Les télomères sont des structures nucléoprotéiques protégeant l’extrémité des chromosomes de la dégradation et assurant la réplication de l’ADN terminal. En effet, de nombreuses protéines de réplication sont impliquées dans le maintien de ces structures, comme le complexe RPA (Replication Protein A). Ce complexe très conservé chez les eucaryotes se fixe à l’ADN simple brin et est impliqué dans la réplication, les mécanismes de recombinaison et la réparation de l’ADN. Chez S.pombe, la mutation ponctuelle de la sous-unité RPA1 (Rpa1-D223Y) provoque le raccourcissement des télomères. Dans cette étude, nous montrons que cette mutation provoque l’accumulation de structures aberrantes de haut poids moléculaire aux télomères corrélant avec une présence persistante de Polα aux télomères suggérant une accumulation de structures sur le brin riche en G. Nous avons pu mettre en évidence que la surexpression d’hélicases de la famille Pif1 incluant S.cerevisiae Pif1 et PIF1 humain ainsi que Pfh1 (S.pombe) sont capable de restaurer une longueur de télomères sauvage dans mutant rpa1-D223Y. Ces résultats suggèrent que RPA pourrait empêcher l’accumulation de G4 au niveau du brin retardé télomérique afin de faciliter l’élongation des télomères par la télomérase. De plus, des expériences in vitro ont montré que la mutation correspondante de RPA1 humain réduisait spécifiquement l’affinité de RPA pour le simple brin télomérique humain dans les conditions ou il forme des G4.Enfin l’étude de la stabilité de séquences répétées formant des G4 (minisatellite CEB25), chez S.pombe, a permis de renforcer l’hypothèse selon laquelle RPA pourrait empêcher la formation ou aiderait à la résolution de G4. / Telomeres are nucleoprotein structures that protect chromosome ends from degradation and ensure replication of the terminal DNA. In fact, many of replication proteins are involved in telomere maintenance, like RPA (Replication Protein A). RPA is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination and repair. In S. pombe a mutation in the largest RPA subunit (Rpa1-D223Y) leads to substantial telomere shortening. In this study, we found that the D223Y mutation leads to the accumulation of aberrant secondary structures at telomeres. The presence of these secondary DNA structures correlates with a high association of Polα with telomeres suggesting that this mutation impairs lagging strand (G-rich) telomere replication. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G-quadruplex, human PIF1 and Phf1 (homolog of Pif1 in S.pombe) rescue the telomeric length defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G-quadruplex. We propose that RPA prevents the formation of G-quadruplex structures at lagging strand telomeres to facilitate telomerase action at telomeres. Furthermore, the study, in S.pombe, of the stability of G-rich repeat sequences (minisatellite CEB25) as known to form G4 enforce the hypothesis that RPA can prevents the formation of G4 or helps to solve this structure.

<div>G-quadruplexes (G4s) are non-canonical secondary structures formed in single-stranded guanine-rich nucleic acid sequences, such as those found in oncogene promoters and telomeres. <i>MYC</i>, one of the most critical oncogenes, has a DNA G4 (MycG4) in its proximal promoter region that functions as a transcriptional silencer. MycG4 is very stable and the pathological activation of <i>MYC</i> requires its active unfolding. However, it remains unclear what drives MycG4 unfolding in cancer cells. We have studied the interactions of DDX5 with the MycG4 at both molecular and cellular levels and discovered that DDX5 actively unfolds the MycG4 and is involved in the <i>MYC</i> gene transcriptional regulation, which is described in the first part of this dissertation. DDX5 is extremely proficient at unfolding the MycG4 and ATP hydrolysis is not directly coupled to the G4-unfolding of DDX5. In cancer cells, DDX5 is enriched at the <i>MYC</i> promoter and activates <i>MYC</i> transcription. G4-interactive small molecules inhibit the DDX5 interaction with the <i>MYC</i> promoter and DDX5-mediated <i>MYC</i> activation. The second part of this dissertation describes the study of interactions of indenoisoquinoline anticancer drugs with MycG4. The MycG4 transcriptional silencer is a very attractive therapeutic target. Compounds that bind and stabilize the MycG4 have been shown to repress <i>MYC</i> gene transcription and are antitumorigenic. Indenoisoquinolines are human topoisomerase I inhibitors in clinical testing. However, some indenoisoquinolines with potent anticancer activity do not exhibit strong topoisomerase I inhibition, suggesting a separate mechanism of action. Our studies show that indenoisoquinolines strongly bind and stabilize MycG4 and lower <i>MYC</i> levels in cancer cells. Moreover, the analysis of indenoisoquinoline analogues for their <i>MYC</i> inhibitory activity, topoisomerase I inhibitory activity, and anticancer activity reveals a synergistic effect of <i>MYC</i> inhibition and topoisomerase I inhibition on anticancer activity. Besides the MycG4, human telomeric G4s are also attractive targets for anticancer drugs due to their ability to inhibit telomere extension in cancer cells. The last part of this dissertation reviews two recent solution structural studies on small molecule complexes with the hybrid-2 telomeric G4 and the hybrid-1 telomeric G4. Structural information of those complexes can advance the design of telomeric G4-interactive small molecules in the cancer therapeutic areas.</div>

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18101号 / 理博第3979号 / 新制||理||1574(附属図書館) / 30959 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 藤井 紀子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

No description available.

Molecular recognition, particularly as it applies to strong binding interactions between complementary ligand/receptor molecules in solution, is important in such varied areas as molecular biology, pharmacology, synthetic chemistry, and chemical detection. Strong binding is the additive result of a number of specific, weak, non-covalent interactions occurring between complementary molecules. This dissertation reports on the energetics of forming complexes between small molecules and model DNA constructs. Ligands included cationic and metallated cationic porphyrins and polyheterocyclic ruthenium compounds. DNA receptors included double stranded B-DNAs (hairpin and short linear sequences) as well G-quadruplex DNAs. Thermodynamic data were collected using isothermal titration calorimetry, circular dichroism spectropolarimetry, ultraviolet-visible spectroscopy, and mass spectrometry. The measured thermodynamic parameters included the changes in free energy, enthalpy and entropy for ligand/receptor complex formation as well as the stoichiometry of the stable complexes. The first section of this dissertation reports that the binding of cationic porphyrins to model G-quadruplex DNA may proceed through two pathways, end stacking and intercalation. Modulating the number of pyridinium groups on a pyridinium substituted porphyrin yielded differing binding thermodynamics leading to the understanding that a balance of surface area, charge, and geometry affect the ability of a porphyrin to bind to G-quadruplex DNA. Further investigations into the binding of metallated porphyrins developed the understanding that the geometry of the central metal ion affected not only the thermodynamics but could also inhibit the intercalative mode. It was previously shown that the high affinity binding for binuclear polyheterocyclic ruthenium compounds proceeds through an intercalative mode. To further understand the binding process and the structureunction relationship of the ligand components, the binding of smaller mononuclear complexes that were representative of portions of the binuclear complex was examined in this dissertation. While limiting the intercalative ability lowered the binding affinity, the mononuclear complex with the full intercalating bridge was able bind to DNA with a higher affinity than the binuclear complex. These studies have been successful in part in determining the contributions of numerous weak interactions including: charge (Coulombic interactions), H-bonding, hydrophobic interactions, and solvent structure (solvation changes), to the overall energetics of this molecular recognition process. The first section of this dissertation reports that the binding of cationic porphyrins to model G-quadruplex DNA may proceed through two pathways, end stacking and intercalation. Modulating the number of pyridinium groups on a pyridinium substituted porphyrin yielded differing binding thermodynamics leading to the understanding that a balance of surface area, charge, and geometry affect the ability of a porphyrin to bind to G-quadruplex DNA. Further investigations into the binding of metallated porphyrins developed the understanding that the geometry of the central metal ion affected not only the thermodynamics but could also inhibit the intercalative mode. It was previously shown that the high affinity binding for binuclear polyheterocyclic ruthenium compounds proceeds through intercalation. To further understand the binding process and the structureunction relationship of the ligand components, the binding of smaller mononuclear complexes that were representative of portions of the binuclear complex was examined in this dissertation. While limiting the intercalative ability lowered the binding affinity, the mononuclear complex with the full intercalating bridge was able bind to DNA with a higher affinity than the binuclear complex. These studies have been successful in part in determining the contributions of numerous weak interactions including: charge (Coulombic interactions), H-bonding, hydrophobic interactions, and solvent structure (solvation changes), to the overall energetics of this molecular recognition process.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24125号 / 理博第4853号 / 新制||理||1694(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)准教授 板東 俊和, 教授 深井 周也, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第24326号 / エネ博第454号 / 新制||エネ||85(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 教授 森井 孝, 教授 杤尾 豪人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

Peptide hormones are important biomolecules that transduce downstream effects such as cell proliferation, regulation, and gene expression. Their levels have been upregulated in various disorders such as cancer, yet detection methods are lacking. We designed two split aptamer-based assays for the detection of a peptide hormone – Somatostatin (SST) – with different signal readouts: fluorescent readout based on light-up aptamers and the colorimetric readout of ABTS peroxidation from a G-quadruplex. We used an already selected split-aptamer –SSTA5–for SST for our designs and we had expected the developed detection systems to exhibit detection and quantification capabilities that would hopefully allow their use for SST monitoring in clinical samples. However, our experiments did not support the hypothesis of this project and SST was not able to be detected using either of our fluorescent or colorimetric methods. To determine if the SSTA5 aptamer could bind SST appropriately, Förster resonance energy transfer (FRET) was used. We verified that there was no energy transfer between two covalently-attached light-sensitive molecules (one attached to each part of the split SSTA5 aptamer); thus, we theorize that the aptamer does not hybridize in the presence of the tetra decapeptide SST. Therefore selection of another, more appropriate, aptamer for SST will be needed for further aptameric-based detection methods. Once this is accomplished, our methodologies could be re-applied for detection of SST which could lead to real-time detection of essential hormonal levels in patients.

No description available.