Numerous short flapped DNA structures are created during the semi-discontinuous replication. These toxic intermediates are quickly resolved to produce a fully intact duplex of replicated DNA. Structure specific nuclease are key to resolving these structures, and show a high degree of specificity for their cognate substrate structures while being essentially insensitive to nucleotide sequence. Herein I demonstrate through confocal based single molecule experiments that the 5’ structure specific nuclease Flap Endonuclease 1 (FEN1) achieves its substrate specificity by coupling the bending of DNA substrate with structuring of the active site in a way that non-cognate structures binding is significantly destabilized and enzymatic features are incapable of structuring in the absence of particular substrate features, in particular a single nucleotide 3’ flap the FEN1 induces in nearly all DNA substrates. Debate remained over whether DNA was bound via a conformational capture or induced fit mechanism, and so I proceed to investigate the dynamics of the DNA itself in solution. Conclusions about conformational capture or induced fit remain elusive, however I did determine that DNA structures are rigidified by charge repulsion, an effect lessened by the salt concentration, which functions to shield the negative charge of DNA from itself. Additionally unstacking of the DNA in nicked structures incurs a significant free energy penalty, which FEN1 overcomes by its hydrophobic wedge motif, lending credence to an induced fit mechanism.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13085号 / 論医博第2126号 / 新制||医||1021(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 渡邊 直樹, 教授 岩田 想, 教授 富樫 かおり / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20243号 / 医博第4202号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 原田 浩, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

Discovering, screening, and associating changes in DNA sequence are important to a broad range of disciplines and play a central role in Forensic Science. The typical types of changes include sequence variations [single nucleotide polymorphisms (SNP)] and length variations [short tandem repeats (STR)]. The steps for forensic DNA sample processing are similar for both types of changes but diverge at the point of detection. A number of approaches are being explored for SNP genotyping while STR analysis primarily consists of size-based analysis by capillary electrophoresis. Limitations exist for all current detection methods that pose significant impacts to forensic analysis. Bi-allelic SNPs result in three possible genotypes with a minimal amount of information generated per marker. Limitations for SNP analysis are due to the inability to amplify a suitable number of SNP markers from low DNA content samples to provide an appropriate level of discrimination. Multi-allelic STR markers are currently the marker of choice for forensic typing but a variety of experimental artifacts are possible that consist of either biology or technology related causes. Molecular genotyping methods developed across other disciplines have potential to alleviate some of these shortcomings but no current approach is capable of genotyping both SNP and STR loci with a single chemistry. The need for a more effective, efficient, and generalized approach led to development of a unique method called Dye Probe Fluorescence Resonance Energy Transfer (dpFRET) and determination of its suitability for forensic analysis. The development phase of the research consisted of synthetic testing to establish proof of concept for the chemistry followed by polymerase chain reaction (PCR) based assays to demonstrate real world applications. Following successful development, the boundaries and limitations for the technology were established (sensitivity, allelic dropout, mixed samples) and efforts were made to improve the approach. In the process, parallel testing for other fields including molecular pathology and conservation biology were incorporated to explore potential widespread application of this new approach. The overall goal of this project was to develop and explore the limitations for a unique approach to genotyping both SNPs and STRs. A majority of the work involved development of the method itself with the ultimate objective of application for forensic science. The focus of this project was to address and alleviate some of the shortcomings of current approaches that result in potential limitations for forensic analysis. It is expected that future applications of this technology might impact a wide range of disciplines to aid in discovery, screening and association of changes in DNA sequence.

No description available.

The LuxR family of proteins serves as quorum-sensing transcriptional regulators in proteobacteria. At high population densities, a small acyl-homoserine lactone (AHL) molecule, produced by a LuxI homologue, accumulates in the environment. The LuxR proteins bind to their respective AHL when the ligand accumulates to sufficient levels. Once bound to AHL, the holoproteins usually become functional as transcriptional activators. However, there is a subset of LuxR homologues, the EsaR subfamily, which is active without the AHL ligand and becomes inactivated once bound to it. EsaR is the best understood member of this subfamily. It controls virulence in the corn pathogen Pantoea stewartii ssp. stewartii. Solubility issues have previously limited structural studies of LuxR homologues as the proteins could not be purified without the AHL ligand. A soluble recombinant EsaR protein, HMGE, is biologically active and can be purified in the absence and presence of AHL, unlike most other LuxR homologues. Using HMGE, amino acid substitutions and Förster resonance energy transfer (FRET), experimental methods were designed for determining the dimerization interface of EsaR and for testing the hypothesis that EsaR undergoes a conformational shift when presented with the AHL ligand. To identify residues of the dimerization interface, heterodimerization assays were designed, involving either coexpression or coincubation of wild-type EsaR and variant HMGE proteins. In this assay, the inability of the proteins to copurify by nickel affinity chromatography would indicate that the modified residue(s) are important for dimerization of EsaR. To determine the conformational change that EsaR undergoes when bound to the AHL ligand, a FRET assay was developed to estimate the distances between amino acid residues in the absence and presence of AHL. Future work will have to include a few modifications to the methods and/or control experiments. This study provides the basis upon which the present methods can be further developed and later used for structural studies of EsaR. / Master of Science

Les récepteurs couplés aux protéines G forment une grande famille de récepteurs transmembranaires. De nombreuses études montrent que ces récepteurs présenteraient une tendance à interagir entre eux et à former des oligomères. Ces structures sont toutefois sujettes à controverse. En effet, très peu d'éléments permettent d'affirmer que ces oligomères existeraient dans les tissus natifs, la plupart des caractérisations se faisant en systèmes hétérologues. Nous avons donc développé une approche basée d'une part sur l'utilisation de ligands fluorescents pour marquer les récepteurs dans leur environnement natif et d'autre part sur le FRET (Fluorescence Resonance Energy Transfer) en temps résolu en utilisant des cryptates de lanthanides, en particulier le Lumi4-Tb. Nous avons ainsi pu montrer et publier l'existence d'oligomères du récepteur de l'ocytocine dans la glande mammaire. Le protocole de cette étude a aussi été publié et a été validé pour la mise en évidence d'hétéro-oligomères, plus précisément entre les récepteurs V1a et V2 de la vasopressine. La poursuite de l'étude de ce phénomène dans les tissus natifs nous a poussés à développer notre propre dispositif de microscopie FRET en temps résolu. Ce dispositif est basé sur un microscope en champ large auquel nous avons ajouté une source laser pour l'excitation pulsée et une caméra CCD Multigate pour la détection. Nous en présentons ici les premiers résultats ainsi que sa validation pour l'utilisation de multiples fluorophores accepteurs avec une contamination minimale par le Lumi4-Tb. Enfin, nous proposons un modèle pharmacologique montrant l'utilisation de ligands bivalents pour étudier le couplage des oligomères. / G-protein coupled receptors form a very large family of transmembrane receptors. Numerous studies have shown that these receptors showed a tendency to interact and form oligomers. These structures are however the matter of great debate. Indeed, very few elements allow us to maintain that these oligomers could exist in native tissues, most studies being carried out in heterologous systems. We have therefore developed an approach based for one part on the use of fluorescent ligands to label receptors in their native environment, and on the other part on time-resolved FRET (Fluorescence Resonance Energy Transfer) by using lanthanide cryptates, more specifically Lumi4-Tb. We have thus been able to show and publish the existence of oxytocin receptor oligomers in the mammary gland. The protocol used for this study was also published and validated for the study of hetero-oligomers, more specifically between vasopressin V1a and V2 receptors. Following on our study of oligomers in native tissues, we have developed our own setup to perform time-resolved FRET microscopy. This setup is based on a wide field microscope to which we added a laser source for the pulsed excitation and a Multigate CCD camera for imaging. We are here presenting the first results as well as its validation for the use of multiple acceptor fluorophores with minimal bleed through from the Lumi4-Tb. Lastly, we propose a pharmacological model showing the use of bivalent ligands to study oligomer coupling.

Les protéines kinases A (PKA) sont des enzymes qui catalysent la phosphorylation de résidus sérine ou thréonine. L’activité des PKA peut être mesurée in cellulo grâce aux biosenseurs AKAR (A-Kinase Activity Reporter). AKAR est composé de 4 modules: la séquence substrat des PKA, un domaine de liaison aux acides aminés, et 2 protéines fluorescentes pouvant interagir par FRET (Förster Resonant Energy Transfer). La phosphorylation de la séquence consensus par la PKA et l’interaction de l’acide aminé phosphorylé avec le module senseur provoque une modification de la conformation d’AKAR et une augmentation du FRET entre les 2 protéines fluorescentes.L’objectif initial de ce travail était de produire un biosenseur AKAR basé sur un nouveau couple de protéines fluorescentes plus performantes, et insensibles au pH. Ce biosenseur devant à terme être utilisable en imagerie ratiométrique, et en FLIM (Fluorescence Lifetime Imaging Microscopy). Ainsi nous avons mis au point une nouvelle version d’AKAR: AqAKARCit. Cette version exploite la protéine Aquamarine, l'une des meilleures protéines cyan disponibles aujourd'hui, avec un rendement quantique de 89%, un déclin de fluorescence mono-exponentiel, et une insensibilité au pH dans tout le domaine physiologique. Les résultats obtenus en FLIM avec cet AqAKARCit ont permis des améliorations notables en stabilité et sensibilité.Une version baptisée AqAKARTagRFP a été construite, dans laquelle l’accepteur est une protéine fluorescente orange permettant une meilleure séparation spectrale entre donneur et accepteur et insensible aux variations de pH. Les étapes de caractérisations de AqAKARTagRFP ont été accomplies en FLIM, et en ratiométrie. AqAKARTagRFP permet d'obtenir de bonnes réponses en FRET par ratiométrie, mais reste difficilement utilisable en FLIM en raison d'une dynamique de réponse limitée. La sensibilité du biosenseur a été améliorée par une modification de l'ancrage de l'Aquamarine. Les modifications apportées à cette version nommée AqEAKARTagRFP la place au niveau des biosenseurs AKAR les plus performants actuellement disponibles.Les mesures de sensibilité au pH d’ AqEAKARTagRFP réalisées en FLIM ont révélé une insensibilité au pH du biosenseur sur une étendue de pH jamais atteinte jusqu’à présent. Cependant, en ratiométrie, on note malgré tout une sensibilité détectable aux pHs fortement acides (pH ≤ 6), ce qui ne permettra pas de l'utiliser pour l'imagerie ratiométrique de compartiments cellulaires acides. Un contrôle négatif non phosphorylable AqEAKARmutTagRFP a été étudié. Ce contrôle présente les mêmes variations de signal en réponse à des changements imposés de pH intracellulaire, révélant que ces variations sont indépendantes de l’activité PKA.L'étude de tandems CFP-Cit et Aq-Cit dépourvus de la partie senseur nous à permis d'analyser le comportement de FRET des couples cyan/jaune en fonction du pH. Un modèle décrivant ce comportement a été créé et appliqué à AKAR.Les expériences complémentaires faites sur CFPAKARCit sont en accord avec nos simulations mais la construction AqAKARCit révèle du FRET résiduel à pH acide que notre modèle numérique ne prévoit pas. Une sensibilité aux pH acides de la partie senseur d’AKAR qui provoquerait un changement de conformation du biosenseur et une augmentation de FRET pourrait expliquer ce phénomène.Ce travail de thèse a permis la mise au point d’un nouveau couple de protéines fluorescentes par le FRET insensibles au pH. Ce couple va permettre une meilleure caractérisation des sensibilités des biosenseurs existants comme nous l’avons montré avec AKAR. Ce couple de protéines fluorescentes pourra également être utilisé dans des compartiments cellulaires acides, par exemple pour étudier des interactions protéine/protéine. Enfin, grâce à une meilleure séparation spectrale en excitation et en émission, ce couple peut être utilisé dans des applications plus exigeantes comme la microscopie biphotonique. / Protein kinase A (PKA) are enzymes which catalyze the phosphorylation of serine or threonine residues. The activity of PKA can be measured in cellulo through AKAR biosensors (A-Kinase Activity Reporter). AKAR consists of 4 modules: the PKA substrate sequence, a phospho-amino binding domain, and two fluorescent proteins that can interact by FRET (Förster Resonant Energy Transfer). After action of the PKA, the interaction between the phophorylated amino acid and the phospho-amino binding domain causes a change in the conformation of AKAR and an increase in FRET between the two fluorescent proteins.The initial objective of this work was to produce an AKAR biosensor based on a new pair of improved fluorescent proteins, and insensitive to pH. This biosensor to eventually be used in ratiometric imaging and FLIM (Fluorescence Lifetime Imaging Microscopy). So we developed a new version of AKAR: AqAKARCit. This version uses the Aquamarine protein, one of the best cyan proteins available today, with a quantum yield of 89%, a near mono-exponential fluorescence decay, and insensitive to pH throughout the physiological range. The results obtained with this AqAKARCit allowed significant improvements in stability and sensitivity.A version called AqAKARTagRFP was built, in which the acceptor is an orange fluorescent protein allowing better spectral separation between donor and acceptor and insensitive to pH variations. The characterization of AqAKARTagRFP was performed in FLIM, and ratiometry. AqAKARTagRFP provides good answers in FRET by ratiometry but remains difficult to use in FLIM due to limited dynamic responses. The sensitivity of the biosensor has been improved by modification of the anchoring of Aquamarine. Changes to this version named AqEAKARTagRFP place it at the most efficient AKAR biosensors currently available.The pH-responsive measures of AqEAKARTagRFP made in FLIM showed insensitivity to pH on a range never reached so far. However, in ratiometry, there is still a detectable sensitivity to highly acidic pHs (pH ≤ 6), which will not allow to use it for ratiometric imaging of cellular acidic compartments. A negative unphosphorylatable control AqEAKARmutTagRFP was studied. This control presents the same signal variations in response to changes imposed on intracellular pH, revealing that these variations are independent of PKA activity.The study of the CFP-Cit and Aq-Cit tandems devoid of the sensor part allowed us to analyze the behavior of cyan / yellow FRET pairs regarding the pH. A model describing this behavior was created and applied to AKAR. Additional experiments on CFPAKARCit are in agreement with our simulations but AqAKARCit reveals residual FRET at acidic pHs that our numerical model does not predict. A sensitivity to acidic pH of the sensor module of AKAR which would cause a conformational change in the biosensor and an increase in FRET could explain this phenomenon.This thesis has allowed the development of a new pair of fluorescent proteins for FRET imaging insensitive to pH. This couple will allow better characterization of existing biosensors sensibilities as we have shown with AKAR. This pair of fluorescent proteins may also be used in acidic cellular compartments, for example to study protein / protein interactions. Finally, through improved spectral separation in excitation and in emission, this pair can be used in more demanding applications such as biphoton microscopy.