Spelling suggestions: "subject:"ultrabasic""
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Apurinic/apyrimidinic endonuclease 1 (APE1) is dispensable for activation-induced cytidine deaminase (AID)-dependent somatic hypermutation in the immunoglobulin gene / Apurinic/apyrimidinic endonuclease 1 (APE1) はactivation-induced cytidine deaminase (AID)依存的な免疫グロブリン遺伝子の体細胞変異には必須ではないIslam, Helena 27 July 2020 (has links)
付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22697号 / 医科博第112号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 竹内 理, 教授 髙折 晃史, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Towards observing the encounter of the T7 DNA replication fork with a lesion site at the Single molecule levelShirbini, Afnan 05 1900 (has links)
Single-molecule DNA flow-stretching assays have been a powerful approach to study various aspects on the mechanism of DNA replication for more than a decade. This technique depends on flow-induced force on a bead attached to a surface-tethered DNA. The difference in the elastic property between double-strand DNA (long) and single-strand DNA (short) at low regime force allows the observation of the beads motion when the dsDNA is converted to ssDNA by the replisome machinery during DNA replication. Here, I aim to develop an assay to track in real-time the encounter of the bacteriophage T7 replisome with abasic lesion site inserted on the leading strand template. I optimized methods to construct the DNA substrate that contains the abasic site and established the T7 leading strand synthesis at the single molecule level. I also optimized various control experiments to remove any interference from the nonspecific interactions of the DNA with the surface. My work established the foundation to image the encounter of the T7 replisome with abasic site and to characterize how the interactions between the helicase and the polymerase could influence the polymerase proofreading ability and its direct bypass of this highly common DNA damage type.
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Photophysical Properties and Applications of Fluorescent Probes in Studying DNA Conformation and DynamicsJanuary 2015 (has links)
abstract: Fluorescence spectroscopy is a popular technique that has been particularly useful in probing biological systems, especially with the invention of single molecule fluorescence. For example, Förster resonance energy transfer (FRET) is one tool that has been helpful in probing distances and conformational changes in biomolecules. In this work, important properties necessary in the quantification of FRET were investigated while FRET was also applied to gain insight into the dynamics of biological molecules. In particular, dynamics of damaged DNA was investigated. While damages in DNA are known to affect DNA structure, what remains unclear is how the presence of a lesion, or multiple lesions, affects the flexibility of DNA, especially in relation to damage recognition by repair enzymes. DNA conformational dynamics was probed by combining FRET and fluorescence anisotropy along with biochemical assays. The focus of this work was to investigate the relationship between dynamics and enzymatic repair. In addition, to properly quantify fluorescence and FRET data, photophysical phenomena of fluorophores, such as blinking, needs to be understood. The triplet formation of the single molecule dye TAMRA and the photoisomerization yield of two different modifications of the single molecule cyanine dye Cy3 were examined spectroscopically to aid in accurate data interpretation. The combination of the biophysical and physiochemical studies illustrates how fluorescence spectroscopy can be used to answer biological questions. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015
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Encounter of T7 Replisome with Abasic DNA LesionAlhudhali, Lubna F. 11 1900 (has links)
In order to monitor the T7 replisome fate upon encountering abasic lesion, I optimized
a single molecule flow stretching assay where the replisome encounters either abasic site or undamaged site inserted at 3.5 kilobases from the replication fork. The obtained events were categorized into three groups; bypass, restart and permanent stop. The results showed 52% bypass, 39% pause and 9% stop upon encountering the abasic lesion. The pause duration in the restart events was found to be ten times longer than the undamaged one.
Moreover, an ensemble experiment was performed, and the results were slightly consistent with regard to the bypass percentage (70%) but the stoppage percentage was significantly higher in the ensemble replication reaction (30%). Further investigations were made and it was found that the rate of the T7 replisome increases after bypassing the abasic lesion.
To inquire more about this rate switch and the difference between the single molecule and ensemble results, another unwinding experiment was performed where only gp4 (helicase) was used from the replisome. Interestingly, the rate of DNA unwinding by gp4 was similar to the rate observed after the replisome bypasses the lesion. We hypothesize that the polymerase is stalled at the abasic site and its interaction with the helicase is lost.
Consequently, the helicase and the polymerase will uncouple where the helicase continues unwinding the DNA to result in a higher observed rate after bypassing the abasic site.
Additional studies will be performed in the future to directly observe the helicase and
polymerase uncoupling upon encountering the lesion.
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Ligands macrocycliques de sites abasiques en tant qu'inhibiteurs de la réparation de l'ADN : Synthèse, études biochimiques et biologiques / Macrocyclic ligands for DNA abasic sites as inhibitors of DNA repair : Synthesis, biochemical and biological studiesCaron, Coralie 18 October 2019 (has links)
Dans le contexte de la chimiothérapie, la réparation de l’ADN réduit les dommages induits par les agents alkylants de l’ADN dont le témozolomide (TMZ), conduisant à la chimiorésistance. Une des voies principales de réparation de l’ADN est la voie par excision de base (BER) au sein de laquelle une enzyme clée, APE1 (endonucléase AP 1), clive les sites abasiques générés suite aux traitements par les agents alkylants et initie la réparation de la coupure simple-brin. Ce mécanisme représente une source majeure de chimiorésistance dans certains cancers. Plusieurs études ont ainsi validé la voie BER et plus particulièrement APE1 comme une cible importante dans le but d’améliorer l’efficacité des agents anticancéreux; pour ces raisons, de nombreux inhibiteurs d’APE1 ont été développés. Cependant, à la place d’une inhibition directe de l’enzyme, une stratégie alternative consiste à cibler le substrat de cette dernière : les sites abasiques. Les composés macrocycliques de type naphtalénophane ont montré la capacité à se lier fortement et sélectivement aux sites abasiques. Ce processus interfère avec la reconnaissance de ces derniers par APE1 et conduit in vitro à deux effets : l’inhibition du clivage enzymatique d’APE1 et le clivage du site AP par les macrocycles par un mécanisme différent de celui d'APE1, de type β-élimination. Ainsi, une nouvelle série de naphtalénophanes fonctionnalisés, composée de neuf nouveaux dérivés, a été synthétisée et étudiée. La plupart des macrocycles démontre la capacité à se lier fortement et sélectivement aux sites abasiques de l’ADN ainsi qu’à inhiber l’activité d’APE1 in vitro, avec des constantes d’inhibition s'étalant de 39 nM à 25 µM. De plus, l’activité d’inhibition d’APE1 par les ligands, caractérisée par les valeurs de Kı, a pu être corrélée avec leur affinité et leur sélectivité pour les sites abasiques. La structure moléculaire des macrocycles montre une forte influence sur l’activité de clivage de ces derniers pouvant conduire à une abolition ou à une très haute activité de clivage des sites abasiques. De façon intéressante, la formation d’un adduit covalent ADN – ligand avec un des macrocycles a été caractérisée. Enfin, l’activité biologique des naphtalénophanes sur la lignée cellulaire de glioblastome T98G résistante au TMZ a été étudiée. La plupart des ligands affiche une cytotoxicité élevée, avec des GI₅₀ de l’ordre du micromolaire. De plus, un remarquable effet synergique lors du traitement des cellules avec le TMZ et le MMS en combinaison avec un ligand (2,7-BisNP-O4Me) a été démontré. Ce macrocycle augmente également le nombre de sites abasiques et le nombre de coupures double-brins après un co-traitement cellulaire avec les agents alkylants suggérant ainsi l'inhibition d'APE1 attendue. Ces résultats mettent ainsi en évidence le fort intérêt thérapeutique de ce composé. / In the context of chemotherapy, DNA repair reduces the DNA damage induced by DNA-alkylating drugs such as temozolomide, leading to chemoresistance. One of the most important pathways of DNA repair is Base Excision Repair (BER), where a key enzyme, APE1 (AP endonuclease 1), cleaves abasic sites generated following treatment with DNA-alkylating drugs and initiates the repair of the single-strand break. The DNA repair activity of APE1 was identified as the major source of chemoresistance in certain cancers. Several studies validated the BER pathway and, particularly, the APE1 enzyme as important drug targets for improvement the efficacy of anti-cancer drugs; for this reason, several APE1 inhibitors have been developed. However, instead of direct inhibition of the enzyme, an alternative strategy can rely on targeting its substrate: the AP sites in DNA. Macrocyclic compounds, namely naphthalenophanes, show a strong and selective binding to abasic sites in the DNA. This process interferes with the recognition of the latter by APE1 and leads in vitro to two effects: inhibition of the APE1-induced DNA cleavage and macrocycle-induced DNA cleavage by a mechanism different from that of APE1, namely β-elimination. Herein, a novel serie of functionalized naphthalenophanes, composed of nine novel derivatives, has been synthesized and studied. Most ligands demonstrate a strong and selective binding to AP-sites in DNA and an inhibition of APE1 activity in vitro, with inhibitory constants from 39 nM to 25 µM. Moreover, the inhibitory activity of ligands, as characterized by Kı values, could be directly related to their affinity and selectivity to AP-sites. The molecular design of macrocycles has a crucial influence on their intrinsic AP-site cleavage activity leading either to total abolition, or to an exceptionally high AP-site cleavage activity. Interestingly, an unprecedented formation of a covalent DNA-ligand adduct with one of the ligands have been characterized. Finally, the biological activity of naphthalenophanes was assessed in the TMZ-resistant glioblastoma cell line T98G. Most compounds are highly active, with GI₅₀ values in sub-micromolar or low-micromolar range. In addition, a remarkable synergic effect upon co-treatment of TMZ or MMS with one ligand (2,7-BisNP-O4Me) was demonstrated. This ligand was found to increase the number of AP-sites and the number of double-strands break in DNA upon co-treatment with TMZ and MMS suggesting APE1 inhibition as excepted. These observations highlight the hight therapeutic interest of this compound.
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A kinetic and biochemical approach to understanding the mechanisms of novel DNA polymerasesFiala, Kevin 14 September 2007 (has links)
No description available.
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The role of the associated 3' to 5' exonuclease activity and processivity factor (UL42) or herpes simplex virus type 1 DNA polymerase on the fidelity of DNA replicationSong, Liping 19 May 2004 (has links)
No description available.
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Novel Methods for Synthesis of High Quality OligonucleotidesSemenyuk, Andrey January 2006 (has links)
<p>The first part of the work describes a procedure of oligonucleotide purification using a reversed-phase cartridge. The developed method employs a very efficient yet mild oligonucleotide detritylation on the cartridge support allowing fast purification of oligonucleotides regardless of their 5´-modification. Thiol- and amino-modified oligonuc-leotides were detritylated and purified with the same high efficiency as non-modified oligonucleotides. The method enables fast, parallel and automated purification of many oligonucleotide probes that was not possible before. In combination with the method of removal of tritylated failure fragments oligonucleotides were produced with purity superior to that of oligonucleotides purified using RP HPLC.</p><p>In the second part of the present study a method of solid-phase RNA synthesis using 2´-tert-butyldithiomethyl (2´-O-DTM) is discussed. The stability of the DTM group during oligonucleotide assembly and deprotection in ammonia, together with its ability for rapid deprotection under mild conditions, allowed the synthesis of RNA with the quality similar to that of synthetic DNA oligonucleotides. The advantage of the 2´-O-DTM group is that it is completely orthogonal to all protecting groups used for the traditional solid-phase DNA synthesis. Therefore, the synthesis can be performed using a standard DNA synthesis procedure – no changes are needed for the product assembly. RNA oligonucleotides synthesized with retained 5´-terminal trityl group can be subjected to a cartridge-based purification using the procedure described in the first part of the study. The phosphoramidite synthesis was optimized for a large scale preparation and gives versatility for introduction of other alkyldithiomethyl groups according to the preference to their certain properties.</p><p>The third part of the thesis describes the synthesis of a dithiomethyl linker and its utility for reversible conjugation of oligonucleotides. A dithiomethyl group, cleavable under mild conditions, was introduced onto 3´-OH of tritylated nucleosides via 3´-O-methylthiomethyl derivatives. The influence of different alkyl substituents on the disulfide bond stability was investigated, and stable analogues were employed in oligosyntheses. Two applications were developed using the present linker: 1) purification of oligonucleotides linked to the solid support; and 2) cartridge-based purification of tritylated oligonucleotides having an additional hydrophobic group on their 3´- terminus.</p>
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Novel Methods for Synthesis of High Quality OligonucleotidesSemenyuk, Andrey January 2006 (has links)
The first part of the work describes a procedure of oligonucleotide purification using a reversed-phase cartridge. The developed method employs a very efficient yet mild oligonucleotide detritylation on the cartridge support allowing fast purification of oligonucleotides regardless of their 5´-modification. Thiol- and amino-modified oligonuc-leotides were detritylated and purified with the same high efficiency as non-modified oligonucleotides. The method enables fast, parallel and automated purification of many oligonucleotide probes that was not possible before. In combination with the method of removal of tritylated failure fragments oligonucleotides were produced with purity superior to that of oligonucleotides purified using RP HPLC. In the second part of the present study a method of solid-phase RNA synthesis using 2´-tert-butyldithiomethyl (2´-O-DTM) is discussed. The stability of the DTM group during oligonucleotide assembly and deprotection in ammonia, together with its ability for rapid deprotection under mild conditions, allowed the synthesis of RNA with the quality similar to that of synthetic DNA oligonucleotides. The advantage of the 2´-O-DTM group is that it is completely orthogonal to all protecting groups used for the traditional solid-phase DNA synthesis. Therefore, the synthesis can be performed using a standard DNA synthesis procedure – no changes are needed for the product assembly. RNA oligonucleotides synthesized with retained 5´-terminal trityl group can be subjected to a cartridge-based purification using the procedure described in the first part of the study. The phosphoramidite synthesis was optimized for a large scale preparation and gives versatility for introduction of other alkyldithiomethyl groups according to the preference to their certain properties. The third part of the thesis describes the synthesis of a dithiomethyl linker and its utility for reversible conjugation of oligonucleotides. A dithiomethyl group, cleavable under mild conditions, was introduced onto 3´-OH of tritylated nucleosides via 3´-O-methylthiomethyl derivatives. The influence of different alkyl substituents on the disulfide bond stability was investigated, and stable analogues were employed in oligosyntheses. Two applications were developed using the present linker: 1) purification of oligonucleotides linked to the solid support; and 2) cartridge-based purification of tritylated oligonucleotides having an additional hydrophobic group on their 3´- terminus.
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Molecule recognition of nucleic acids, nucleosides, nucleotides, and their derivativesLiu, Wanbo 01 January 2013 (has links) (PDF)
It has long been known that the efficiency of anticancer drugs is limited by the emergence of resistance due to the evolving repair of such DNA lesions in malignant cells. Therefore, development of pharmaceutical agents, which can interfere with the DNA repair pathways, may represent a novel approach to enhance the cytotoxic effects of chemotherapy by reducing drug resistance. Abasic sites (AP sites) are the key intermediates in the BER pathway and promising targets for BER inhibition. In chapter 2, we report the synthesis of two small molecules specifically targeting at AP sites and the evaluation of their activity in terms of interstrand crosslinking formation. Our results show no covalent adduct is induced, which is due to the weak DNA binding affinity. In chapter 3, we try to use TFOs to deliver the interstrand crosslinking moiety to the AP site in a sequence specific manner. Two modified phosphoramidites were synthesized and incorporated into the 5' end of TFOs. The activity was evaluated by using various biophysical and biochemical experiments. The work reported in chapter 4 is focused on the G-quadruplex structure formed in the guanine rich telomeric sequence. Many studies have shown G4 ligands can induce and stabilize G-quadruplex within telomere region and inhibit the activity of telomerase that is overexpressed in 80-90% of cancer cells. Our results indicate that phenanthroline based metal complexes, Ni(Phen) 2 , have strong binding affinity and selectivity towards G-quadruplex over duplex DNA. The effect of Ni(Phen) 2 on telomerase activity and cytotoxicity towards cancer cells was also investigated. Calixarenes containing DNA building units such as nucleotides, nucleosides, and nucleobases have recently aroused much interest because of their versatile applications. In chapter 5, we report the synthesis of calix[4]arenes ( 5.11-5.14 ) functionalized with a single nucleobase (thymine, adenine, guanine, or cytosine) at the upper rim via click chemistry. Their complexation with alkali metal ions was examined using MALDI-TOF mass spectrometry and their molecular interactions were determined using 1 H NMR. All calix[4]arene derivatives show good complexation with alkali metal ions with apparent selectivity. The results also reveal that nucleobase-calix[4]arenes are capable of self-association in CDC1 3 and calix[4]arenes bearing complementary nucleobases can bind to each other via base pairing.
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