Tesi sul tema "Mitochondrial DNA replication"

A mouse model was generated that accumulates multiple point mutations throughout the mitochondrial genome, due to an exonuclease deficient DNA polymerase. The work described here has focused on studying the structure of the mitochondrial genome of these mice and has shown that these mice suffer an increase in replication stalling. Breakage of stalled molecules at specific points, leads to the generation of a linear 11 kb DNA molecule and aborts replication. It is proposed here that these numerous rounds of futile replication lead to ‘cellular exhaustion’ and therefore the premature ageing phenotype of the mutator mouse. 5-15% of mtDNA molecules contain a third strand of DNA, 7S DNA, in the non-coding region, forming a displacement (D) loop structure. Here two-dimensional agarose gel electrophoresis and circular reverse transcription of PCR indicate that an additional strand of RNA is present in the NCR of the opposite orientation to 7S DNA. Therefore, this region may form a DNA-RNA bubble structure of D/R-loops rather than just a simple D-loop. Mitochondrial DNA is organised into nucleoid complexes with a number of DNA binding proteins. One of these proteins, ATAD3B, has been demonstrated to have a preference for binding to D-loop structures. Therefore, a fragment of recombinant ATAD3B has been studied to determine further details of its binding properties and inform future studies in living cells. This work has identified the Q149R polymorphism as a variant that increases the ability of the protein to bind single and double stranded DNA, with only minor affects on D-loop binding. It is proposed that this protein has a role in the regulation of mtDNA topology.

All eukaryotic cells possess mitochondrial DNA (mtDNA), which is maternally inherited through the oocyte, its replication being regulated by nuclear-encoded replication factors. It was hypothesised that mtDNA replication is highly regulated in oocytes, pre-implantation embryos and embryonic stem cells (ESCs) and that this may be disrupted following nuclear transfer (NT). MtDNA copy number decreased between 2-cell and 8-cell staged porcine embryos and increased between the morula and expanded blastocyst stages, coinciding with increased expression of mtDNA replication factors. Competent porcine oocytes replicated their mtDNA prior to and during in vitro maturation to produce and maintain the 100000 mtDNA copies required for fertilisation. Those oocytes in which mtDNA replication was delayed had reduced developmental ability. Expression of pluripotency-associated genes decreased as murine ESCs differentiated into embryoid bodies, although expression of mtDNA replication factors did not increase until the stage equivalent to organogenesis. Cross-species NT embryos in which the donor cell-derived mtDNA was replicated produced decreased developmental outcomes compared to those in which no mtDNA replication took place. Disruption of the strict regulation of mtDNA replication that occurs during early embryogenesis, as is likely following NT, may therefore contribute to the reduced developmental ability of embryos produced using such techniques.

The function of homologous DNA recombination in human mitochondria has been a topic of ongoing debate for many years, with implications for fields ranging from DNA repair and mitochondrial disease to population genetics. While genetic and biochemical evidence supports the presence of a mitochondrial recombination activity, the purpose for this activity and the proteins involved have remained elusive. The work presented in this thesis was designed to evaluate the mitochondrial localization of the major recombinase protein in human cells, Rad51, as well as determine what function it plays in the maintenance of mitochondrial DNA (mtDNA) copy number that is critical for production of chemical energy through aerobic respiration. The combination of subcellular fractionation with immunoblotting and immunoprecipitation approaches used in this study clearly demonstrates that Rad51 is a bona fide mitochondrial protein that localizes to the matrix compartment following oxidative stress, where it physically interacts with mtDNA. Rad51 was found to be critical for mtDNA copy number maintenance under stress conditions. This requirement for Rad51 was found to be completely dependent on ongoing mtDNA replication, as treatment with the DNA polymerase gamma (Pol ϒ) inhibitor, ddC, suppresses both recruitment of Rad51 to the mitochondria following the addition of stress, as well as the mtDNA degradation observed when Rad51 has been depleted from the cell. The data presented here support a model in which oxidative stress induces a three-part response: (1) The recruitment of repair factors including Rad51 to the mitochondrial matrix, (2) the activation of mtDNA degradation systems to eliminate extensively or persistently damaged mtDNA, and (3) the increase in mtDNA replication in order to maintain copy number. The stress-induced decrease in mtDNA copy number observed when Rad51 is depleted is likely the result of failure to stabilize or repair replication forks that encounter blocking lesions resulting in further damaged to the mtDNA and its eventual degradation.

Regulation of mtDNA content is critical to normal human health and is often abnormal in mitochondrial diseases. Only a proportion of a female’s mtDNA is used to populate the oocytes she forms during embryogenesis. This mtDNA bottleneck can cause rapid shifts in heteroplasmy levels between generations in families with mtDNA mutations. The heteroplasmy levels of several mtDNA mutations were analysed from mother-child pairs using previously validated pyrosequencing assays. Analysis of the shifts in heteroplasmy caused by the mtDNA bottleneck reveals that the inheritance of the pathogenic tRNA mutations m.3243A>G and m.8344A>G do not show selection bias. However, the distribution of m.8993T>G (in MT-ATP6) in offspring suggests this mutation is selected for during the mtDNA bottleneck. This finding is in agreement with meta-analysis performed on previously published data, which also reveals biased inheritance of the LHON mutations m.11778G>A and m.3460G>A, in Complex I genes. Selection for these mutations explains the higher prevalence of homoplasmy, as fixation can occur within fewer generations. Crucial to formation of the mtDNA bottleneck is the dramatic increase in mtDNA replication rate that allows primordial germ cells (PGCs) to repopulate their mitochondrial genomes. Gene expression analysis during pre-implantation embryonic development has not revealed any other biological processes associated with regulation of mtDNA copy number. Upregulation of Lrpprc correlates with the expression of transcription factors as pre-implantation development progresses at this stage. The limited number of PGCs in the early stages of post-implantation development prevented sufficient quantity of high-quality RNA to be isolated for use with gene expression analysis. The reduction of mtDNA copy number observed during the mtDNA bottleneck was modelled in myoblasts and fibroblasts, using ddC, a reverse transcriptase inhibitor. Although some gene expression changes were induced during repopulation of mtDNA, these were limited to below 3-fold change. Forcing reliance on oxidative phosphorylation through culture with galactose media could not increase the rate of mtDNA replication or induce greater gene expression responses. Ragged-red fibres (RRFs) are a common hallmark of many mitochondrial diseases, caused by proliferation of mitochondria in the subsarcolemmal region of muscle tissue. Multiplex immunohistochemistry allowed fibre typing and identification of RRFs in muscle tissue from a patient with the m.8344A>G mutation. A novel technique was iii established to ensure extraction of intact high-quality RNA from laser-microdissected muscle fibres. The RNA was used in a pilot microarray experiment to study the differences not only between control and patient tissue, but specifically within RRF, and has identified a multitude of potential signalling pathways involved in mitochondrial biogenesis and formation of RRFs. This thesis reveals the complexity of mtDNA copy number regulation and, the critical involvement it has to human health and the inheritance and pathogenicity of mitochondrial diseases.

Mitochondrial DNA encodes thirteen protein subunits in the oxidative phosphorylation system (OXPHOS) that is responsible for cellular energy production. Mitochondrial disorders have been identified to be associated with mtDNA mutations. However, the molecular mechanisms of specific mtDNA mutations are still being explored in order to establish causative links. This study tries to elucidate the mutational effects of mtDNA on OXPHOS complex activities and cell growths. Using mouse 3T3 fibroblasts as a cell model, single-cell clones with different growth rates were isolated. The entire mtDNA genome was sequenced for mutations. The enzymatic activities of OXPHOS complex I to V were analysed. Three growth patterns represented by five clones were identified. Three clones (clone #2, #3, and #6) had the shortest doubling times (11.5 - 14.9 hours). Clone #1 had a medium growth rate (19.2 hous); and clone #5 had a significantly slow growth rate (22 hours). MtDNA sequencing results revealed that clone #5 had several heteroplasmic mutations (one in 16S rRNA, two in tRNAser (UCN), three in tRNAasp, one in tRNAlys, one in COI, five in COII, and one in ATPase8) while the other four clones showed sequence homology. Enzymatic analyses showed that on average clone #5 had significantly low complex III, IV, and V activities (p < 0.05). Changes in biochemical properties and protein structure were analyzed to deduct possible mechanisms for reduced respiration. In conclusion, the slow growth rate is associated with reduced OXPHOS enzyme functions. It is most likely that the combination of COI and COII mutations resulted in the reduction of complex IV function. It is still unclear whether the ATPase8 mutation (T7869A) in the non-conserved region alone can have such a pronounced phenotypic effect. A reduction in complex III also cannot be explained since there were no mutations in the only mtDNA-encoded complex III gene, but it is possible that there are mutations in the nDNA-encoded complex III genes. Mutations in tRNA and rRNA genes may also be responsible for reduced protein syntheses and consequently reduced OXPHOS activities. It is unclear why complex I activity was not affected. Although the mutational effect of individual mtDNA mutation observed cannot be clearly identified, this study establishes a correlation between mtDNA mutation and cell energy production and growth.

Au cours de la croissance des levures, la cellule doit dupliquer sont génome nucléaire et mitochondrial, le processus de réplication est bien moins étudié dans les mitochondries. Néanmoins, si de multiples ADN polymérases sont impliquées dans les processus de réplication et de réparation dans le noyau, il est considéré jusqu’à aujourd’hui qu’une seule ADN polymérase est impliquée dans ces processus dans la mitochondrie. Des résultats récents mettent en exergue le fait que la situation est bien plus compliquée qu’il n’y apparait au départ. Pour élucider le processus de réplication dans la mitochondrie de levure, j’ai focalisé mon intérêt à tenter de purifier et de caractériser le complexe de réplication. Ce travail était important à développer étant donné la découverte au laboratoire d’une seconde ADN polymérase supplémentaire à la polymérase gamma, dans les mitochondries de levure. Une première partie de ma thèse a été de m’investir afin d’obtenir suffisamment de protéines dans le but d’une identification par spectrométrie de masse, compte tenu de la faible proportion des ADN polymérases dans la cellule et en particulier dans la mitochondrie. Nous avons démontré que cette polymérase est codée par le gène unique POL1. Par des techniques d’ultracentrifugation et d’analyse biochimiques, j’ai réussi à isoler et caractériser un complexe de réplication mitochondrial. Des techniques d’exclusion chromatographiques ont permis d’attribuer une masse native à ce complexe. Sa composition a été étudiée grâce à des colonnes ioniques et hydrophobes, une autre méthode d’analyse repose sur l’utilisation de colonnes d’affinité afin de reconstituer in-vitro les interactions existant entre plusieurs protéines présumées impliquées. Ainsi, un réseau d’interactions impliquant les deux ADN polymérases mitochondriales avec cinq autres protéines a été reconstitué. La masse native de différentes formes stables de ce complexe se situent à 500 kDa ou au-delà de 1 MDa
During yeast growth, cells must duplicate their nuclear and mitochondrial DNA. The replication process involved is less studied in mitochondria. Nevertheless, if multiple DNA polymerases are implicated in the nuclear replication and repair mechanisms, until now it is believed that only one DNA polymerase is involved in these processes in mitochondria. Recent results pointed out that the situation is more complicated than preliminary believed. To elucidate the replication process in yeast mitochondria I focused my interest in attempts to purify and characterize the replication complexes. This work was important to develop in accord with the discovery in the laboratory of a second DNA polymerase in addition to the polymerase gamma in yeast mitochondria. One first part of my thesis was to hardly purify enough of this enzyme to be allowed to identify it by mass spectrometry as the DNA polymerase alpha, encoded by the unique POL1 gene. By ultracentrifugation and biochemical techniques, I succeeded to purify the complex. Exclusion chromatographies were managed to elucidate the native mass of this complex. In addition ionic and hydrophobic chromatographic columns were carried out to determine its composition. Another way to study the complex was the reconstitution in vitro of the interactions happening with some usual suspect proteins with the help of chromatographic affinity columns. I reconstituted partly an interactions model network, including the two mitochondrial DNA polymerases and 5 others proteins implicated in replication. I determined the mass of different stable forms of the isolated complexes, around 500 kDa and over 1 MDa

Chez les vertebres, l'adn mitochondrial est dna bicatenaire (15 kb). Un intermediaire de replication possedant dune boucle de deplacement (boucle d) contient un brin h neosynthetise de taille fixe et est trouve en proportion variable dans les mitochondries. Le quart du genome mitochondrial de xenopus laevis a ete sequence: la region boucle d, 4 genes d'arn de transfert, le gene de l'apocytochrome b et celui de l'arn 125. La sequence nucleotique indique que l'organisation genetique de cette region est identique a celles des mammiferes. La cartographie au nucleotide pres du brin h de la boucle d a mis en evidence 2 familles de brin h ayant une extremite 3' commune, des structures secondaires sont potentiellement inapliquees dans la terminaison des brins h de la boucle d. Une proteine de 21,5 k da a ete isolee, se fixe a l'adn dans la region origine de replication et semble jouer un role regulateur dans la replication ou l'expression de l'adn mitochondrial. La replication du brin h peut etre observee dans un systeme de synthese d'adn "in vitro" (adn polymerase indispensable)

La mitochondrie est une organelle essentielle possédant son propre ADN circulaire. Cet ADN peut présenter des mutations et/ou des délétions, consécutives à l’exposition à différents types de dommages ou en raison de protéines mutées. Ces mutations ou délétions sont impliquées dans de nombreuses pathologies, dont les cancers, et le vieillissement. Leur apparition peut survenir notamment lors de la réplication ou de la réparation. A ce jour, la réplication et la réparation mitochondriales ne sont pas encore bien élucidées. L’objectif de ce projet est donc de mieux en appréhender les mécanismes et de mieux comprendre l’émergence d’anomalies en nous intéressant plus particulièrement à une délétion appelée « Common Deletion ». Ce travail reposait sur l’hypothèse que cette délétion put résulter d’une mauvaise réparation de cassure(s) double-brin et/ou d’une erreur durant la réplication de l’ADN mitochondrial. L’analyse de ces résultats révèle que la formation de la « Common Deletion » ne nécessite qu’une seule cassure double-brin proche des séquences répétées entourant cette dernière et implique les protéines de la réplication de l’ADN mitochondrial. Ainsi, ce travail permet de mieux saisir les mécanismes de réplication et de réparation assurant la stabilité de l’ADN mitochondrial. Un second projet a été de proposer un modèle d’étude in vitro des topoisomérases en utilisant des minicercles d’ADN permettant la visualisation du complexe covalent, étape clef de la réaction de relaxation de ces enzymes
Mitochondria is an essential organelle with its own circular DNA. This DNA may exhibit mutations and/or deletions, as a result of exposure to different types of damage or due to mutated proteins. These mutations or deletions are involved in many pathologies, including cancers, and aging. They may occur during replication or repair. For now, mitochondrial replication and repair have not yet been fully elucidated. The objective of this project is therefore to better understand the mechanisms and the emergence of anomalies by focusing on a deletion called "Common Deletion". This work was based on the assumption that this deletion could result from poor repair of double-strand break(s) and/or error during mitochondrial DNA replication. Analysis of these results reveals that the formation of the "Common Deletion" requires only a single double-strand break close to the repeated sequences surrounding the latter and involves the proteins of mitochondrial DNA replication. Thus, this work makes it possible to better understand the mechanisms of replication and repair ensuring the stability of mitochondrial DNA. A second project was to propose an in vitro model for topoisomerases using DNA minicircles allowing visualization of the covalent complex, a key step in the relaxation reaction of these enzymes

Deficiencies of the oxidative phosphorylation system (OXPHOS) that consists of five enzyme complexes (I-IV) lead to a diversity of cellular consequences. This includes altered Ca2+ homeostasis, reduced ATP production and increased ROS (reactive oxygen species) production. One of the secondary consequences of such deficiencies is the adaptive transcriptional responses of several mitochondrial- and nuclear-encoded genes involved in OXPHOS biogenesis. Additionally, several other genes that are involved in several other functions, such as metallothioneins (MTs), are differentially expressed. In this study we investigated two hypotheses: firstly, that in complex I deficient cells the increased expression of MTs, specifically MT1B and MT2A, has a protective effect against ROS-related consequences of a complex I deficiency. The second hypothesis stated that genes involved in mitochondrial replication and transcription are differentially expressed in OXPHOS deficient cell lines. Firstly, the expression and role of metallothioneins (MTs) in an in vitro complex I deficient model was investigated. The increased expression of different MT isoforms in the presence of the complex I inhibitor rotenone in HeLa cells was confirmed. In this complex I deficient model overexpression of MT1B and especially MT2A isoforms also protected against ROS, mtPTP opening, apoptosis and ROS-induced necrosis. This data supports the hypothesis that increased expression of MT2A has a protective effect against the death-causing cellular consequences of rotenonetreated HeLa cells. Secondly, we investigated the differential expression of selected mitochondrial- and nuclear genes involved in OXPHOS function and regulation. Two experimental in vitro models were developed and utilized in the study. Firstly, a transient siRNA knockdown model of the NDUFS3 subunit of complex I in 143B cells was developed, characterized and introduced. Then the effect of the knockdown on several biochemical parameters (ROS and ATP levels), mtDNA copy number, total mtRNA levels, and RNA levels of several nuclear- and mitochondrial-encoded transcripts encoding structural as well as functional proteins was determined. Additionally, to investigate the effect of stable OXPHOS deficiency, stable shRNA knockdown models of the NDUFS3 subunit of complex I, as well as the Rieske subunit of complex III were introduced and characterized. The second hypothesis about the effect of OXPHOS deficiencies on mtDNA replication and transcription could not, without a doubt, be supported or contradicted by the data. It was determined from the data that an OXPHOS deficiency, which does not result in increased ROS levels, does not significantly affect the regulation of mtDNA replication/transcription or nuclear OXPHOS gene transcription. However, when OXPHOS deficiency was accompanied by increased ROS levels, some structural mitochondrial-encoded transcripts and regulatory nuclear-encoded transcripts were up-regulated, specifically ND6, D-loop, DNApol and TFB2M. Nonetheless, increased ROS production in the presence of OXPHOS deficiency is probably not exclusively responsible for responses of all regulatory proteins involved in mtDNA replication/transcription in vitro. Additionally, this compensatory regulation might be more dependent on mtDNA transcription than mtDNA copy number, and the data showed that TFB2M might be a key regulatory protein involved early in this mechanism before any other regulatory proteins are affected.
Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.

Plant mitochondrial genomes are large and complex, and the mechanisms for maintaining mitochondrial DNA (mtDNA) remain unclear. Arabidopsis thaliana has two DNA polymerase genes, polIA and polIB, that have been shown to be dual localized to mitochondria and chloroplasts but are unequally expressed within primary plant tissues involved in cell division or cell expansion. PolIB expression is observed at higher levels in both shoot and root apexes, suggesting a possible role in organelle DNA replication in rapidly dividing or expanding cells. It is proposed that both polIA and polIB are required for mtDNA replication under wild type conditions. An Arabidopsis T-DNA polIB mutant has a 30% reduction in mtDNA levels but also a 70% induction in polIA gene expression. The polIB mutant shows an increase relative to wild type plants in the number of mitochondria that are significantly smaller in relative size, observed within hypocotyl epidermis cells that have a reduced rate of cell expansion. These mutants exhibit a significant increase in gene expression for components of mitorespiration and photosynthesis, and there is evidence for an increase in both light to dark (transitional) and light respiration levels. There is not a significant difference in dark adjusted total respiration between mutant and wild type plants. Chloroplast numbers are not significantly different in isolated mesophyll protoplasts, but mesophyll cells from the mutant are significantly smaller than wild type. PolIB mutants exhibit a three-day delay in chloroplast development but after 7dpi (days post-imbibition) there is no difference in relative plastid DNA levels between the mutant and wild type. Overall, the polIB mutant exhibits an adjustment in cell homeostasis, which enables the maintenance of functional mitochondria but at the cost of normal cell expansion rates.

An approach was made to enrich isolated mitochondrial replication intermediates (mtRIs) for intact RNA/DNA hybrids. The S9.6 antibody preferentially binds RNA/DNA hybrids over other nucleic acid substrates, and so it was applied to highly purified preparations of mtDNA. mtRIs bound by the antibody were found to be enriched for RNase H sensitive mtRIs with blocked restriction sites, which are hallmarks of the RITOLS mechanism of replication. Conversely, mtRIs not bound by the antibody were RNase H resistant and cleaved on all branches by REs, consistent with products of strand-coupled DNA synthesis. Therefore, the S9.6 antibody based separation of mtRIs substantiates the earlier proposal of two classes of mtRI. To determine the role of RNase H1 in vivo, Rnaseh1 conditional knockout mice were generated and mouse embryo fibroblasts (MEFs) cultured. The addition of tamoxifen to the culture media results in both Rnaseh1 alleles being excised. In other experiments, wildtype and mutant versions of human RNase H1 were over-expressed in HEK293 cells. Mitochondrial DNA derived from these varied sources was analyzed by 1D-AGE, 2D-AGE, ligation mediated PCR (LM-PCR) and ribonuclease protection assay (RPA). Compared to control samples, the analysis found persistent RNA primers attached to the 5’ ends of nascent H- and L-strand molecules, combined with altered RNA-DNA transition sites. These nascent strands were sometimes incorporated into daughter molecules and ligated to upstream nascent DNA. These molecules were particularly unstable during subsequent rounds of replication and ultimately the mtDNA copy number was depleted upon modulation of RNase H1 levels. These data suggest that RNase H1 plays essential roles in generating and clearing RNAs that act as primers of DNA replication. RNase H1 primer processing is needed not only for initiation of replication, but also late in the replication cycle.

Le processus de vieillissement correspond à un déclin fonctionnel des tissus en fonction du temps. On considère généralement que la sénescence cellulaire, l'attrition des télomères et le dysfonctionnement mitochondrial contribuent au processus de vieillissement. La sénescence cellulaire est un état permanent d'arrêt du cycle cellulaire, caractérisé par des changements dans la structure de la chromatine et l'activation d'un phénotype pro-inflammatoire. Les télomères sont les structures situées à l'extrémité des chromosomes et sont protégés par un complexe protéique composé de six protéines (TRF1, TRF2, RAP1, TPP1, TIN2 et POT1) appelé shelterin. Il existe de plus en plus de preuves de l'existence de liens multiples entre la fonction mitochondriale et les télomères. Le dysfonctionnement mitochondrial entraîne une augmentation des niveaux d'espèces réactives de l'oxygène (ROS), qui provoquent un raccourcissement des télomères, et ce raccourcissement peut induire un dysfonctionnement mitochondrial par le biais de l'activation de p53. Des études récentes ont suggéré que la télomérase et certaines sous-unités de la shelterin régulent la fonction mitochondriale indépendamment de leur rôle télomérique, peut-être par le biais de leur localisation directe dans les mitochondries. Par exemple, la transcriptase inverse de la télomérase (TERT) se localise dans les mitochondries et protège l'ADN mitochondrial (ADNmt) dans les neurones en réduisant les niveaux de ROS. La protéine shelterin TIN2 se trouve dans les mitochondries où elle régule la phosphorylation oxydative. TRF2 régule l'expression de la sirtuine mitochondriale SIRT3 dans les cellules musculaires squelettiques. Dans l'ensemble, ces résultats suggèrent une boucle de rétroaction positive entre le dysfonctionnement des télomères et des mitochondries et soulèvent la question de savoir comment le lien entre les télomères et les mitochondries contribue à la sénescence.Pour répondre à cette question, nous avons étudié les fonctions de toutes les sous-unités de shelterin dans les mitochondries en utilisant des cellules de fibroblastes embryonnaires de souris (MEF). Nous avons étudié leur rôle dans le métabolisme mitochondrial et leur implication dans la réplication de l'ADN mitochondrial (ADNmt) en utilisant une technique d'analyse in situ de la réplication de l'ADN mitochondrial (MIRA). Nous avons montré que TIN2, TPP1 et TRF2 affectent le métabolisme mitochondrial, mais que seule la déplétion de TRF2 a un effet néfaste sur la réplication de l'ADNmt. Fait important, nous avons constaté que TRF2 était localisé dans les mitochondries en utilisant diverses techniques, y compris la microscopie électronique. Nous avons approfondi la caractérisation des différents domaines de TRF2 et découvert que le domaine N-terminal de TRF2 (domaine B) était nécessaire et suffisant pour sa localisation mitochondriale et pour son rôle dans la réplication de l'ADNmt. Ce domaine a déjà été impliqué dans la reconnaissance des intermédiaires de réplication, où il les protège de la dégradation par les nucléases les intermédiaires réplicatifs de l'ADN. Nous avons également constaté que les niveaux de TRF2 diminuaient au fur et à mesure que les MEF entraient en sénescence et que l'expression ectopique de TRF2 était suffisante pour maintenir les niveaux de réplication de l'ADNmt au même niveau que ceux des jeunes MEF. Dans l'ensemble, nos résultats démontrent que la protéine shelterin TRF2 régule la réplication mitochondriale au cours de la sénescence
The aging process has been defined as a time-dependent functional decline in tissue functions. Cellular senescence, telomere attrition, and mitochondrial dysfunction are generally considered to contribute to the aging process. Cellular senescence is a permanent state of cell cycle arrest, and it is characterized by changes in chromatin structure and the activation of a pro-inflammatory phenotype. Telomeres are the structures located at the ends of chromosomes and are protected by a protein complex composed of six proteins (TRF1, TRF2, RAP1, TPP1, TIN2 and POT1) called shelterin. There is increasing evidence of multiple links between mitochondrial function and telomeres. Mitochondrial dysfunction leads to increased levels of reactive oxygen species (ROS), which cause telomere shortening, and this shortening can induce mitochondrial dysfunction through p53 activation. Recent studies have suggested that telomerase and some shelterin subunits regulate mitochondrial function independently of their telomeric role, perhaps through their direct localization to the mitochondria. For instance, telomerase reverse transcriptase (TERT) localizes to mitochondria and protects mitochondrial DNA (mtDNA) in neurons by reducing ROS levels. The shelterin protein TIN2 is found at mitochondria where it regulates oxidative phosphorylation. TRF2 regulates the expression of the mitochondrial sirtuin SIRT3 in skeletal muscle cells. Altogether, these findings suggest a positive feedback loop between telomere and mitochondrial dysfunction and raise the question of how the telomere-mitochondria connection contributes to senescence.To address this question, we investigated the functions of all shelterin subunits in mitochondria using mouse embryonic fibroblast cells (MEFs). We investigated their role in mitochondrial metabolism and their implication in mitochondrial DNA (mtDNA) replication by using the in-situ analysis of mitochondrial DNA replication (MIRA) assay. We showed that TIN2, TPP1 and TRF2 affect mitochondrial metabolism, but only TRF2 depletion has a detrimental effect on mtDNA replication. Importantly, we found that TRF2 was located at mitochondria by using a variety of techniques, including electron microscopy. We went deeper into the characterization of the different domains of TRF2 and found that the N-terminal domain of TRF2 (B domain) was required and sufficient for its mitochondrial location and for its role in mtDNA replication. This domain has previously been implicated in the recognition of replication intermediates, where it protects them from nuclease degradation in a sequence-independent manner. We also found that TRF2 levels decreased as the MEFs entered senescence and that ectopic expression of TRF2 was sufficient to maintain mtDNA replication levels as those of young MEFs. Collectively, our results demonstrate that the shelterin protein TRF2 regulates mitochondrial replication during senescence

Plants have two organelles outside the nucleus which carry their own DNA, mitochondria and chloroplasts. These organelles are descendants of bacteria that were engulfed by their host according to the endosymbiotic theory. Over time, DNA has been exchanged between these organelles and the nucleus. Two polymerases, DNA Polymerases Gamma I and II, are encoded in the nucleus and remain under nuclear control, but are transported into the mitochondria and chloroplasts. DNA polymerases gamma I and II are two organelle polymerases which have been studied through sequence analysis and shown to localize to both mitochondria and chloroplasts. Little has been done to characterize the activities of these polymerases. Work in tobacco showed the homology of these polymerases to each other and to DNA Polymerase I in bacteria. They have been characterized as being part of the DNA Polymerase A family of polymerases. In my research I have studied the effect of T-DNA insertions within the DNA Polymerase Gamma I and II genes. Since these DNA Polymerases are targeted to the mitochondria and chloroplasts, I studied the effect of knocking out these genes. A plant heterozygous for an insert in DNA Polymerase Gamma I grows slightly slower than wild type plants with an approximately 20% reduction in mitochondrial and chloroplast DNA copy number. A plant homozygous for an insert in this same gene has a drastic phenotype with stunted plants that grow to around 1 inch tall, with floral stems, and have an approximately 50-55% reduction in mitochondrial and chloroplast DNA copy number. Wild type plants can grow to a height of 12-18 inches with floral stems as a comparison. A plant heterozygous for an insert in the DNA Polymerase Gamma II gene grows slightly slower than wild type plants and has an approximately 15% reduction in mitochondrial DNA copy number and a 50% reduction in chloroplast DNA copy number. These plants also produce much less seed than do other mutants and wild type plants.

博士
國防醫學院
生命科學研究所
104
Background: Mitochondria play important roles in providing metabolic energy and key metabolites for synthesis of cellular building blocks. Mitochondria have additional functions in other cellular processes, including programmed cell death and aging. A previous study revealed Drosophila mitochondrial topoisomerase III alpha (Top3α) contributes to the maintenance of the mitochondrial genome and male germ-line stem cells. However, the involvement of mitochondrial Top3α in the mitochondrion-mediated aging process remains unclear. We used both the M1L flies and mice model to study the function of Top3α protein that lacks the mitochondrial import sequence and is thus present in cell nuclei but not in mitochondria. The Drosophila model system to examine the role of mitochondrial Top3α in the aging of fruit flies. The mice model to points a role of Top3 in early embryogenesis development. Results: Here, we reported that M1L flies exhibit mitochondrial defects which affect the aging process. First, we observed that M1L flies have a shorter life span, which was correlated with a significant reduction in the mitochondrial DNA copy number, the mitochondrial membrane potential, and ATP content compared with those of both wildtype and transgene-rescued flies of the same age. Second, we performed a mobility assay and electron microscopic analysis to demonstrate that the locomotion defect and mitophagy of M1L flies were enhanced with age, as compared with the controls. We showed that the correlation between the mtDNA deletion level and aging in M1L flies resembles what was reported in mammalian systems. Finally, we observed that the M1L homozygous mutants are embryonic lethal at E8.5 day.

It is well known that the mitochondrial genome has a much higher spontaneous mutation rate than the nuclear genome. mtDNA mutations have been identified in association with many diseases and aging. mtDNA replication continues throughout the cell cycle, even in post-mitotic cells. Therefore, a constant supply of nucleotides is required for replication and maintenance of the mitochondrial genome. However, it is not clear how dNTPs arise within mitochondria nor how mitochondrial dNTP pools are regulated. Recent evidence suggests that abnormal mitochondrial nucleoside and nucleotide metabolism is associated with several human diseases. Clearly, to uncover the pathogenesis of these diseases and the mechanisms of mitochondrial mutagenesis, information is needed regarding dNTP biosynthesis and maintenance within mitochondria, and biochemical consequences of disordered mitochondrial dNTP metabolism. The studies described in this thesis provide important insight into these questions. First, we found that a distinctive form of ribonucleotide reductase is associated with mammalian liver mitochondria, indicating the presence of de novo pathway for dNTP synthesis within mitochondria. Second, we found that long term thymidine treatment could induce mtDNA deletions and the mitochondrial dNTP pool changes resulting from thymidine treatment could account for the spectrum of mtDNA point mutations found in Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients. These results support the proposed pathogenesis of this disease. Third, we found that normal intramitochondrial dNTP pools in rat tissues are highly asymmetric, and in vitro fidelity studies show that these imbalanced pools can stimulate base substitution and frameshift mutations, with a substitution pattern that correlates with mitochondrial substitution mutations in vivo. These findings suggest that normal intramitochondrial dNTP pool asymmetries could contribute to mitochondrial mutagenesis and mitochondrial diseases. Last, Amish lethal microcephaly (MCPHA) has been proposed to be caused by insufficient transport of dNTPs into mitochondria resulting from a loss-of-function mutation in the gene encoding a mitochondrial deoxynucleotide carrier (DNC). We found that there are no significant changes of intramitochondrial dNTP levels in both a MCPHA patient's lymphoblasts with a missense point mutation in Dnc gene and the homozygous mutant cells extracted from Dnc gene knockout mouse embryos. These results do not support the proposed pathogenesis of this disease and indicate that the DNC protein does not play a crucial role in the maintenance of intramitochondrial dNTP pools.
Graduation date: 2005

The human mitochondrial DNA polymerase (Pol γ) catalyzes mitochondrial DNA synthesis, and thus is essential for the integrity of the organelle. Mutations of Pol γ have been implicated in more than 150 human diseases. Reduced Pol γ activity caused by inhibition of anti-HIV drugs targeted to HIV reverse transcriptase confers major drug toxicity. To illustrate the structural basis for mtDNA replication and facilitate rational design of antiviral drugs, I have determined the crystal structure of human Pol γ holoenzyme. The structure reveals heterotrimer architecture of Pol γ holoenzyme with a monomeric catalytic subunit Pol γA, and a dimeric processivity factor Pol γB. While the polymerase and exonuclease domains in Pol γA present high structural homology with the other members of the DNA Pol I family, the spacer between the two functional domains shows a unique fold, and constitutes the subunit interface. The structure suggests a novel mechanism for Pol γ’s high processivity of DNA replication. Furthermore, the structure reveals dissimilarity in the active sites between Pol γ and HIV RT, thereby indicating an exploitable space for design of less toxic anti-HIV drugs. Interestingly, the structure shows an asymmetric subunit interaction, that is, one monomer of dimeric Pol γB primarily participates in interactions with Pol γA. To understand the roles of each Pol γB monomer, I generated a monomeric human Pol γB variant by disrupting the dimeric interface of the subunit. Comparative studies of this variant and dimeric wild-type Pol γB reveal that each monomer in the dimeric Pol γB makes a distinct contribution to processivity: one monomer (proximal to Pol γA) increases DNA binding affinity whereas the other monomer (distal to Pol γA) enhances the rate of polymerization. The pol γ holoenzyme structure also gives a rationale to establish the genotypic-phenotypic relationship of many disease-implicated mutations, especially for those located outside of the conserved pol or exo domains. Using the structure as a guide, I characterized a substitution of Pol γA residue R232 that is located at the subunit interface but far from either active sites. Kinetic analyses reveal that the mutation has no effect on intrinsic Pol γA activity, but shows functional defects in the holoenzyme, including decreased polymerase activity and increased exonuclease activity, as well as reduced discrimination between mismatched and corrected base pair. Results provide a molecular rationale for the Pol γA-R232 substitution mediated mitochondrial diseases.
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Indiana University-Purdue University Indianapolis (IUPUI)
DNA double strand break (DSB) is one of the most threatening of all types of DNA damages as it leads to a complete breakage of the chromosome. The cell has evolved several mechanisms to repair DSBs, one of which is break-induced replication (BIR). BIR repair of DSBs occurs through invasion of one end of the broken chromosome into a homologous template followed by processive replication of DNA from the donor molecule. BIR is a key cellular process and is implicated in the restart of collapsed replication forks and several chromosomal instabilities. Recently, our lab demonstrated that the fidelity of DNA synthesis associated with BIR in yeast Saccharomyces Cerevisiae is extremely low. The level of frameshift mutations associated with BIR is 1000-fold higher as compared to normal DNA replication. This work demonstrates that BIR stimulates base substitution mutations, which comprise 90% of all point mutations, making them 400-1400 times more frequent than during S-phase DNA replication. We show that DNA Polymerase δ proofreading corrects many of the base substitutions in BIR. Further, we demonstrate that Pif1, a 5’-3’ DNA helicase, is responsible for making BIR efficient and also highly mutagenic. Pif1p is responsible for the majority of BIR mutagenesis not only close to the DSB site, where BIR is less stable but also at chromosomal regions far away from the DSB break site, where BIR is fast, processive and stable. This work further reveals that, at positions close to the DSB, BIR mutagenesis in the absence of Pif1 depends on Rev3, the catalytic subunit of translesion DNA Polymerase ζ. We observe that mutations promoted by Pol ζ are often complex and propose that they are generated by a Pol ζ- led template switching mechanism. These complex mutations were also found to be frequently associated with gross chromosomal rearrangements. Finally we demonstrate that BIR is carried out by unusual conservative mode of DNA synthesis. Based on this study, we speculate that the unusual mode of DNA synthesis associated with BIR leads to various kinds of genomic instability including mutations and chromosomal rearrangements.

Doctoral Thesis in Pharmaceutical Sciences with specialization in Pharmaceutical Chemistry, presented to the Faculty of Pharmacy of the University of Coimbra.
Over the past few decades, tremendous progress has been made in the understanding, prevention and treatment of cancer. However, despite this progress, the incidence of cancer appears to be increasing. Therefore, there is an urgent need for the development of new chemotherapeutic agents with improved selectivity, efficacy and safety profiles. Numerous studies have highlighted the enormous anticancer potential of triterpenoids. In particular, pentacyclic triterpenoids have been shown to modulate multiple intracellular signaling pathways and exert chemopreventive and antitumor activities in various in vitro and in vivo model systems. Madecassic acid, a pentacyclic triterpenoid of plant origin, has been reported to possess a variety of pharmacological activities. However, only a few studies have attempted to explore the therapeutic potential of this natural compound, particularly regarding to its anticancer activity. In light of this observation, a series of new semi-synthetic derivatives of madecassic acid were designed, synthesized and evaluated for their in vitro cytotoxic activities, to identify promising lead compounds for the development of new anticancer drug therapies. The preparation of new madecassic acid derivatives was designed to follow three main synthetic strategies. The first one focused mainly on the functionalization of the C-2, C-3, C-6 and C-23 hydroxyl groups and the C-28 carboxylic acid. The second one aimed at the conversion of the 6-membered ring into a 5-membered ring with an α,β-unsaturated aldehyde substituent. Finally, the third semi-synthetic strategy was based on the introduction of different substituents at the C-2 position of the pentameric ring. All synthesized compounds were fully characterized using infrared radiation, nuclear magnetic resonance and mass spectrometry techniques, and their anticancer activity was evaluated against the US National Cancer Institute's 60 human cancer cell line (NCI-60) panel using the sulforhodamine blue colorimetric assay. Several analogs exhibited broad-spectrum cytotoxic activities over all nine tumor types represented in the panel, with more potent antiproliferative activities observed against select cancer cell lines, including multidrug-resistant phenotypes. Among them, compound 3.30, a cyclic enol ether derivative bearing a 2-furoyl moiety at C-3, exhibited sub-µM potencies against 26 different tumor cell lines from the NCI-60 panel. The mechanism of action of this compound was predicted by CellMinerTM bioinformatic analysis and confirmed by biochemical and cell-based experiments to involve inhibition of the DNA replication process and disruption of mitochondrial membrane potential. Furthermore, compounds 4.3, 4.7 and 4.10 displayed potent and highly differential antiproliferative activity against 80% of the tumor cells harboring the B-RafV600E mutation within the nanomolar range. Structure-activity analysis revealed that a 5-membered A-ring containing an α,β-unsaturated aldehyde substituted at C-23 with a 2-furoyl group seems to be crucial to produce this particular growth inhibition signature. In silico analysis of the cytotoxicity pattern of these compounds identified two highly correlated clinically approved drugs with known B-RafV600E inhibitory activity. Follow-up analysis revealed inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway through the reduction of rapidly accelerated fibrosarcoma (Raf) protein levels is a key mechanism of action of these compounds. Among these derivatives, compound 4.10 was the most potent compound in suppressing tumor growth of B-RafV600E-mutant cell lines and displayed the highest reduction of Raf protein levels. The 23-methanesulfonyloxy derivatives 5.5 and 5.7 showed different modes of action with broad cytotoxicity seen for compound 5.5 but some selectivity of cellular response seen for compound 5.7. CellMinerTM analysis revealed that compound 5.5 elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a potentially novel mechanism of anticancer action; whereas it identified the tyrosyl-DNA phosphodiesterase 1 (Tdp1) as a potential target of compound 5.7. Taken together, this work contributed to a deeper understanding of structure-activity relationship and chemical reactivity of madecassic acid derivatives. Moreover, it demonstrated the remarkable potential of madecassic acid derivatives, such as compounds 3.30, 4.10, 5.5 and 5.7, as promising leads for the development of new cancer therapies.
Nas últimas décadas tem-se assistido a um tremendo progresso na compreensão, prevenção e tratamento do cancro. No entanto, apesar deste progresso, a incidência do cancro parece estar a aumentar. Há, portanto, uma necessidade premente de desenvolver novos agentes anticancerígenos com melhores perfis de seletividade, eficácia e segurança. Vários estudos têm destacado o enorme potencial anticancerígeno dos triterpenóides. Em particular, os triterpenóides pentacíclicos parecem modular múltiplas vias de sinalização intracelular e exercer atividades quimiopreventivas e antitumorais em vários modelos in vitro e in vivo. O ácido madecássico, um triterpenóide pentacíclico de origem vegetal, demonstrou possuir uma ampla variedade de atividades farmacológicas. No entanto, poucos estudos tentaram explorar o potencial terapêutico deste composto natural, principalmente no que diz respeito à sua atividade anticancerígena. Perante este enquadramento, uma série de novos derivados semi-sintéticos do ácido madecássico foi idealizada e sintetizada e as suas atividades citotóxicas foram avaliadas in vitro, com o objetivo de identificar leads promissores para o desenvolvimento de novas terapias anticancerígenas. A preparação de novos derivados do ácido madecássico foi baseada em três principais estratégias sintéticas. A primeira focou-se principalmente na funcionalização dos grupos hidroxilos a C-2, C-3, C-6 e C-23 e do ácido carboxílico a C-28. A segunda visou a conversão do anel A de seis membros num anel de cinco membros com um substituinte aldeído α,β-insaturado. Finalmente, a terceira estratégia semi-sintética baseou-se na introdução de diferentes substituintes na posição C-2 do anel pentamérico. Todos os compostos sintetizados foram totalmente caracterizados usando técnicas de radiação infravermelha, ressonância magnética nuclear e espectrometria de massa, e as suas atividades anticancerígenas foram avaliadas num painel de 60 linhas celulares tumorais humanas do Instituto Nacional do Cancro dos Estados Unidos (NCI-60) utilizando o ensaio colorimétrico da sulforodamina B. Vários análogos exibiram atividades citotóxicas de amplo espectro nos nove tipos de tumores representados no painel, com atividades antiproliferativas mais promissoras observadas em linhas celulares selecionadas, incluindo fenótipos multirresistentes a fármacos. Entre estes, o composto 3.30, um derivado éter enólico cíclico com um substituinte 2-furoílo em C-3, exibiu actividades sub-µM em 26 linhas de células tumorais do painel NCI-60. O mecanismo de ação deste composto foi determinado utilizando a ferramenta bioinformática CellMinerTM e confirmado por experiências bioquímicas e celulares, envolvendo a inibição do processo de replicação do ADN e a alteração do potencial da membrana mitocondrial. Além disso, os compostos 4.3, 4.7 e 4.10 exibiram atividade antiproliferativa promissora e altamente diferencial dentro da gama nanomolar em 80% das células tumorais avaliadas que possuem a mutação B-RafV600E. Uma análise da relação estrutura-atividade revelou que um anel de cinco membros contendo um aldeído α,β-insaturado substituído em C-23 por um grupo 2-furoílo parece ser crucial para produzir esta assinatura específica na inibição de crescimento. A análise in silico do padrão de citotoxicidade destes compostos identificou dois fármacos clinicamente aprovados altamente correlacionados e com reconhecida atividade inibidora do B-RafV600E. Uma análise posterior revelou que a inibição da via de sinalização da quinase regulada por sinal extracelular (ERK) através da redução dos níveis de proteína do fibrossarcoma rapidamente acelerado (Raf) é um mecanismo de ação chave desses compostos. Entre estes derivados, o composto 4.10 foi o mais potente na supressão do crescimento tumoral de linhas celulares mutantes B-RafV600E e exibiu a maior redução dos níveis de proteína Raf. Os derivados de 23-metanossulfoniloxi 5.5 e 5.7 exibiram diferentes modos de ação, com ampla citotoxicidade observada para o composto 5.5, mas alguma seletividade de resposta celular observada para o composto 5.7. A análise dos resultados obtidos com recurso ao CellMinerTM revelou que o composto 5.5 provocou um perfil único de inibição de crescimento nas linhas celulares tumorais, indicando um mecanismo de ação anticancerígena potencialmente novo, enquanto identificou a tirosil-ADN fosfodiesterase 1 (Tdp1) como um potencial alvo do composto 5.7. Em conclusão, este trabalho contribuiu para uma compreensão mais aprofundada da relação estrutura-atividade e reatividade química dos derivados do ácido madecássico. Além disso, demonstrou o notável potencial dos derivados do ácido madecássico, em particular o dos compostos 3.30, 4.10, 5.5 e 5.7, como leads promissores no desenvolvimento de novas terapias contra o cancro.

Comparativement au génome contenu dans le noyau de la cellule de plante, nos connaissances des génomes des deux organelles de cette cellule, soit le plastide et la mitochondrie, sont encore très limitées. En effet, un nombre très restreint de facteurs impliqués dans la réplication et la réparation de l’ADN de ces compartiments ont été identifiés à ce jour. Au cours de notre étude, nous avons démontré l’implication de la famille de protéines Whirly dans le maintien de la stabilité des génomes des organelles. Des plantes mutantes pour des gènes Whirly chez Arabidopsis thaliana et Zea mays montrent en effet une augmentation du nombre de molécules d’ADN réarrangées dans les plastides. Ces nouvelles molécules sont le résultat d’une forme de recombinaison illégitime nommée microhomology-mediated break-induced replication qui, en temps normal, se produit rarement dans le plastide. Chez un mutant d’Arabidopsis ne possédant plus de protéines Whirly dans les plastides, ces molécules d’ADN peuvent même être amplifiées jusqu’à cinquante fois par rapport au niveau de l’ADN sauvage et causer un phénotype de variégation. L’étude des mutants des gènes Whirly a mené à la mise au point d’un test de sensibilité à un antibiotique, la ciprofloxacine, qui cause des bris double brin spécifiquement au niveau de l’ADN des organelles. Le mutant d’Arabidopsis ne contenant plus de protéines Whirly dans les plastides est plus sensible à ce stress que la plante sauvage. L’agent chimique induit en effet une augmentation du nombre de réarrangements dans le génome du plastide. Bien qu’un autre mutant ne possédant plus de protéines Whirly dans les mitochondries ne soit pas plus sensible à la ciprofloxacine, on retrouve néanmoins plus de réarrangements dans son ADN mitochondrial que dans celui de la plante sauvage. Ces résultats suggèrent donc une implication pour les protéines Whirly dans la réparation des bris double brin de l’ADN des organelles de plantes. Notre étude de la stabilité des génomes des organelles a ensuite conduit à la famille des protéines homologues des polymérases de l’ADN de type I bactérienne. Plusieurs groupes ont en effet suggéré que ces enzymes étaient responsables de la synthèse de l’ADN dans les plastides et les mitochondries. Nous avons apporté la preuve génétique de ce lien grâce à des mutants des deux gènes PolI d’Arabidopsis, qui encodent des protéines hautement similaires. La mutation simultanée des deux gènes est létale et les simples mutants possèdent moins d’ADN dans les organelles des plantes en bas âge, confirmant leur implication dans la réplication de l’ADN. De plus, les mutants du gène PolIB, mais non ceux de PolIA, sont hypersensibles à la ciprofloxacine, suggérant une fonction dans la réparation des bris de l’ADN. En accord avec ce résultat, la mutation combinée du gène PolIB et des gènes des protéines Whirly du plastide produit des plantes avec un phénotype très sévère. En définitive, l’identification de deux nouveaux facteurs impliqués dans le métabolisme de l’ADN des organelles nous permet de proposer un modèle simple pour le maintien de ces deux génomes.
Compared to the nuclear genome, very little is known about the genomes of the two plant cytoplasmic organelles, the plastid and the mitochondria. Indeed, very few factors involved in either the replication or the repair of these genomes have been identified. Here we show the implication of the Whirly protein family in the maintenance of organellar DNA. Indeed, mutations in Whirly genes lead to DNA rearrangements in both Arabidopsis thaliana and Zea mays plastids. These rearrangements are the product of microhomology-mediated break-induced replication that rarely occurs in wild-type plants but increases in absence of Whirly proteins. In a mutant plant devoid of plastidial Whirly proteins, these new DNA molecules can be amplified up to fifty times the normal DNA level and cause a variegated phenotype. In the course of the study of the Whirly mutant plants, we developed a strategy, based on the use of the antibiotic ciprofloxacin, to induce DNA double-strand breaks specifically in plant organelles. The Arabidopsis mutant plants without Whirly proteins in the plastids are more sensitive to the antibiotic ciprofloxacin than wild-type plants. Accordingly, there is a much larger increase in the number of rearranged DNA molecules in the plastids of the mutant plants than in the control plants. Surprisingly, while the mutant plants devoid of Whirly proteins in the mitochondria do not show increased sensitivity to the drug, they do accumulate more rearrangements in their mitochondrial DNA compared to wild-type plants. These results suggest that the Whirly proteins are involved in the repair of DNA double-strand breaks in the plant organelle genomes. Our study of the plant organelle genome stability has lead us to a family of proteins homologous to the DNA polymerase I in bacteria. This family has been proposed to be responsible for most of the DNA-synthesis activity in the plant organelles. We bring genetic proof to support this hypothesis using mutants of the two PolI genes of Arabidopsis. The combined mutation of both genes is lethal and the single mutations cause a decrease in the relative DNA levels in the organelles, thus confirming the involvement of both genes in DNA replication. Interestingly, mutants of the PolIB but not PolIA gene shows increase sensitivity to ciprofloxacin suggesting a function in DNA repair. In line with these results, a cross between a PolIB mutant and the mutant of plastid Whirly genes resulted in plants with severe growth defects and numerous rearrangements in the plastid DNA. In conclusion, we have identified two factors involved in the metabolism of organelle DNA and proposed a simple model of how these genomes are maintained in the plant cell.