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1
Talanian, Robert Vincent. "Development of Selective Inhibitors of DNA Polymerase Delta: A Thesis". eScholarship@UMMS, 1989. https://escholarship.umassmed.edu/gsbs_diss/66.
Testo completoAbstract (sommario):
This thesis is divided into three parts, united by the theme of the development of selective inhibitors of mammalian cell DNA polymerase delta (pol δ). The first part consists of an investigation of the cytotoxic mechanism(s) of certain 2-substituted adenine analogs, selected on the basis of their inhibitory properties towards DNA polymerase alpha (pol α) and mammalian cell DNA synthesis. The second is a direct search for inhibitors of isolated pol δ, and an investigation of inhibitory mechanisms. The third consists of measurement of the effects of a selective pol δ inhibitor on cellular DNA synthesis.
Mechanism of Cytotoxicity of 2-substituted adenine analoqs. The mechanism of inhibition by 2-(p-n-butylanilino)-2'-deoxyadenosine (BuAdA), and related compounds, of Chinese hamster ovary (CHO) cell ([3H]thymidine [3H]TdR) incorporation, was investigated. The potency of the compound could largely be explained by its potency (IC50 = 23 μM) as an inhibitor of CHO cell [3H]TdR uptake. BuAdA inhibited incorporation by CHO cells of [32p]phosphate into DNA relatively weakly, displaying an IC50value of 80 μM.
Differential inhibition of DNA polymerases alpha and delta. Known DNA polymerase inhibitors of a structurally wide range were screened for their ability to inhibit pol δ derived from calf thymus selectively with respect to pol α derived from the same tissue. Pyrophosphate (PPi) and difluoromethanediphosphonate each inhibited pol δ weakly, but with greater potency than pol α. Based on this lead, an expanded series of PPi analogs was screened. Carbonyldiphosphonate (COMDP) inhibited pol δ with a potency (Ki = 1.8 μM) twenty-two times greater than that displayed for pol α. Kinetic studies indicated that COMDP inhibited pol δ competitively with the dNTP specified by the template, but not competitively with the template:primer. Analogous experiments with pol α showed that the compound inhibited that enzyme uncompetitively with the dNTP, and not competitively with the template:primer. COMDP was a weak inhibitor of the 3' → 5' exonuclease activity of pol δ, displaying an IC50value greater than 1 mM.
Inhibition of permeabilized cell DNA synthesis bv a selective pol δ inhibitor. The potency of COMDP as an inhibitor of permeabilized CHO cell DNA synthesis (IC50= 200 μM) did not clearly indicate the participation of pol δ in cellular DNA replication.
Prospectus. The thesis concludes with a prospectus for the development of pol δ inhibitors with improved properties compared to COMDP.
2
Nourizad, Nader. "Recombinant Enzymes in Pyrosequencing Technology". Doctoral thesis, KTH, Biotechnology, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3765.
Testo completoAbstract (sommario):
Pyrosequencing is a DNA sequencing method based on thedetection of released pyrophosphate (PPi) during DNA synthesis.In a cascade of enzymatic reactions, visible light isgenerated, which is proportional to the number of nucleotidesincorporated into the DNA template. When dNTP(s) areincorporated into the DNA template, inorganic PPi is released.The released PPi is converted to ATP by ATP sulfurylase, whichprovides the energy to luciferase to oxidize luciferin andgenerate light. The excess of dNTP(s) and the ATP produced areremoved by the nucleotide degrading enzyme apyrase.
The commercially available enzymes, isolated from nativesources, show batch-tobatch variations in activity and quality,which decrease the efficiency of the Pyrosequencing reaction.Therefore, the aim of the research presented in this thesis wasto develop methods to recombinantly produce the enzymes used inthe Pyrosequencing method. Production of the nucleotidedegrading enzyme apyrase by Pichia pastoris expression system,both in small-scale and in an optimized large-scale bioreactor,is described. ATP sulfurylase, the second enzyme in thePyrosequencing reaction, was produced inEscherichia coli. The protein was purified and utilizedin the Pyrosequencing method. Problems associated with enzymecontamination (NDP kinase) and batch-to-batch variations wereeliminated by the use of the recombinant ATP sulfurylase.
As a first step towards sequencing on chip-format,SSB-(single-strand DNA binding protein)-luciferase and KlenowDNA polymerase-luciferase fusion proteins were generated inorder to immobilize the luciferase onto the DNA template.
The application field for the Pyrosequencing technology wasexpanded by introduction of a new method for clone checking anda new method for template preparation prior the Pyrosequencingreaction.
Keywords:apyrase, Pyrosequencing technology, Zbasictag fusion, luciferase, ATP sulfurylase, dsDNAsequencing, clone checking, Klenow-luciferase, SSB-luciferase,Pichia pastoris, Echerichia coli.
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Evans, Geraint Wyn. "Real-time single-molecule observations of conformational changes in DNA polymerase". Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:fdf11b59-2e58-4174-9219-9d61e4528f65.
Testo completoAbstract (sommario):
Genetic information is encoded in the long sequence of bases which form DNA, which is replicated during cell division by enzymes known as DNA Polymerases. Polymerases replicate DNA extremely accurately to avoid errors which can cause cell death and diseases such as cancer, although the mechanisms behind these extraordinary fidelities are not well understood. A large conformational change in the protein, in which the “fingers" subdomain closes around an incoming nucleotide, is thought to be implicated in these fidelity mechanisms. Here we present an assay to monitor this conformational change in single polymerase molecules, in real-time. We achieve this using total-internal-reflection-fluorescence (TIRF) microscopy to monitor the fluorescence resonance energy transfer (FRET) of an intra-protein dye labelled DNA Polymerase I (KF) as it binds to surface-immobilised DNA. Initially, we investigated the polymerase fingers-conformations during the pre-chemistry polymerisation reaction, resolving forward and backward rates which would be challenging to observe using ensemble techniques. These observations confirmed that KF closes rapidly around complementary nucleotide, but we discovered that the reverse step, fingers-opening, is particularly slow relative to chemistry. These finger kinetics act to remove the influence of the reaction rate-limiting step on fidelity, surprising given decades of investigations have focused on the rate-limiting step as the key determinant of fidelity. We also use our kinetic measurements to quantify contributions of different reaction steps to the macroscopic error rate of the polymerase. Subsequently, we developed our assay to investigate the fingers-conformations across the entire DNA polymerisation reaction. We observed single-nucleotide incorporations, and processive DNA polymerisation at high and low nucleotide concentrations, which suggested heterogeneous nucleotide incorporation rates. The observations demonstrated that the post-chemistry slow step that limits processive polymerisation occurs before post-chemistry fingers-opening, or is accounted for by post-chemistry fingers-opening. We observe a correlation in turn-over kinetics and binary complex kinetics, suggesting that turn-over rates could be limited by the intrinsic dynamics of the binary complex, as seen in other protein systems, although more work is needed on this.
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Showalter, Alexander Keith. "KINETIC STUDIES OF TWO ERROR-PRONE DNA REPAIR ENZYMES: POSSIBLE MECHANISMS FOR VIRAL MUTAGENESIS". Connect to this title online, 2002. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1016207119.
Testo completoAbstract (sommario):
Thesis (Ph. D.)--Ohio State University, 2002.Title from first page of PDF file. Document formatted into pages; contains xii, 97 p.; also contains graphics (some col.). Includes abstract and vita. Advisor: Ming-Daw Tsai, Dept. of Chemistry. Includes bibliographical references (p. 92-97).
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Stephenson, Anthony Aaron. "Mechanistic studies of enzymes involved in DNA transactions". The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1531497128385619.
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Madsen, Susan M. "Divergence in repetitive DNA sequences among three sitopsis wheat species /". free to MU campus, to others for purchase, 1998. http://wwwlib.umi.com/cr/mo/fullcit?p9901260.
Testo completo
7
Arndt, Joseph W. "Characterization and structural determination of metalloenzymes DNA polymerase beta, carboxypeptidase, and acetyl coenzyme-A decarbonylase/synthase /". Columbus, OH : Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1061312369.
Testo completoAbstract (sommario):
Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xxii, 172 p. : ill., some col. Includes abstract and vita. Advisor: Michael K. Chan, Dept. of Chemistry. Includes bibliographical references (p. 165-172).
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Pessoa-Brandão, Luis. "Genetic and molecular studies of Saccharomyces cerevisiae Cdc7-Dbf4 kinase function in DNA damage-induced mutagenesis /". Connect to full text via ProQuest. IP filtered, 2005.
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9
Walker, Alice Rachel. "Computational Simulations of Cancer and Disease-Related Enzymatic Systems Using Molecular Dynamics and Combined Quantum Methods". Thesis, University of North Texas, 2018. https://digital.library.unt.edu/ark:/67531/metadc1157647/.
Testo completoAbstract (sommario):
This work discusses applications of computational simulations to enzymatic systems with a particular focus on the effects of various small perturbations on cancer and disease-related systems. First, we cover the development of carbohydrate-based PET imaging ligands for Galectin-3, which is a protein overexpressed in pancreatic cancer tumors. We uncover several structural features for the ligands that can be used to improve their binding and efficacy.
Second, we discuss the AlkB family of enzymes. AlkB is the E. coli DNA repair protein for alkylation damage, and has human homologues with slightly different functions and substrates. Each has a conserved active site with a catalytic iron and a coordinating His...His...Asp triad. We have applied molecular dynamics (MD) to investigate the effect of a novel single nucleotide polymorphism for AlkBH7, which is correlated with prostate cancer and has an unknown function. We show that the mutation leads to active site distortion, which has been confirmed by experiments.
Thirdly, we investigate the unfolding of hen egg white lysozyme in 90% ethanol solution and low pH, to show the initial steps of unfolding from a native-like state to the disease-associated beta-sheet structure. We compare to mass spectrometry experiments and also show differing pathways based on protonation state. Finally, we discuss three different DNA polymerase systems. DNA polymerases are the primary proteins that replicate DNA during cell division, and have various extra or specific functions. We look at a proofreading-deficient DNA polymerase III mutant, the effects of solvent on DNA polymerase IV's ability to bypass bulky DNA adducts, and a variety of mutations on DNA polymerase kappa.
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O'Hanlon, Karen Ann. "Studies on the enzyme DNA-dependent RNA polymerase". Thesis, University of Reading, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266340.
Testo completo
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Faria, Surian Guerios. "Estabelecimento e caracterização de um banco de células de trabalho para produção da enzima Taq DNA polimerase". Universidade Tecnológica Federal do Paraná, 2017. http://repositorio.utfpr.edu.br/jspui/handle/1/2729.
Testo completoAbstract (sommario):
O Instituto de Biologia Molecular do Paraná (IBMP) produz kits de diagnóstico para o Sistema Único de Saúde (SUS), que consistem em testes moleculares, realizados por reação em cadeia da polimerase (polymerase chain reaction - PCR). Essa reação ocorre pela atividade da enzima Taq DNA Polimerase (Taq). O IBMP fabrica a Taq a partir do cultivo de células bacterianas, em conformidade às Boas Práticas de Fabricação (BPF) e pretende produzi-la a partir de um banco de células provindas de um mesmo clone visando o aumento da homogeneidade e reprodutibilidade do processo produtivo. O objetivo do presente trabalho é estabelecer um banco de células de trabalho (BCT) e avaliar sua estabilidade para a produção da enzima Taq, partindo de um banco de células mestre (BCM) estabelecido em Bio-Manguinhos. O estabelecimento do BCT consiste em cultivar colônias do BCM, caracterizá-las, avaliar desempenho, e eleger células adequadas para compor o BCT. O estudo da estabilidade contempla testes para comprovar que as células retêm a capacidade de produzir a Taq ao longo do tempo (i.e., viabilidade celular, estabilidade plasmídica, cinética de crescimento, indução da expressão, extração e dosagem do DNA plasmidial, análise de restrição e avaliação plasmidial). O critério decisivo para a seleção de um dos clones para compor o BCT foi o resultado da cinética de crescimento. A estabilidade foi estudada em 10 avaliações realizadas mês a mês. Nelas, a viabilidade celular foi superior a 1,0 x 106 UFC/mL, mostrando que as células permanecem com capacidade de realizar seu metabolismo e reprodução. O crescimento até a fase exponencial se deu entre 6 e 7 horas de cultivo, com taxa específica de crescimento superior a 0,4 min-1 . Os cultivos acrescidos de 1 mM de IPTG expressaram a enzima Taq DNA Polimerase, revelando a qualificação das células para o fim proposto. A estabilidade plasmídica foi superior a 90%, indicando que o plasmídeo pBioMTaq se mantém estável e com boa capacidade de replicação. A concentração plasmidial média foi de 338 ng/μL, e em todos os meses foi maior que 100 ng/μL. Na análise de restrição os plasmídeos foram corretamente clivados e na avaliação plasmidial houve adequada amplificação do fragmento de 500 pb, demonstrando que o plasmídeo se mantém íntegro e estável, com sequências compatíveis com sua construção. O BCT foi testado como substrato para um processo fabril típico do IBMP de produção da Taq DNA polimerase, se apresentando satisfatório em todas as etapas em que foi avaliado. Esses resultados indicam que o banco estabelecido se manteve estável ao longo de 10 meses e está apto a ser utilizado como protótipo para um BCT em conformidade às BPF.The Molecular Biology Institute of Paraná (Instituto de Biologia Molecular do Paraná - IBMP) produces diagnostic kits for the Brazilian Unified National Health System (Sistema Único de Saúde - SUS), that consist in molecular tests, carried out by polymerase chain reaction (PCR). This reaction is performed by the enzyme Taq DNA Polymerase (Taq). The IBMP manufactures Taq from bacterial cell culture in accordance with Good Manufacturing Practices (GMP) and intends to produce it from a cell bank using cells from the same clone in order to increase the homogeneity and reproducibility of the production process. The objective of the present work is to establish a working cell bank (WCB) and to evaluate its stability for the production of the Taq enzyme, starting from a master cell bank (MCB) established in BioManguinhos. The WCB establishment consists of cultivating MCB colonies, characterizing them, evaluating performance, and electing proper cells to compose the WCT. The stability study contemplates tests to prove that cells retain the ability to produce Taq over time (i.e., cell viability, plasmid stability, growth kinetics, expression induction, plasmid DNA extraction and dosage, restriction analysis and plasmid evaluation). The decisive discretion for the selection of one of the clones to compose BCT was the result of growth kinetics. Stability was studied in 10 month-to-month evaluations. In them, cell viability was higher than 1,0 x 106 CFU/mL, showing that the cells remain capable of performing their metabolism and reproduction. Growth to the exponential phase occurred between 6 and 7 hours of culture, with a specific growth rate greater than 0.4 min-1 . The cultures with 1 mM IPTG expressed Taq DNA polymerase enzyme, revealing the qualification of the cells to the intended purpose. The plasmid stability was greater than 90%, indicating that the plasmid pBioMTaq remains stable and has good replication ability. The mean plasmid concentration was 338 ng/μL, and in all months it was greater than 100 ng/μL. In the restriction analysis the plasmids were correctly cleaved and in the plasmid evaluation there was adequate amplification of the 500 bp fragment, demonstrating that the plasmid remains intact and stable, with compatible sequences with its construction. The WCB was tested as substrate for a typical IBMP production process of Taq DNA polymerase, presenting satisfactory in all the stages in which it was evaluated. These results indicate that the established bank remained stable over 10 months and is apt to be used as a prototype for a WCB in compliance with GMP.
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Boulton, Sallyanne. "Biological effects of novel poly (adenosine diphosphate ribose) polymerase inhibitors". Thesis, University of Newcastle Upon Tyne, 1995. http://hdl.handle.net/10443/1004.
Testo completoAbstract (sommario):
Poly(ADP-ribose) polymerase (PADPRP) is a nuclear enzyme with a well documented role in DNA repair. Inhibitors of PADPRP, (e.g. 3' substituted benzamides) potentiate the cytotoxicity of a wide range of antitumour drugs. The results presented in this thesis represent, to the best of my knowledge, the first comprehensive and quantitative assessment of the ability of a range of P ADPRP inhibitors to modulate the cellular responses to damaging agents. Two novel PADPRP inhibitors, 8-hydroxy-2-methyl quinazolin-4(3H)-one (NU1025) and 3,4 dihydro-5-methoxyisoquinolin-1-(2H)-one (PD 128763) were compared with two "classical" PADPRP inhibitors, 3-aminobenzamide (3AB) and benzamide (BZ). The relative potencies for 3AB, BZ, NU1025 and PD 128763 as PADPRP inhibitors in vitro were 1.0, ~1.0, ~43 and ~53 respectively. All compounds potentiated the growth inhibition and cytotoxicity of the monofunctional alkylating agent temozolomide (TM) in L1210 cells. For example, 10/-lM NUI025 and PD 128763 gave dose enhancement factors (DEF) of ~2 at 100/0 survival, whereas ImM 3AB and 0.5mM BZ where required to give similar DEF values. Cellular NADl- levels were depleted up to 50% by 1-2mM TM and this depletion was completely prevented by coincubation with 50-100µM PD 128763 and 1-3mM 3AB. TM induced DNA single strand break levels were increased in a concentration dependent manner by the P ADPRP inhibitors. Overall, the relative potencies for ability of the compounds to potentiate TM induced growth inhibition, cytotoxicity and DNA single strand breaks showed good correlation with those determined in an in vitro inhibition study, with both NU1025 and PD 128763 exhibiting ~60 fold increased inhibitory activity as compared to 3AB. The PADPRP inhibitors per se did not effect the growth or survival of the L 121 0 cells, nor increase DNA strand breakage. NAD+ is the substrate for PADPRP. A L1210 cell line made resistant to tiazofurin (TZ) utilising a step wise selection protocol was shown to be deficient in nicotinamide mononucleotide adenyl transferase (NMNAT) , the final enzyme required for NAD+ biosynthesis. The consequences of a reduced NMNAT activity (<3% of the parental line ) and an ~40% reduction in intracellular NAD+ levels were determined. The resistant cells showed an ~3 fold increased sensitivity to TM as compared to the parental cells. Upon coincubation with increasing concentrations of NU1025 in the presence of a fixed concentration of TM, growth inhibition was potentiated ~70 fold in the resistant cells but only ~10 fold in the parental cell line, demonstrating the reduced level of competition between NAD+ and NUI025 for PADPRP. However, DNA single strand breaks were increased in the resistant compared to the parental cell line only when NU1025 was coincubated with TM. In contrast, in the presence of the PADPRP inhibitors alone, equivalent growth inhibitory effects were observed in each of the cell lines, suggesting inhibition of PADPRP was not the cytotoxic effector. The ~40% NAD+ depletion observed could therefore suggest, that NAD+ levels in the resistant cells were reduced to, or near to the KmNAD+ for PADPRP.
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Estrela, Mariely Cordeiro. "Avaliação de uma análise automatizada para determinação de atividade enzimática". Universidade Tecnológica Federal do Paraná, 2017. http://repositorio.utfpr.edu.br/jspui/handle/1/2875.
Testo completoAbstract (sommario):
O Instituto de Biologia Molecular do Paraná (IBMP) atua na produção de insumos para detecção de doenças. Em parceria com Bio-Manguinhos (Fiocruz) é atualmente responsável pelo fornecimento do módulo de amplificação do KIT NAT Brasileiro para o diagnóstico molecular de HIV (AIDS), HCV (Hepatite C) e HBV (Hepatite B), entre outros produtos para diagnóstico in vitro. O teste molecular consiste basicamente, na amplificação do material genético do vírus (DNA ou RNA) através da técnica de PCR (reação em cadeia pela polimerase) em tempo real, que possibilita a detecção do agente patógeno a partir de pequenas quantidades de ácido nucleico presente na amostra. A reação de PCR ocorre pela atividade da Taq DNA Polimerase, uma enzima termostável amplamente utilizada para replicação seletiva de fragmentos de DNA. Esta enzima foi isolada a partir de uma bactéria termofílica, denominada Thermus aquaticus e é produzida pelo IBMP, sendo considerada um insumo de alta criticidade. Uma das etapas de controle do processo produtivo dessa enzima é a avaliação do extrato bruto enzimático e a determinação da atividade da enzima purificada. O método de quantificação consiste em avaliar a atividade enzimática através da metodologia de PCR convencional, seguida por uma análise do perfil eletroforético das amostras em gel de agarose. No entanto, a metodologia empregada atualmente apresenta uma grande subjetividade, visto que a interpretação dos resultados pode sofrer variações quando analisados por diferentes operadores. O objetivo do presente trabalho é avaliar a implementação de uma análise automatizada dos resultados através do processamento digital de imagens, que além de facilitar sobremaneira as rotinas laboratoriais, pode ser a chave para resultados com maior grau de precisão e repetibilidade, eliminando assim o viés subjetivo do analista. A nova metodologia de análise implica em menor interferência do analista na interpretação dos resultados. O método proposto foi testado em um conjunto de imagens e os resultados obtidos foram comparados com os valores da análise manual atualmente utilizada. Os resultados foram considerados promissores, pois a análise automatizada, além de reduzir significativamente o tempo de análise, possibilita uma padronização dos resultados.The Molecular Biology Institute of Paraná (IBMP) acts in the production of inputs for detection of diseases. In partnership with Bio-Manguinhos (Fiocruz), it is currently responsible for manufacturing the amplification module of the Brazilian NAT KIT for HIV (AIDS), HCV (Hepatitis C) and HBV (Hepatitis B), besides other products for molecular diagnostics. The molecular test basically consists of amplifying the genetic material of the virus (DNA or RNA) through the real-time PCR (polymerase chain reaction) technique, which enables detection of the pathogen from small amounts of nucleic acid present in the sample. The PCR reaction occurs by the activity of Taq DNA Polymerase, a thermostable enzyme widely used for selective replication of DNA fragments. This enzyme was isolated from a thermophilic bacterium, called Thermus aquaticus and is produced by the IBMP, being considered an input of high criticality. One of the steps in controlling the production process of this enzyme is the evaluation of the enzymatic extract and the determination of the activity of the purified enzyme. The quantification method consists in evaluating the enzymatic activity through the conventional PCR methodology, followed by an analysis of the electrophoretic profile of the agarose gel samples. However, the methodology currently used presents a great subjectivity, since the interpretation of results can suffer variations when analyzed by different operators. The objective of the present work is to evaluate the implementation of an automated analysis of the results through digital image processing, which in addition to facilitating the laboratory routines, can be the key to results with a greater degree of precision and repeatability, thus eliminating the subjective bias of the analyst. The new methodology of analysis implies less interference of the analyst in the interpretation of the results. The proposed method was tested in a set of images and the obtained results were compared with the values of the manual analysis currently used. The results were considered promising because the automated analysis, besides significantly reducing the analysis time, allows a standardization of the results.
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Duran, Harry Leo. "The site specific mutagenic efficiency of the alkylated DNA base, O⁴-ethylthymine : interactions of deoxynuleotide triphosphates, polymerases and repair enzymes in gap misrepair mutagenesis /". The Ohio State University, 1985. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487261919109649.
Testo completo
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Caramia, Sara. "Purification and preliminary structural characterization by NMR spectroscopy of the "HoLaMa" DNA polymerase". Master's thesis, Alma Mater Studiorum - Università di Bologna, 2017. http://amslaurea.unibo.it/14420/.
Testo completoAbstract (sommario):
HoLaMa is a Klenow sub-fragment lacking the 3’-5’ exonuclease domain, whose gene codes for residues 515-928 of Escherichia coli DNA polymerase I. The enzyme was designed in a previous study starting from different Klenow enzymes, with the aim of studying a mini-DNA polymerase with NMR spectroscopy. In the present work, we studied a new purification protocol for the production of HoLaMa in order to obtain an appropriate quantity for NMR analysis. We tested three different purification procedure and at the end, we collected 5.8 mg of HoLaMa (Volume 1.8 mL, concentration 67 μM). After the purification, we started the study of HoLaMa by NMR spectroscopy, focusing on the nature of enzyme-substrate interactions and studying the kinetics of the reaction. Our preliminary studies were designed to understand the characteristic NMR signals of HoLaMa under different conditions of temperature and buffer; finally, we also focused our analysis on the interactions between protein, DNA and nucleotides.
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Ren, Yaou. "Trinucleotide Repeat Instability Modulated by DNA Repair Enzymes and Cofactors". FIU Digital Commons, 2018. https://digitalcommons.fiu.edu/etd/3762.
Testo completoAbstract (sommario):
Trinucleotide repeat (TNR) instability including repeat expansions and repeat deletions is the cause of more than 40 inherited incurable neurodegenerative diseases and cancer. TNR instability is associated with DNA damage and base excision repair (BER). In this dissertation research, we explored the mechanisms of BER-mediated TNR instability via biochemical analysis of the BER protein activities, DNA structures, protein-protein interaction, and protein-DNA interaction by reconstructing BER in vitro using synthesized oligonucleotide TNR substrates and purified human proteins. First, we evaluated a germline DNA polymerase β (pol β) polymorphic variant, pol βR137Q, in leading TNR instability-mediated cancers or neurodegenerative diseases. We find that the pol βR137Q has slightly weaker DNA synthesis activity compared to that of wild-type (WT) pol β. Because of the similar abilities between pol βR137Q and WT pol β in bypassing a template loop structure, both pol βR137Q and WT pol β induces similar amount of repeat deletion. We conclude that the slightly weaker DNA synthesis activity of pol βR137Q does not alter the TNR instability compared to that of WT pol β, suggesting that the pol βR137Q carriers do not have an altered risk in developing TNR instability-mediated human diseases. We then investigated the role of DNA synthesis activities of DNA polymerases in modulating TNR instability. We find that pol βY265C and pol ν with very weak DNA synthesis activities predominantly promote TNR deletions. We identify that the sequences of TNRs may also affect DNA synthesis and alter the outcomes of TNR instability. By inhibiting the DNA synthesis activity of pol β using a pol β inhibitor, we find that the outcome of TNR instability is shifted toward repeat deletions. The results provide the direct evidence that DNA synthesis activity of DNA polymerases can be utilized as a potential therapeutic target for treating TNR expansion diseases. Finally, we explored the role of post-translational modification (PTM) of proliferating cell nuclear antigen (PCNA) on TNR instability. We find that ubiquitinated PCNA (ub-PCNA) stimulates Fanconi associated nuclease 1 (FAN1) 5’-3’ exonucleolytic activities directly on hairpin structures, coordinating flap endonuclease 1 (FEN1) in removing difficult secondary structures, thereby suppressing TNR expansions. The results suggest a role of mono-ubiquitination of PCNA in maintaining TNR stability by regulating nucleases switching. Our results suggest enzymatic activities of DNA polymerases and nucleases and the regulation of the activities by PTM play important roles in BER-mediated TNR instability. This research provides the molecular basis for future development of new therapeutic strategies for prevention and treatment of TNR-mediated neurodegenerative diseases.
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Niu, Jia. "Translation of DNA into Evolvable Sequence-Defined Synthetic Polymers". Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11351.
Testo completoAbstract (sommario):
Laboratory directed evolution have enabled the discovery of numerous functional natural and synthetic macromolecules with tailor-made functions. However, approaches that use enzymes to effect the crucial translation from an information carrier molecule such as DNA or RNA to synthetic polymers are limited to producing close analogs of nucleic acids, either due to a strict requirement to hybridize with a nucleic acid template or as a consequence of the limited substrate scope of polymerase enzymes.Chemistry and Chemical Biology
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Torella, Joseph Peter. "Confocal single-molecule fluorescence as a tool for investigating biomolecular dynamics in vitro and in vivo". Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:f57d1984-8db9-4d79-b333-f1be507ca3bf.
Testo completoAbstract (sommario):
Confocal single-molecule fluorescence is a powerful tool for monitoring conformational dynamics, and has provided new insight into the enzymatic activities of complex biological molecules such as DNA and RNA polymerases. Though useful, such studies are typically qualitative in nature, and performed almost exclusively in highly purified, in vitro settings. In this work, I focus on improving the methodology of confocal single-molecule fluorescence in two broad ways: (i) by enabling the quantitative identification of molecular dynamics in proteins and nucleic acids in vitro, and (ii) developing the tools needed to perform these analyses in vivo. Toward the first goal, and together with several colleagues, I have developed three novel methods for the quantitative identification of dynamics in biomolecules: (i) Burst Variance Analysis (BVA), which unambiguously identifies dynamics in single-molecule FRET experiments; (ii) Dynamic Probability Density Analysis (PDA), which hypothesis-tests specific kinetic models against smFRET data and extracts rate information; and (iii) a novel molecular counting method useful for studying single-molecule thermodynamics. We validated these methods against Monte Carlo simulations and experimental DNA controls, and demonstrated their practical application in vitro by analyzing the “fingers-closing” conformational change in E.coli DNA Polymerase I; these studies identified unexpected conformational flexibility which may be important to the fidelity of DNA synthesis. To enable similar studies in the context of a living cell, we generated a nuclease-resistant DNA analogue of the Green Fluorescent Protein, or “Green Fluorescent DNA,” and developed an electroporation method to efficiently transfer it into the cytoplasm of E.coli. We demonstrate in vivo confocal detection of smFRET from this construct, which is both bright and photostable in the cellular milieu. In combination with PDA, BVA and our novel molecular counting method, this Green Fluorescent DNA should enable the characterization of DNA and protein-DNA dynamics in living cells, at the single-molecule level. I conclude by discussing the ways in which these methods may be useful in investigating the dynamics of processes such as transcription, translation and recombination, both in vitro and in vivo.
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Rentergent, Julius. "Time course analysis of complex enzyme systems". Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/time-course-analysis-of-complex-enzyme-systems(1c44f0cf-188d-4cd7-ab2d-012da27646a8).html.
Testo completoAbstract (sommario):
In studies of enzyme kinetics, reaction time courses are often condensed into a single set of initial rates describing the rate at the start of the reaction. This set is then analysed with the Henri-Michaelis-Menten equation. However, this process necessarily removes information from experimental data and diminishes its statistical significance due to a reduction of available data points. Further, if the examined system does not approach steady-state rapidly, the application of the steady-state-assumption can lead to flawed conclusions. Here, the analysis of two complex enzyme systems by numerical integration of kinetic rate equations is demonstrated. DNA polymerase catalyses the synthesis of DNA in a reaction that involves two substrates, DNA template and dNTP, both of which are highly heterogeneous in nature. The tight binding of DNA to DNA polymerase and its polymer properties prohibit the application of the initial-rate approach. By combining an explicit DNA binding step with a steady-state dNTP incorporation on a template of finite length, the DNA binding parameters and the dNTP incorporation steady-state parameters were estimated from processive polymerisation data in a global regression analysis. This approach is described in Chapter 2 and the results are in good agreement with previously published values. Further properties were investigated in studies of the temperature dependence and solvent isotope dependence of the kinetics. The processive polymerisation of DNA template was monitored using the fluorophore PicoGreen in a simple and inexpensive method described in Chapter 3. The catalytic cycle of ethanolamine ammonia lyase involves the homoloysis of the Co-C bond within the intrinsic B12 cofactor. This homolysis results in the formation of a Co(II)-adenosyl radical intermediate, which can be monitored using stopped-flow spectroscopy. The stopped-flow transients observed for EAL and related enzymes have long been difficult to analyse and interpret, possibly due to rapid methyl group rotation on the substrate. In Chapter 4 of this thesis we were able to rationalise this behaviour using numerical integration of the rate equations of a branched 16-state-kinetic model to fit stopped-flow transients in a global regression analysis. We were able to determine some intrinsic rate constants, and showed that the initial hydrogen atom transfer step is unlikely to have an inflated primary kinetic isotope effect, despite previous claims. More generally, this study demonstrates that the numerical integration analysis used here is likely to be applicable to a broad range of enzyme reaction kinetics.
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Srinivasan, Sheila. "The design and synthesis of novel heterocyclic inhibitors of the DNA-repair enzyme, poly(ADP-ribose) polymerase, as potential resistance-modifying agents". Thesis, University of Newcastle Upon Tyne, 1997. http://hdl.handle.net/10443/1022.
Testo completoAbstract (sommario):
The abundant nuclear enzyme, poly(ADP-ribose) polymerase (PARP) is activated by DNA strand breaks and catalyses the transfer of ADP-ribose moieties from its substrate, nicotinamide adenine dinucleotide (NAD), to various histone- or non-histone nuclear acceptor proteins. The net result is the formation of long, homopolymeric chains, the exact purpose of which is not clearly understood. Since this process is thought to facilitate DNA repair, the PARP enzyme represents a possible therapeutic target. A well known mechanism by which tumours become resistant to anticancer treatments is increased DNA repair. Inhibition of PARP may thus be a strategy for the potentiation of DNA-damaging agents and PARP inhibitors may function as resistance-modifying agents in conjunction with cancer chemotherapeutic agents. The aim of this research was to design and synthesise novel heterocyclic inhibitors of PARP, based on the existing knowledge of structure-activity requirements. A great deal of information has already been gathered from the use of early inhibitors, such as nicotinamide and 3-aminobenzamide (3AB). However, these inhibitors lack potency, specificity for the enzyme, and aqueous solubility, and so are limited in their use as clinical agents. A novel series of quinazolin-4-[3H]-ones (structure A) has been synthesised by a highyielding, reproducible pathway, including derivatives bearing electron-withdrawing and electron-donating substituents in the 2-position. These derivatives exhibit excellent in vitro PARP inhibitory activity, with IC50 values in the micromolar concentration range, and the selected compound, NU1025 (8-hydroxy-2-methylquinazolin-4-[3H]-one) has been shown to potentiate the effects of a range of mechanistically diverse anticancer agents, including y-irradiation. A water-soluble phosphate prodrug derivative of NU1025 has been synthesised and this shows promising enzyme-mediated conversion to the parent compound in plasma. A second series of 1H-benzimidazole-4-carboxamides (structure B) has been synthesised bearing mono-, di- or trisubstituted aryl rings in the 2-position. Biological evaluation of this series has shown that these derivatives are among the most potent PARP inhibitors reported to date, with K; values in the low nanomolar concentration range.
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Raper, Austin T. "Mechanistic Studies of DNA Replication, Lesion Bypass, and Editing". The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1529576739391675.
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Pochet, Sylvie. "Synthèse d'oligodésoxynucléotides comportant des sites ambigus ou apuriniques et de sondes ancrées à un support solide". Paris 6, 1986. http://www.theses.fr/1986PA066065.
Testo completoAbstract (sommario):
Synthèse et étude par spectroscopies RMN, IR et DC d'oligodésoxyribonucléotides contenant un résidu sans base: coexistence des conformations b et z sur un même duplex. Elaboration d'une base universelle dans la série des nucléosides pyrimidiques. Synthèse d'oligodésoxynucléotides ancrés à un support solide; applications.
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Nemakonde, Avhashoni Agnes. "Efficacy of different DNA polymerase enzymes in PCR amplification of forensic bovine DNA". Diss., 2012. http://hdl.handle.net/2263/27077.
Testo completoAbstract (sommario):
DNA profiling of exhibits that originate from forensic stock theft cases is routinely used as a tool to link suspects to the crime or scene. DNA derived from aged or degraded samples is often highly fragmented which compromises the efficiency for obtaining a complete genotypic profile using PCR. Conventional polymerases such as Taq, lack certain repair mechanisms for use on degraded DNA templates. New generation polymerases are known to have high fidelity characteristics. The aim of this study was to determine the efficiency of Restorase®, a novel DNA polymerase blend that is known to repair damaged DNA and the FastStart High Fidelity PCR System enzymes, on degraded forensic bovine samples using PCR-based methodology. Bovine meat samples were subjected to different degrees of degradation in the sun and in the shade during summer and winter seasons. DNA was extracted, subjected to PCR amplification using 16 bovine microsatellites and genotypes were generated for analyses. Rapid degradation of samples was observed during winter while during summer samples tend to dry out. Restorase® exhibited high enzyme activity on degraded samples as compared with FastStart and Taq DNA polymerase. Some of the markers that failed to be successfully amplified by Taq polymerase, such as ETH10 and SPS115 were recovered using Restorase®. Markers such as BM1818, BM2113, ETH3, INRA23 and TGLA227 remained active throughout the experiment using all the enzymes, and therefore can form a basis of the bovine marker panel. Restorase® was found to be an alternative enzyme for use in bovine forensic analysis. CopyrightDissertation (MSc)--University of Pretoria, 2012.
Animal and Wildlife Sciences
unrestricted
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Pillay, Sarveshni. "Improvement of thermostability of a fungal xylanase using error-prone polymerase chain reaction (EpPCR)". Thesis, 2007. http://hdl.handle.net/10321/310.
Testo completoAbstract (sommario):
Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 vi, 92 leavesInterest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.
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Zhang, Yongchao Heller Adam. "Amperometric DNA sensing using wired enzyme based electrodes". 2003. http://wwwlib.umi.com/cr/utexas/fullcit?p3122806.
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Zhang, Yongchao. "Amperometric DNA sensing using wired enzyme based electrodes". Thesis, 2003. http://hdl.handle.net/2152/1090.
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Kranaster, Ramon [Verfasser]. "DNA polymerase activity on solid support : from diagnostics to directed enzyme evolution / vorgelegt von Ramon Kranaster". 2010. http://d-nb.info/1007476508/34.
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Magee, Wendy Colleen. "The mechanism of action of cidofovir and (S)-9-(3-hydroxy-2-phosphonomethoxypropyl) adenine against viral polymerases". 2009. http://hdl.handle.net/10048/534.
Testo completoAbstract (sommario):
Thesis (Ph.D.)--University of Alberta, 2009.Title from pdf file main screen (viewed on Sept. 18, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Virology, Medical Microbiology and Immunology, University of Alberta." Includes bibliographical references.
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Jeřábek, Petr. "Teoretická studie enzymů spojených s procesem karcinogeneze: DNA polymerázy β a cytochromů P450". Doctoral thesis, 2012. http://www.nusl.cz/ntk/nusl-330411.
Testo completoAbstract (sommario):
Present doctoral thesis contributed to understanding of mechanistic principles of two enzymes participating in the process of carcinogenesis; DNA polymerase (pol ) and cytochromes P450 (CYP). Pol is part of the DNA base-excision repair mechanism (BER). The primary role of pol in, the BER mechanism, is inserting a new nucleotide into a DNA strand according to Watson-Crick base pairing rules. Pol plays an important role in the process of carcinogenesis, approximately 30 % of human tumors express pol mutants. The ability of pol to discriminate between "right" and "wrong" nucleotide during the insertion process is called fidelity. We employed computational methods to elucidate molecular basis of the fidelity of pol . First, the relative free energy calculation method LRA was employed to compare differences in free energies between the "right" and "wrong" nucleotide during its insertion into DNA. The results indicated a better stabilization of transition-state of the nucleophilic substitution catalyzed by pol in the case of the "right" versus "wrong" nucleotide. This difference resulted in an 80-fold contribution to its fidelity. Further, computational methods FEP and LIE were used to examine how mutations effect fidelity of pol . Results were than correlated with experimental data...
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Strerath, Michael [Verfasser]. "Manipulation der Selektivität von DNA-Polymerasen : chemisch modifizierte Substrate und optimierte Enzyme zur Analyse und Anwendung / vorgelegt von Michael Strerath". 2006. http://d-nb.info/984611452/34.
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