the genes responsible for human mtDNA stability, POLG, is exploited in this study. POLG encodes the human mitochondrial polymerase gamma. In human, POLG mutations are a major cause of mitochondrial disorders including hepatic insufficiency; Alpers syndrome, progressive external ophthalmoplegia, sensory neuropathy and ataxia. They are also associated with Parkinsonism. Currently, there is no effective and disease-specific therapy for these diseases. Based on the conservation of mitochondrial function from yeast to human, we used Saccharomyces cerevisiae and Caenorhabditis elegans as first pass filters to identify chemical compounds that suppresses mtDNA instability in cultured fibroblasts of a POLG-deficient patient. We found three potential candidates, MRS2, MRS3 and MRS4, from a chemical screening of nearly 2000 compounds in yeast. MRS3 is the most efficacious in stabilizing mtDNA in yeast, filamentous fungi, worm and patient fibroblasts. This unsuspected compound, clofilium tosylate (CLO), belongs to a class of antiarrhythmic agents for cardiovascular disease. Two other antiarrhythmic agents (FDA-approved) sharing common pharmacological properties and chemical structure with CLO also show potential benefit for POLG deficiency in C. elegans. Using a chemogenomic approach in yeast, we also discovered that a mitochondrial fission actor Fis1 is implicated in the mechanism of action of CLO. Fis1 is important for cellular viability in a slightly toxic concentration of CLO and is required for the mtDNA stabilizing potency of CLO. Our findings provide evidence of the first mtDNA-stabilizing compound that may be an effective pharmacological alternative for the treatment of POLG-related diseases and uncover a new connection between the mitochondrial fission process and mtDNA replication. / Ketidakstabilan DNA mitokondria (mtDNA) dalam bentuk pengurangan kopi mtDNA di dalam sel (ketidakstabilan kuantitatif), atau pun dalam bentuk delesi pada sekuens mtDNA (ketidakstabilan kualitatif) merupakan salah satu penyebab penyakit mitokondria. Salah satu gen yang bertanggung jawab dalam menjamin kestabilan mtDNA adalah POLG. Gen POLG mengkode protein polimerase gamma pada manusia, yang mereplikasi dan mereparasi mtDNA di dalam mitokondria. Mutasi pada gen POLG dapat menyebabkan penyakit kelainan mitokondria pada manusia, seperti gagal ginjal, sindrom Alpers, Progressive External Ophtalmoplegia, neuropati sensorial, ataxia dan bisa dikaitkan dalam beberapa gejala Parkinsonisme. Saat ini, belum ada terapi obat yang dapat mengatasi penyakit – penyakit tersebut. Berdasarkan kesamaan evolutif dari ragi hingga manusia, pada studi ini kami menggunakan Saccharomyces cerevisiae dan Caenorhabditis elegans untuk mengidentifikasi molekul obat yang berpotensi mengatasi ketidakstabilan mtDNA dari fibroblas pasien manusia yang memiliki mutasi gen POLG. Kami mengidentifikasi tiga kandidat potensial, yakni MRS2, MRS3 dan MRS4 dari penapisan kurang lebih 2000 molekul obat dengan menggunakan ragi. MRS3 adalah kandidat yang paling berkhasiat dan mampu mengatasi ketidakstabilan mtDNA pada ragi, Podospora, cacing dan fibroblas manusia. MRS3 adalah alias bagi clofilium tosylate (CLO), sebuah molekul antiaritmia untuk penyakit kardiovaskuler. Pada studi ini, kami juga menguji aktifitas dua molekul antiaritmia lain yang tergabung dalam kelas yang sama dengan CLO, dan menemukan bahwa kedua molekul ini juga berpotensi mengatasi defisit POLG pada cacing C. elegans. Dengan menggunakan metode kemogenomik pada ragi, kami juga mengidentifikasi sebuah aktor prosesus pembelahan mitokondria, Fis1, yang berpotensi terlibat dalam mekanisme seluler CLO. Fis1 dibutuhkan untuk: (1) kelangsungan hidup ragi pada konsentrasi toksik CLO dan (2) efek CLO dalam menstabilkan mtDNA pada ragi. Keseluruhan studi ini membuktikan potensi CLO sebagai molekul penstabil mtDNA yang pertama, yang dapat dikembangkan sebagai salah satu alternatif terapi obat untuk penyakit – penyakit mitokondria terkait mutasi POLG. Melalui studi ini, juga diungkap adanya hubungan antara kestabilan mtDNA dan prosesus pembelahan mitokondria.

As mitocôndrias desempenham um papel fundamental na sobrevivência e morte celular. Alterações do DNA mitocondrial (DNAmt) - como, por exemplo, amplificação, mutação homoplásmica, deleção e depleção -, bem como suas implicações clínico-patológicas, tem sido analisadas em inúmeras neoplasias humanas. No intuito de se pesquisar alterações mitocondriais associadas à tumorigênese, o presente trabalho teve como objetivos analisar a expressão de genes implicados no metabolismo energético e envolvidos na replicação e transcrição mitocondriais, quantificar o número de organelas mitocondriais e de cópias de DNAmt e analisar a expressão dos genes em astrocitomas de diferentes graus de malignidade (23 OMS grau I, 26 grau II, 18 grau III e 84 grau IV ou GBM) em relação ao tecido cerebral não tumoral (22 amostras). As expressões relativas dos genes selecionados, bem como as quantificações relativa e absoluta do DNA mitocondrial, foram realizadas por PCR em tempo real. O aumento de expressão relativa de genes-chave da via glicolítica, alterações nos níveis de expressão dos genes do ciclo dos ácidos tricarboxílicos e hipoexpressão de genes da fosforilação oxidativa detectados corroboraram o efeito Warburg. Foi demonstrado que a redução do número de cópias do DNAmt está associada com o grau de malignidade dos astrocitomas difusamente infiltrativos, sendo GBM o mais depletado e independente do número de organelas. As médias observadas para tecido não tumoral, astrocitoma grau I, grau II, grau III e GBM foram, respectivamente, 1,28, 0,26, 0,45, 0,42 e 0,17. Níveis aumentados de expressão relativa dos genes dos fatores de transcrição mitocondriais A (TFAM), B1 (TFB1M), B2 (TFB2M) e da subunidade catalítica da polimerase mitocondrial (POLG) foram detectados em todos os graus de astrocitomas, exceto TFB2M em astrocitoma grau II. Embora exista forte correlação entre os fatores de transcrição mitocondriais, somente os níveis de expressão de POLG se correlacionaram inversamente com o número de cópias de DNAmt. A expressão elevada de TFAM está associada a uma maior sobrevida no grupo de pacientes com GBM, interpretada como compensatório. As hiperexpressões de TFAM e POLG estão relacionadas a um melhor prognóstico em pacientes com GBM. Embora nossos achados da disfunção do metabolismo intermediário e depleção do DNAmt em astrocitomas corroborem a literatura, ainda não está bem esclarecida sua implicação na iniciação e manutenção da transformação maligna. Investigações futuras são necessárias para o esclarecimento destas questões. / Mitochondria has a key role in cell survival and death. Mitochondrial DNA (mtDNA) alterations, for example, amplification, homoplasmic mutation, deletion and depletion, and their clinical and pathological implications have been analyzed in human malignancies. In order to search for mitochondrial alterations associated to tumorigenesis, this study aimed to analyze the expression levels of genes involved in energetic metabolism, and in mitochondrial replication and transcription, to quantify the number of mitochondrial organelle and mtDNA copy number in astrocytomas of different grades of malignancy (23 WHO grade I, 26 grade II, 18 grade III and 84 grade IV or GBM) related to non-neoplastic brain tissue (22 samples). The relative expression level of the selected genes as well as the relative and absolute quantification of mtDNA were performed by real-time PCR. Relative expression increase of glycolytic pathway key genes, change of citric acid cycle genes and hipoexpression of oxidative phosphorylation genes were detected, and confirmed the presence of Warburg effect. The reduced mtDNA copy number was associated to the grade of malignancy of diffusely infiltrating astrocytoma, being GBM the most depleted, and not related to parallel decrease in the number of organelle. The mean mtDNA copy number for non neoplastic tissue, astrocytoma grade I, grade II, grade III and GBM were respectively 1.28, 0.26, 0.45, 0.42 and 0.17. The increased relative gene expression of mitochondrial transcription factor A (TFAM), B1 (TFB1M), B2 (TFB2M) and the catalytic subunit of mitochondrial polymerase (POLG) were observed in all grades of astrocytoma, except TFB2M in grade II astrocytoma. Although a strong correlation was observed among the mitochondrial transcription factors, only the expression level of POLG correlated inversely to the mtDNA copy number. The overexpression of TFAM was associated with long-term survival in the GBM patients and interpreted as compensatory. TFAM and POLG overexpressions were related to better prognosis in GBM patients. Although our findings concerning the impairment of intermediary metabolism and depletion of mtDNA in astrocytomas confirmed previous reports, their role in initiation or maintenance of malignant transformation were not fully understood. Further investigations are needed to clarify these issues.

As mitocôndrias desempenham um papel fundamental na sobrevivência e morte celular. Alterações do DNA mitocondrial (DNAmt) - como, por exemplo, amplificação, mutação homoplásmica, deleção e depleção -, bem como suas implicações clínico-patológicas, tem sido analisadas em inúmeras neoplasias humanas. No intuito de se pesquisar alterações mitocondriais associadas à tumorigênese, o presente trabalho teve como objetivos analisar a expressão de genes implicados no metabolismo energético e envolvidos na replicação e transcrição mitocondriais, quantificar o número de organelas mitocondriais e de cópias de DNAmt e analisar a expressão dos genes em astrocitomas de diferentes graus de malignidade (23 OMS grau I, 26 grau II, 18 grau III e 84 grau IV ou GBM) em relação ao tecido cerebral não tumoral (22 amostras). As expressões relativas dos genes selecionados, bem como as quantificações relativa e absoluta do DNA mitocondrial, foram realizadas por PCR em tempo real. O aumento de expressão relativa de genes-chave da via glicolítica, alterações nos níveis de expressão dos genes do ciclo dos ácidos tricarboxílicos e hipoexpressão de genes da fosforilação oxidativa detectados corroboraram o efeito Warburg. Foi demonstrado que a redução do número de cópias do DNAmt está associada com o grau de malignidade dos astrocitomas difusamente infiltrativos, sendo GBM o mais depletado e independente do número de organelas. As médias observadas para tecido não tumoral, astrocitoma grau I, grau II, grau III e GBM foram, respectivamente, 1,28, 0,26, 0,45, 0,42 e 0,17. Níveis aumentados de expressão relativa dos genes dos fatores de transcrição mitocondriais A (TFAM), B1 (TFB1M), B2 (TFB2M) e da subunidade catalítica da polimerase mitocondrial (POLG) foram detectados em todos os graus de astrocitomas, exceto TFB2M em astrocitoma grau II. Embora exista forte correlação entre os fatores de transcrição mitocondriais, somente os níveis de expressão de POLG se correlacionaram inversamente com o número de cópias de DNAmt. A expressão elevada de TFAM está associada a uma maior sobrevida no grupo de pacientes com GBM, interpretada como compensatório. As hiperexpressões de TFAM e POLG estão relacionadas a um melhor prognóstico em pacientes com GBM. Embora nossos achados da disfunção do metabolismo intermediário e depleção do DNAmt em astrocitomas corroborem a literatura, ainda não está bem esclarecida sua implicação na iniciação e manutenção da transformação maligna. Investigações futuras são necessárias para o esclarecimento destas questões. / Mitochondria has a key role in cell survival and death. Mitochondrial DNA (mtDNA) alterations, for example, amplification, homoplasmic mutation, deletion and depletion, and their clinical and pathological implications have been analyzed in human malignancies. In order to search for mitochondrial alterations associated to tumorigenesis, this study aimed to analyze the expression levels of genes involved in energetic metabolism, and in mitochondrial replication and transcription, to quantify the number of mitochondrial organelle and mtDNA copy number in astrocytomas of different grades of malignancy (23 WHO grade I, 26 grade II, 18 grade III and 84 grade IV or GBM) related to non-neoplastic brain tissue (22 samples). The relative expression level of the selected genes as well as the relative and absolute quantification of mtDNA were performed by real-time PCR. Relative expression increase of glycolytic pathway key genes, change of citric acid cycle genes and hipoexpression of oxidative phosphorylation genes were detected, and confirmed the presence of Warburg effect. The reduced mtDNA copy number was associated to the grade of malignancy of diffusely infiltrating astrocytoma, being GBM the most depleted, and not related to parallel decrease in the number of organelle. The mean mtDNA copy number for non neoplastic tissue, astrocytoma grade I, grade II, grade III and GBM were respectively 1.28, 0.26, 0.45, 0.42 and 0.17. The increased relative gene expression of mitochondrial transcription factor A (TFAM), B1 (TFB1M), B2 (TFB2M) and the catalytic subunit of mitochondrial polymerase (POLG) were observed in all grades of astrocytoma, except TFB2M in grade II astrocytoma. Although a strong correlation was observed among the mitochondrial transcription factors, only the expression level of POLG correlated inversely to the mtDNA copy number. The overexpression of TFAM was associated with long-term survival in the GBM patients and interpreted as compensatory. TFAM and POLG overexpressions were related to better prognosis in GBM patients. Although our findings concerning the impairment of intermediary metabolism and depletion of mtDNA in astrocytomas confirmed previous reports, their role in initiation or maintenance of malignant transformation were not fully understood. Further investigations are needed to clarify these issues.