Mastreviruses (family Geminiviridae) are plant-infecting viruses with circular single-stranded (ss) DNA genomes (~2.7kb). The genus Mastrevirus is comprised of thirty-two species which are transmitted by leafhoppers belonging to the genus Cicadulina. Mastreviruses are widely distributed and have been found in the Middle East, Europe, Asia, Australia, Africa and surrounding islands. Only one species, dragonfly-associated mastrevirus has so far been identified in the Americas, isolated from a dragonfly in Puerto Rico. Species can be group based on the host(s) they infect, those which infect monocotyledonous (monocot) plants and those which infect dicotyledonous (dicot) plants. In recent years many new mastrevirus species have been discovered. Several of these new discoveries can largely been attributed to the development of new molecular tools. The current state of sequencing platforms has made it affordable and easier to characterise mastreviruses at a genome level thus allowing scientists to delve deeper into understanding the dynamics of mastreviruses. A few mastrevirus species have been identified as important agricultural pathogens and as a result have been the focus of much of the mastrevirus research. Maize streak virus, strain A (MSV-A) has been the most extensively studied due to the devastating impact it has on maize production in Africa. Studies have shown that MSV-A likely emerged as a pathogen of maize less than 250 years following introduction of maize in Africa by early European settlers. There is compelling evidence to suggest that MSV-A is likely the result of recombination events between wild grass adapted MSV strains. It therefore is equally important to monitor viruses infecting non-cultivated plants in order to gain a greater understanding of the epidemiological dynamics of mastreviruses, which in turn is essential for implementing disease management strategies. The objective of the research undertaken as part of this PhD thesis was to investigate global mastrevirus dynamics focusing on diversity, host and geographic ranges, mechanisms of evolution, phylogeography and possible origins of these viruses. In addition to this a viral metagenomic approach was used in order to identify novel mastreviruses or mastrevirus-like present in New Zealand. The dynamics of the monocot-infecting mastreviruses are investigated in Chapter Two and Three. The work described in these two chapters focus mainly on mastreviruses which infect non-cultivated grasses in Africa and Australia, a total of 161 full mastrevirus genomes were recovered collectively in the two studies. Chapter Two reveals a high level of mastrevirus diversity present in Australia with the discovery of four new species and several new strains of previously characterised species. An extensive sampling effort in Africa undertaken in Chapter Three reveals a broader host range and geographic distribution of the African monocot-infecting mastreviruses than previously documented. Mosaic patterns of recombination are evident among both the Australian and African monocot-infecting mastreviruses. In Chapters Four, Five and Six a comprehensive investigation was undertaken focusing on the dicot-infecting mastreviruses. The study undertaken in Chapter Four entailed the recovery of 49 full mastrevirus genomes from Australia, the Middle East, Africa, Turkey and the Indian Subcontinent to investigate the diversity of dicot-infecting mastreviruses from a global context. Analyses revealed a high degree of CpCDV strain diversity and extended the known geographic range of CpCDV. For the first time phylogeographic analysis was able to investigate the origins of the dicot-infecting mastreviruses. Results revealed the likely origin of the most recent common ancestor (MRCA) of these viruses is likely closer to Australia than anywhere else that dicot-infecting mastreviruses have been sampled and illuminated a supported series of historical movements following the emergence of the MRCA. In Chapter Five two novel mastreviruses Australian-like mastreviruses were isolated from chickpea material from Pakistan. A comprehensive analysis of CpCDV isolates in the major pulse growing regions of Sudan in Chapter Six reveals that this region harbours a high degree of strain diversity. Complex patterns of intra-species recombination indicate these strains are evidently circulating in these regions and infecting the same hosts, driving the emergence of new CpCDV strains. Collectively the results discussed in Chapters Two through Six extended the current knowledge of mastrevirus diversity. The natural host range of many mastreviruses has proven to be more extensive than previously documented, with many species having overlapping host ranges and hence these hosts could be acting as ‘mixing vessels’ enabling inter-species recombination. Patterns of recombination and selection were observed in both the monocot-infecting and the dicot-infecting mastreviruses further elucidating the mechanisms these viruses employ to evolve rapidly. Extensive sampling in a wide range of geographic regions provides insights into the true geographic range of species such as MSV and CpCDV. Given that mastreviruses have been able to move globally and Australia has been identified as a major mastrevirus diversity hotspot it is conceivable that mastreviruses are also present in New Zealand. In Chapter Seven and Eight this is explored by using a viral metagenomic approach to investigate the ssDNA viral populations associated with wild grasses and sewage material in New Zealand. Although no mastreviruses were recovered, this endeavour resulted in the discovery of more than 50 novel circular Rep-encoding ssDNA (CRESS DNA) viruses associated with non-cultivated grasses and treated sewage material, many of which are similar to mastreviruses and other geminiviruses. These discoveries expand current knowledge on the diversity of ssDNA viruses present in New Zealand and further highlight this viral metagenomic approach as an effective method for ssDNA virus discovery. Overall the results discussed in this thesis provide insights into mastrevirus diversity and dynamics as well as revealing a wealth of novel CRESS DNA viruses, some of which share similarities to geminiviruses.

No description available.

Thesis (M.Sc. (Tropical Medicine))--Mahidol University, 2003.

Au cours de la réplication de l’ADN, les cellules rencontrent régulièrement des obstacles d’origine endogène et exogène qui peuvent mettre en péril la réplication des génomes et menacer la duplication et ségrégation des chromosomes en mitose. La Recombinaison Homologue (RH) a un rôle bien caractérisé dans la réparation des cassures double-brin. Par contre, son rôle dans la protection et le redémarrage des fourches de réplication est moins bien caractérisé. Il a été montré par l’équipe que le redémarrage des fourches bloquées par la RH dépend de la formation d’ADN simple-brin et pas d’une cassure double-brin.Afin d’étudier les mécanismes par lesquels la RH contribue au sauvetage des fourches de réplication bloquées, un système permettant de bloquer localement la progression d’une seule fourche de réplication a été utilisé. Cet essai génétique a permis de montrer que le redémarrage de fourches bloquées par la RH est associé à une synthèse d’ADN fautive suite à des événements de glissement de la polymérase au niveau de micro-homologies. Un marqueur génétique a été associé à la barrière de réplication afin de mesurer l’efficacité de redémarrage des fourches bloquées et d’étudier l’étape de résection (i.e formation de l’ADN simple brin) dans différents fonds génétiques.Dans ce travail, le rôle de facteurs impliqués dans la résection a été étudié dans le contexte d’un blocage de fourche de réplication. Comme pour la réparation de cassures double-brin, la résection des fourches bloquées se fait en deux étapes : résection initiale et extensive. La résection initiale, de faible portée, dépend du complexe MRN (Mre11/Rad50/Nbs1) et Ctp1. A cette étape, la dégradation de l’ADN néosynthétisé se fait sur une distance de 110 bp. Cette résection est suffisante pour permettre de recruter les facteurs de la RH, mais est aussi nécessaire pour que les fourches continuent à être résectées. L’absence de MRN et/ou Ctp1 conduit à un défaut de redémarrage. La résection extensive, qui expose de l’ADN simple brin sur une distance de 0,8 à 1Kb, est largement dépendante de la nucléase Exo1. Contrairement à la résection initiale, la résection extensive n’est pas critique pour le redémarrage des fourches par la RH.De façon intéressante, le facteur Ku, connu pour être impliqué dans la jonction d’extrémités non-homologue, a un rôle dans le contrôle de la résection initiale et extensive et dans l’optimisation du redémarrage des fourches bloquées. Plus précisément, en absence de Ku, de l’ADN simple-brin s’accumule en amont des fourches bloquées, et la dynamique de redémarrage est affaiblie, mais pas abolie. Globalement, ces résultats clarifient une étape cruciale dans le redémarrage des fourches par la RH : la résection. / On a regular basis, cells encounter endogenous and exogenous replication stresses that jeopardize the progression of replication forks, thus threatening both the accuracy of chromosome duplication and their segregation during mitosis. Homologous recombination (HR) has a well-known role in repairing DNA double strand breaks (DSB). Other less acknowledged functions of HR are to protect and restart impeded forks. As it was previously reported by the team, restarting replication forks by HR requires the exposure of a single-stranded gap through fork resection, and not a DSB, to allow the recruitment of recombination factors.To study the effects of HR in blocked replication forks, a conditional fork barrier (RFB) was used to terminally-arrest replication at a specific locus. This construct allowed to determine that replication restart by HR is error-prone, leading to replication forks liable to slippage at micro-homology. A genetic reporter assay was placed in the vicinity of the RFB to allow the efficiency of replication restart and the step of resection to be quantified.In here, we explored factors involved in the formation of ssDNA gaps at halted replication forks. Similarly to DSB repair, resection in fork restart occurs in two steps. The initial resection is performed by MRN (Mre11/Rad50/NBS1) and Ctp1. This small degradation of approximately 110 bp of newly synthetized strands is sufficient to recruit HR factors and is required to promote the subsequent resection. The absence of either MRN or Ctp1 leads to defective replication restart by HR. The extensive resection (about 0.8-1Kb in size) is largely dependent on the nuclease Exo1, and it is not required for efficient fork restart.Interestingly, the non-homologous end-joining factor Ku was found to have a role in orchestrating initial and extensive resection and fine-tuning fork restart. Specifically, in the absence of Ku, ssDNA accumulates at the terminally-arrested replication forks, and fork restart dynamics is decreased, but not abolished. Overall, these results shed light on a delicate step of replication fork recovery by homologous recombination: resection.

Background The Red proteins of lambda phage mediate probably the simplest and most efficient homologous recombination reactions yet described. However the mechanism of dsDNA recombination remains undefined. Results Here we show that the Red proteins can act via full length single stranded intermediates to establish single stranded heteroduplexes at the replication fork. We created asymmetrically digestible dsDNA substrates by exploiting the fact that Redα exonuclease activity requires a 5' phosphorylated end, or is blocked by phosphothioates. Using these substrates, we found that the most efficient configuration for dsDNA recombination occurred when the strand that can prime Okazaki-like synthesis contained both homology regions on the same ssDNA molecule. Furthermore, we show that Red recombination requires replication of the target molecule. Conclusions Hence we propose a new model for dsDNA recombination, termed 'beta' recombination, based on the formation of ssDNA heteroduplexes at the replication fork. Implications of the model were tested using (i) an in situ assay for recombination, which showed that recombination generated mixed wild type and recombinant colonies; and (ii) the predicted asymmetries of the homology arms, which showed that recombination is more sensitive to non-homologies attached to 5' than 3' ends. Whereas beta recombination can generate deletions in target BACs of at least 50 kb at about the same efficiency as small deletions, the converse event of insertion is very sensitive to increasing size. Insertions up to 3 kb are most efficiently achieved using beta recombination, however at greater sizes, an alternative Red-mediated mechanism(s) appears to be equally efficient. These findings define a new intermediate in homologous recombination, which also has practical implications for recombineering with the Red proteins.

Human RPA (replication protein A), a single-stranded DNA-binding protein, is required for many cellular pathways including DNA repair, recombination and replication. However, the role of RPA in nucleotide excision repair remains elusive. In the present study, we have systematically examined the binding of RPA to a battery of well-defined ssDNA (single-stranded DNA) substrates using fluorescence spectroscopy. These substrates contain adducts of (6-4) photoproducts, N-acetyl-2-aminofluorene-, 1-amino-pyrene-, BPDE (benzo[a]pyrene diol epoxide)- and fluorescein that are different in many aspects such as molecular structure and size, DNA disruption mode (e.g. base stacking or non-stacking), as well as chemical properties. Our results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA and that the affinity of RPA for damaged ssDNA depends on the type of adduct. Interestingly, the bulkier lesions have a greater effect. With a fluorescent base-stacking bulky adduct, (+)-cis-anti-BPDE-dG, we demonstrated that, on binding of RPA. the fluorescence of BPDE-ssDNA was significantly enhanced by up to 8-9-fold. This indicated that the stacking between the BPDE adduct and its neighbouring ssDNA bases had been disrupted and there was a lack of substantial direct contacts between the protein residues and the lesion itself. For RPA interaction with short damaged ssDNA, we propose that, on RPA binding, the modified base of ssDNA is looped out from the surface of the protein, permitting proper contacts of RPA with the remaining unmodified bases.

Les interactions ARN-protéine interviennent dans de nombreux processus cellulaires fondamentaux. L'obtention de détails à l'échelle atomique de ces interactions nous éclaire sur leurs fonctions, mais permet également d'envisager la conception rationnelle de ligands pouvant les moduler. Lorsque les deux techniques majeures que sont la RMN et la cristallographie aux rayons X ne permettent pas d'obtenir une structure 3D entre les deux partenaires, des approches de docking peuvent être utilisées pour apporter des modèles. L'application de ces approches aux complexes ARN-protéine se heurtent cependant à une difficulté. Ces complexes résultent en effet souvent de la liaison spécifique d'une courte séquence d'ARN simple-brin (ARNsb) à sa protéine cible. Hors, la flexibilité inhérente aux segments simples-brins impose dans une approche classique de docking d'explorer un large ensemble de leur espace conformationnel. L'objectif du projet est de contourner cette difficulté par le développement d'une approche de docking dite "par fragments". Ce dernier s'est fait à partir de domaines de liaison à l'ARN très représentés dans le monde du vivant. Les résultats ont montré une excellente capacité prédictive de l'approche à partir de la séquence de l'ARN. Ils ont de plus montré un potentiel intéressant dans la prédiction de séquences d'ARN simple-brin préférentiellement reconnues par des domaines de liaisons à l'ARN. / RNA-protein interactions mediate numerous fundamental cellular processes. Atomic scale details of these interactions shed light on their functions but can also allow the rational design of ligands that could modulate them. NMR and X-ray crystallography are the 2 main techniques used to resolve 3D highresolution structures between two interacting molecules. Docking approaches can also be utilized to give models as an alternative. However, the application of these approaches to RNA-protein complexes is hampered by an issue. RNA-protein interactions often relies on the specific recognition of a short singlestranded RNA (ssRNA) sequence by the protein. The inherent flexibility of the ssRNA segment would impose, in a classical docking approach, to explore their resulting large conformation space which is not computationally reliable. The goal of this project is to overcome this barrier by using a fragment-based docking approach. This approach developed from some of the most represented RNA-binding domains showed excellent results in the prediction of the ssRNA-protein binding mode from the RNA sequence and also a great potential to predict preferential RNA binding sequences.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12821号 / 論農博第2794号 / 新制||農||1025(附属図書館) / 学位論文||H26||N4816(農学部図書室) / 31308 / (主査)教授 河田 照雄, 教授 保川 清, 准教授 橋本 渉 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

<p>Bacteria of the genus <em>Deinococcus</em> are perhaps the most resilient life forms ever discovered, demonstrating extreme resistance to ionizing radiation, ultraviolet radiation, desiccation, and a variety of mutagenic chemical agents. The most studied member of this genus, <em>D. radiodurans</em>, has been observed to rapidly reassemble its genome following severe fragmentation by hundreds of γ-radiation induced double-strand DNA breaks. Amongst the numerous factors contributing to DNA repair, a single-stranded annealing protein, DdrB, is believed to play an important role during the initial phases of recovery. The work described in this thesis represents the first structural characterization of DdrB, revealing a novel fold for single-stranded DNA binding. Together with biochemical data delineating the DNA-binding interface, two crystal structures of the DdrB/ssDNA complex were also solved, providing a comprehensive illustration of this interaction. Quaternary assemblies observed in these crystal structures also informed on the potential contribution of higher-order nucleoprotein complexes to the function of DdrB in single-stranded annealing. Most significantly, a face-to-face assembly of DdrB/ssDNA complexes provided insight into the mechanism by which DdrB mediates annealing of DNA, which may represent a common mechanism shared by other single-stranded annealing proteins.<strong></strong></p> / Doctor of Philosophy (PhD)

<p>This thesis is based on six papers, Papers I-VI, focusing on the interplay between the stabilizing elements of nucleic acids self-assembly; hydrogen bonding, stacking and solvent effects. In Paper I we investigate how the substitution of the O4' for CH₂ in the sugar moiety of adenosine (2'-deoxyaristeromycin) at the A⁶ position of the Dickerson-Drew dodecamer makes the two modified bases exist in a dynamic equilibrium between Hoogsteen and Watson-Crick base pairing in the NMR time scale. Paper II is a structural study of the incorporation of 1-(1',3'-<i>O</i>-anhydro-<i>β</i>-D-psicofuranosyl)thymine in the T⁷ position of the Dickerson-Drew dodecamer. NMR constrained molecular dynamics and hydration studies show the base-base distortions caused by the introduction of a North-type locked sugar in an otherwise B-type DNA•DNA duplex. Paper III shows that the stacking distortion caused by the 1-(1',3'-<i>O</i>-anhydro-<i>β</i>-D-psicofuranosyl)thymine building block perturbs the charge transfer similar to a DNA mismatch. Paper IV highlights how the sequence context affects the physico-chemical properties, monitored by the p<i>K</i><i>a</i> of guanine itself as well as how the charge perturbation is experienced by the neighboring bases, in ssDNA and ssRNA. Paper V focuses on the differences between the structural equilibria of single-stranded ssDNA and ssRNA. Directional differences in single-stranded stacking between ssDNA and ssRNA are identified and provide a basis to explain directional differences in p<i>K</i><i>a</i> modulation and dangling-end stabilization. In Paper VI the thermodynamic gains of dangling ends on DNA and RNA core duplexes are found to correlate with the X-ray geometries of dangling nucleobases relative to the hydrogen bonds of the closing base pairs.</p>