<p> Three pieces of work are contained in this thesis. OGRe is a relational database that stores mitochondrial genomes of animals. The database has been operational for approximately five years and the number of genomes in the database has expanded to over 1000 in this period. However, sometimes, new genomes can not be added to the database because of small errors in the source ffies. Several improvements to the update method and the organizational structure of OGRe have been done, which are presented in the first part of this thesis. </p> <p> The second part of this thesis is a study on codon usage in mitochondrial genomes of mammals and fish. Codon usage bias can be caused by mutation and translational selection. In this study, we use some statistical tests and likelihood-based tests to determine which factors are most important in causing codon bias in mitochondrial genomes of mammals and fish. It is found that codon usage patterns seem to be determined principally by complex context-dependent mutational effects. </p> <p> The third part of this thesis is a phylogenetic study of 159 avian species obtained using mitochondrial rRNA sequences that were provided by Dr. van Tuinen. In this study, two methods are used: one considers sites of sequences as independently evolving; the other includes the secondary structure of rRNAs. Unfortunately, the amount of information in the rRNA sequences seems to be insufficient to determine the whole phylogeny of birds. However, our results make it clear that several traditionally defined orders are polyphyletic and therefore need to be redefined. </p> / Thesis / Master of Science (MSc)

Background: Phylogenomic analyses recently became popular to address questions about deep metazoan phylogeny. Ribosomal proteins (RP) dominate many of these analyses or are, in some cases, the only genes included. Despite initial hopes, hylogenomic analyses including tens to hundreds of genes still fail to robustly place many bilaterian taxa. Results: Using the phylogenetic position of myzostomids as an example, we show that phylogenies derived from RP genes and mitochondrial genes produce incongruent results. Whereas the former support a position within a clade of platyzoan taxa, mitochondrial data recovers an annelid affinity, which is strongly supported by the gene order data and is congruent with morphology. Using hypothesis testing, our RP data significantly rejects the annelids affinity, whereas a platyzoan relationship is significantly rejected by the mitochondrial data. Conclusion: We conclude (i) that reliance of a set of markers belonging to a single class of macromolecular complexes might bias the analysis, and (ii) that concatenation of all available data might introduce conflicting signal into phylogenetic analyses. We therefore strongly recommend testing for data incongruence in phylogenomic analyses. Furthermore, judging all available data, we consider the annelid affinity hypothesis more plausible than a possible platyzoan affinity for myzostomids, and suspect long branch attraction is influencing the RP data. However, this hypothesis needs further confirmation by future analyses.

The objective of this study was to characterize the mitochondrial genomes of the species within the genus Ceratocystis and investigate the evolutionary process of the ribosomal RNA cistron found within these fungi. Ceratocystis incorporates a number of pathogenic species affecting a variety of hosts, making the study of these fungi economically significant. The fortuitous identification of a Ceratocystis species, C. manginecans, which contained two different internally transcribed spacer sequence types within the ribosomal rRNA cistron, enabled a study of concerted evolution in this fungus. Using this non-model organism we were able to show empirical evidence for unequal crossing over and gene conversion as the ultimate forces acting on this gene region dictating a concerted evolutionary effect. We suggest that this process is true for all eukaryotes. Using the knowledge drawn from previously characterized and annotated mitochondrial genomes of other eukaryotes, the genomes of three Ceratocystis species, namely Ceratocystis fimbriata, Ceratocystis albifundus and Ceratocystis moniliformis were fully assembled and annotated for comparative analysis. This comparative study addressed the genome size, gene content, tRNA presence as well as intron types and their homing endonucleases found among these three mitochondrial genomes. An interspecies characterization was then undertaken using the mitochondrial genomes of six different C. albifundus isolates from different geographical locations in Africa. Genetic variation and similarities among these isolates supports the previous hypothesis that the origin of this fungus is Southern Africa. It is hoped that the research presented in this thesis will contribute to the improved understanding of the mitochondrial genomes in Ceratocystis species. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Genetics / Unrestricted

Much of our understanding of eukaryotic life has come from studying mitochondrial DNA, giving rise to leading hypotheses in evolution. To enable these studies, efficient algorithms are needed to interpret, analyze, and draw relevant conclusions from the available mitochondrial sequence data. The central theme of this work is to provide such algorithms for two biological problems in mitogenomes. The key element of both methods is the de Bruijn graph. Small sequence segments of length k, called k-mers, of the genomes represented in the graph form the vertices. Two vertices are connected if the suffix of length (k-1) of the first vertex is equal to the prefix of length (k-1) of the second vertex. The edges are thus specified by the (k+1)-mer consisting of the k-prefix of the first vertex and the last character of the second vertex. The first problem is the automated accurate annotation of genes in complete mitochondrial sequences. For this purpose, a new method, called DeGeCI, is presented. The method uses a large collection of mitogenomes whose sequence data is represented as an annotated de Bruijn graph. To annotate an input genome sequence, initially, a subgraph induced by all (k+1)-mers of the sequence is constructed. Unmapped parts of the sequence result in disconnected components in this subgraph, which are bridged in the next step. For this purpose, alternative trails with a high sequence similarity to the respective unmapped subsequences of the input genome are identified in the database graph and added to the subgraph. Using a clustering approach, DeGeCI aggregates annotations contained in the resulting subgraph to obtain gene predictions for the input sequence. The thesis also presents the follow-up version of DeGeCI, which offers additional features and, in contrast to DeGeCI, can be used via a web server front-end. Genome rearrangements, which change the arrangement of the genes in the genome, are particularly common in mitogenomes. The locations in the genomes where the gene order differs are called breakpoints. The second objective of this thesis is to localize these breakpoints in the nucleotide sequences of complete mitochondrial genomes, taking into account possible high substitution rates. A novel method, DeBBI, is presented to address this task. The method constructs a colored de Bruijn graph of the input sequences, where each color is associated with one of the sequences. This graph is searched for certain structures that can be associated with the breakpoint locations. These so-called breakpoint bulges are common paths that branch into two separate paths and rejoin again at another location. One of the branches is short and of a single color, while the other branch is long and color-alternating. Sequence dissimilarities distort these structures by introducing additional branches. To identify the bulges despite these distortions, DeBBI uses a heuristic algorithm.

La propagation de bactéries intracellulaires invasives peut entrainer celle des génomes mitochondriaux qui leur sont liés génétiquement au sein du cytoplasme. Cette sélection par autostop peut conduire à une réduction de la taille efficace (Ne) pour le génome mitochondrial. Elle peut également favoriser l'introgression d'une mitochondrie introduite dans une espèce suite à une hybridation. Le principal objectif de ma thèse est de quantifier ces différents effets, de manière globale, au moyen d'un large échantillonnage d'Arthropodes de Polynésie française. Les événements d'introgressions mitochondriales sont à l'origine de discordances entre les histoires évolutives des génomes mitochondriaux et nucléaires. Afin de rechercher de telles discordances, nous avons développé des marqueurs génomiques nucléaires de type RADseq, permettant de reconstruire l'histoire des populations étudiées. J'ai pu montrer au moyen de simulations que ce type de données pouvait être utilisé pour inférer des relations phylogénétiques entre espèces (Cariou et al. 2013). Des améliorations du protocole RADseq nous ont également permis de démontrer l'applicabilité de cette méthode à de nombreux spécimens au sein de librairies hautement multiplexées (Henri et al. 2015). A partir d'analyses in silico, j'ai par ailleurs évalué l'importance de différents biais liés à l'utilisation de marqueurs RADseq pour estimer les diversités génétiques et proposé une méthode permettant de corriger certains d'entre eux. A partir de ces développements, j'ai pu démontrer que sur 30 espèces de Diptères et de Lépidoptères testées à ce jour, la proximité génétique mitochondriale est systématiquement confirmée par les marqueurs nucléaires, rejetant ainsi l'hypothèse d'une introgression mitochondriale récente. Sur un plus large échantillon, nous avons en revanche mis en évidence une réduction significative du Ne mitochondrial dans les lignées infectées par Wolbachia, suffisante pour réduire le polymorphisme, mais insuffisante pour générer une réduction notable de l'efficacité de la sélection naturelle / The spread of endosymbiotic bacteria can drive that of the linked mitochondrial genomes within the cytoplasm. This hitchhiking selection can lead to a reduction of the effective population size of the mitochondrial genomes (Ne). 1t can also facilitate mitochondrial introgression, following the introduction of exogenous mitochondria in a species by hybridization. The main objective of my thesis is to quantify these different effects, on a global scale, using a large sample of Arthropods. Mitochondrial introgressions can lead to discrepancies between the evolutionary histories of mitochondrial and nuclear genomes. To investigate such patterns, we used RADseq genomic markers, that allow reconstructing population histories, and developed improvements for the library preparation and data analysis. Using in silico experiments, 1 showed that RADseq data is suitable for phylogenetic inferences (Cariou et al. 2013). Adjustments in the RADseq protocol also allowed us to demonstrate the applicability of this method for highly multiplexed libraries (Henri et al. 2015). The impact of various biases related the estimation of population genetic diversity using RADseq was also investigated in silico, which lead me to propose an ABC method to correct some of them. Following these developments, 1 showed on 30 species of Diptera and Lepidoptera that nuclear markers always confirmed the mitochondrial genetic relatedness, ruling out the hypothesis of recent mitochondrial introgressions. On a larger sample, we detected a reduction of the mitochondrial Ne in Wolbachia infected lineages. This reduction caused a significant decrease in the polymorphism of infected populations, but appeared insufficient to reduce the efficacy of natural selection

Les plantes doivent assurer la protection de trois génomes localisés dans le noyau, les chloroplastes et les mitochondries. Si les mécanismes assurant la réparation de l’ADN nucléaire sont relativement bien compris, il n’en va pas de même pour celui des chloroplastes et des mitochondries. Or il est important de bien comprendre ces mécanismes puisque des dommages à l’ADN non ou mal réparés peuvent entraîner des réarrangements dans les génomes. Chez les plantes, de tels réarrangements dans l’ADN mitochondrial ou dans l’ADN chloroplastique peuvent conduire à une perte de vigueur ou à un ralentissement de la croissance. Récemment, notre laboratoire a identifié une famille de protéines, les Whirly, dont les membres se localisent au niveau des mitochondries et des chloroplastes. Ces protéines forment des tétramères qui lient l’ADN monocaténaire et qui accomplissent de nombreuses fonctions associées au métabolisme de l’ADN. Chez Arabidopsis, deux de ces protéines ont été associées au maintien de la stabilité du génome du chloroplaste. On ignore cependant si ces protéines sont impliquées dans la réparation de l’ADN. Notre étude chez Arabidopsis démontre que des cassures bicaténaires de l’ADN sont prises en charge dans les mitochondries et les chloroplastes par une voie de réparation dépendant de très courtes séquences répétées (de cinq à cinquante paires de bases) d’ADN. Nous avons également montré que les protéines Whirly modulent cette voie de réparation. Plus précisément, leur rôle serait de promouvoir une réparation fidèle de l’ADN en empêchant la formation de réarrangements dans les génomes de ces organites. Pour comprendre comment les protéines Whirly sont impliquées dans ce processus, nous avons élucidé la structure cristalline d’un complexe Whirly-ADN. Nous avons ainsi pu montrer que les Whirly lient et protègent l’ADN monocaténaire sans spécificité de séquence. La liaison de l’ADN s’effectue entre les feuillets β de sous-unités contiguës du tétramère. Cette configuration maintient l’ADN sous une forme monocaténaire et empêche son appariement avec des acides nucléiques de séquence complémentaire. Ainsi, les protéines Whirly peuvent empêcher la formation de réarrangements et favoriser une réparation fidèle de l’ADN. Nous avons également montré que, lors de la liaison de très longues séquences d’ADN, les protéines Whirly peuvent s’agencer en superstructures d’hexamères de tétramères, formant ainsi des particules sphériques de douze nanomètres de diamètre. En particulier, nous avons pu démontrer l’importance d’un résidu lysine conservé chez les Whirly de plantes dans le maintien de la stabilité de ces superstructures, dans la liaison coopérative de l’ADN, ainsi que dans la réparation de l’ADN chez Arabidopsis. Globalement, notre étude amène de nouvelles connaissances quant aux mécanismes de réparation de l’ADN dans les organites de plantes ainsi que le rôle des protéines Whirly dans ce processus. / Plants must protect the integrity of three genomes located respectively in the nucleus, the chloroplasts and the mitochondria. Although DNA repair mechanisms in the nucleus are the subject of multiple studies, little attention has been paid to DNA repair mechanisms in chloroplasts and mitochondria. This is unfortunate since mutations in the chloroplast or the mitochondrial genome can lead to altered plant growth and development. Our laboratory has identified a new family of proteins, the Whirlies, whose members are located in plant mitochondria and chloroplasts. These proteins form tetramers that bind single-stranded DNA and play various roles associated with DNA metabolism. In Arabidopsis, two Whirly proteins maintain chloroplast genome stability. Whether or not these proteins are involved in DNA repair has so far not been investigated. Our studies in Arabidopsis demonstrate that DNA double-strand breaks are repaired in both mitochondria and chloroplasts through a microhomology-mediated repair pathway and indicate that Whirly proteins affect this pathway. In particular, the role of Whirly proteins would be to promote accurate repair of organelle DNA by preventing the repair of DNA double-strand breaks by the microhomology-dependant pathway. To understand how Whirly proteins mediate this function, we solved the crystal structure of Whirly-DNA complexes. These structures show that Whirly proteins bind single-stranded DNA with low sequence specificity. The DNA is maintained in an extended conformation between the β-sheets of adjacent protomers, thus preventing spurious annealing with a complementary strand. In turn, this prevents formation of DNA rearrangements and favors accurate DNA repair. We also show that upon binding long ssDNA sequences, Whirly proteins assemble into higher order structures, or hexamers of tetramers, thus forming spherical particles of twelve nanometers in diameter. We also demonstrate that a lysine residue conserved among plant Whirly proteins is important for the stability of these higher order structures as well as for cooperative binding to DNA and for DNA repair. Overall, our study elucidates some of the mechanisms of DNA repair in plant organelles as well as the roles of Whirly proteins in this process.

Les plantes doivent assurer la protection de trois génomes localisés dans le noyau, les chloroplastes et les mitochondries. Si les mécanismes assurant la réparation de l’ADN nucléaire sont relativement bien compris, il n’en va pas de même pour celui des chloroplastes et des mitochondries. Or il est important de bien comprendre ces mécanismes puisque des dommages à l’ADN non ou mal réparés peuvent entraîner des réarrangements dans les génomes. Chez les plantes, de tels réarrangements dans l’ADN mitochondrial ou dans l’ADN chloroplastique peuvent conduire à une perte de vigueur ou à un ralentissement de la croissance. Récemment, notre laboratoire a identifié une famille de protéines, les Whirly, dont les membres se localisent au niveau des mitochondries et des chloroplastes. Ces protéines forment des tétramères qui lient l’ADN monocaténaire et qui accomplissent de nombreuses fonctions associées au métabolisme de l’ADN. Chez Arabidopsis, deux de ces protéines ont été associées au maintien de la stabilité du génome du chloroplaste. On ignore cependant si ces protéines sont impliquées dans la réparation de l’ADN. Notre étude chez Arabidopsis démontre que des cassures bicaténaires de l’ADN sont prises en charge dans les mitochondries et les chloroplastes par une voie de réparation dépendant de très courtes séquences répétées (de cinq à cinquante paires de bases) d’ADN. Nous avons également montré que les protéines Whirly modulent cette voie de réparation. Plus précisément, leur rôle serait de promouvoir une réparation fidèle de l’ADN en empêchant la formation de réarrangements dans les génomes de ces organites. Pour comprendre comment les protéines Whirly sont impliquées dans ce processus, nous avons élucidé la structure cristalline d’un complexe Whirly-ADN. Nous avons ainsi pu montrer que les Whirly lient et protègent l’ADN monocaténaire sans spécificité de séquence. La liaison de l’ADN s’effectue entre les feuillets β de sous-unités contiguës du tétramère. Cette configuration maintient l’ADN sous une forme monocaténaire et empêche son appariement avec des acides nucléiques de séquence complémentaire. Ainsi, les protéines Whirly peuvent empêcher la formation de réarrangements et favoriser une réparation fidèle de l’ADN. Nous avons également montré que, lors de la liaison de très longues séquences d’ADN, les protéines Whirly peuvent s’agencer en superstructures d’hexamères de tétramères, formant ainsi des particules sphériques de douze nanomètres de diamètre. En particulier, nous avons pu démontrer l’importance d’un résidu lysine conservé chez les Whirly de plantes dans le maintien de la stabilité de ces superstructures, dans la liaison coopérative de l’ADN, ainsi que dans la réparation de l’ADN chez Arabidopsis. Globalement, notre étude amène de nouvelles connaissances quant aux mécanismes de réparation de l’ADN dans les organites de plantes ainsi que le rôle des protéines Whirly dans ce processus. / Plants must protect the integrity of three genomes located respectively in the nucleus, the chloroplasts and the mitochondria. Although DNA repair mechanisms in the nucleus are the subject of multiple studies, little attention has been paid to DNA repair mechanisms in chloroplasts and mitochondria. This is unfortunate since mutations in the chloroplast or the mitochondrial genome can lead to altered plant growth and development. Our laboratory has identified a new family of proteins, the Whirlies, whose members are located in plant mitochondria and chloroplasts. These proteins form tetramers that bind single-stranded DNA and play various roles associated with DNA metabolism. In Arabidopsis, two Whirly proteins maintain chloroplast genome stability. Whether or not these proteins are involved in DNA repair has so far not been investigated. Our studies in Arabidopsis demonstrate that DNA double-strand breaks are repaired in both mitochondria and chloroplasts through a microhomology-mediated repair pathway and indicate that Whirly proteins affect this pathway. In particular, the role of Whirly proteins would be to promote accurate repair of organelle DNA by preventing the repair of DNA double-strand breaks by the microhomology-dependant pathway. To understand how Whirly proteins mediate this function, we solved the crystal structure of Whirly-DNA complexes. These structures show that Whirly proteins bind single-stranded DNA with low sequence specificity. The DNA is maintained in an extended conformation between the β-sheets of adjacent protomers, thus preventing spurious annealing with a complementary strand. In turn, this prevents formation of DNA rearrangements and favors accurate DNA repair. We also show that upon binding long ssDNA sequences, Whirly proteins assemble into higher order structures, or hexamers of tetramers, thus forming spherical particles of twelve nanometers in diameter. We also demonstrate that a lysine residue conserved among plant Whirly proteins is important for the stability of these higher order structures as well as for cooperative binding to DNA and for DNA repair. Overall, our study elucidates some of the mechanisms of DNA repair in plant organelles as well as the roles of Whirly proteins in this process.

Maintenance of genomic integrity and stability is of prime importance for the survival of an organism. Upon exposure to different damaging agents, DNA acquires various lesions such as base modifications, single-strand breaks (SSBs), and double-strand breaks (DSBs). Organisms have evolved specific repair pathways in order to efficiently correct such DNA damages. Among various types of DNA damages, DSBs are the most serious when present inside cells. Unrepaired or misrepaired DSBs account for some of the genetic instabilities that lead to secondary chromosomal rearrangements, such as deletions, inversions, and translocations and consequently to cancer predisposition. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. Mitochondrial DNA (mtDNA) deletions identified in humans are flanked by short directly-repeated sequences, however, the mechanism by which these deletions arise are unknown. mtDNA deletions are associated with various types of mitochondrial disorders related to cancer, aging, diabetes, deafness, neurodegenerative disorders, sporadic and inherited diseases. Compared to nuclear DNA (nDNA), mtDNA is highly exposed to oxidative stress due to its proximity to the respiratory chain and the lack of protective histones. DSBs generated by reactive oxygen species, replication stalling or radiation represents a highly dangerous form of damage to both nDNA and mtDNA. However, the repair of DSBs in mitochondria and the proteins involved in this repair are still elusive. Animals deficient for any one of the known Classical-NHEJ factors are immunodeficient. However, DSB repair (DSBR) is not eliminated entirely in these animals suggesting evidence of alternative mechanism, ‘alternative NHEJ’ (A-NHEJ/A-EJ). Several lines of evidence also suggest that alternative and less well-defined backup NHEJ (B-NHEJ) pathways play an important role in physiological and pathological DSBR. We studied NHEJ in different tissue mitochondrial protein extracts using oligomeric DNA substrates which mimics various endogenous DSBs. Results showed A-EJ, as the predominant pathway in mitochondria. Interestingly, immunoprecipitation (IP) studies and specific inhibitor assays suggested, mitochondrial end joining (EJ) was dependent on A-EJ proteins and independent of C-NHEJ proteins. Further, colocalization studies showed A-EJ proteins localize into mitochondria in HeLa cells. More importantly knockdown experiments showed the involvement of DNA LIGASE III in mitochondrial A-EJ. These observations highlight the central role of A-EJ in maintenance of the mammalian mitochondrial genome. By using oligomeric DNA substrates mimicking various endogenous DSBs, NHEJ in different cancer cell lines were studied. We found that the efficiency of NHEJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of NHEJ proteins. Interestingly, cancer cells with lower levels of BCL2 possessed efficient NHEJ and vice versa. Removal of BCL2 by immunoprecipitation and protein fractionation using size exclusion column chromatography showed elevated levels of EJ. Most importantly, the overexpression of BCL2 in vivo or the addition of purified BCL2 in vitro led to the downregulation of NHEJ in cancer cells. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo using immunoprecipitation and immunofluorescence, respectively. Hence, NHEJ in cancer cells is negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute towards increased chromosomal abnormalities in cancer. In summary, our study showed that the efficiency of EJ in cancers could be regulated by the antiapoptotic protein BCL2. However, it may not affect the mechanistic aspect of EJ. BCL2 instead may interfere with EJ by sequestering KU and preventing it from binding to DNA ends. This may help in better understanding towards increased chromosomal abnormalities in cancer. Study of mitochondrial DSBR in mammalian system highlights the central role of microhomology-mediated A-EJ in the maintenance of the mammalian mitochondrial genome and this knowledge will helpful for the development of future therapeutic strategies against variety of mitochondria associated diseases.