Dissertations / Theses on the topic 'DNA-protein complexes'
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1
DiCapua, Elisabeth. "Complexes of recA protein with DNA /." [S.l.] : [s.n.], 1986. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=8212.
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Hammond, Maria. "DNA-Mediated Detection and Profiling of Protein Complexes." Doctoral thesis, Uppsala universitet, Molekylära verktyg, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-204861.
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Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules. Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).
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Berge, Torunn. "Structural analysis of DNA and DNA-protein complexes using atomic force microscopy." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621339.
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Preston, Nicola Susan. "Structure and DNA binding of HMG boxes." Thesis, University of Portsmouth, 1996. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310386.
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Luo, Dan. "Novel crosslinking technologies to assess protein-DNA binding and DNA-DNA complexes for gene delivery and expression /." Connect to resource, 1998. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1114436532.
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Luo, Dan. "Novel crosslinking technologies to assess protein-DNA binding and DNA-DNA complexes for gene delivery and expression." The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1114436532.
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Bielskienė, Kristina. "Analysis of the barley (Hordeum vulgare) tightly bound DNA-protein complexes." Doctoral thesis, Lithuanian Academic Libraries Network (LABT), 2009. http://vddb.library.lt/obj/LT-eLABa-0001:E.02~2009~D_20091202_111955-77123.
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Despite a great deal of research, the functional significance of tightly bound DNA-protein complexes is not yet clear, therefore these complexes are perfect object for pioneering research. Very little is known about plant TBP-DNA complexes.
In this work we investigated barley TBP-DNA complexes from different organs (first leaves, roots and coleoptiles) at different developmental stages. We characterized individual components of tightly bound DNA-proteins complexes: polypeptides (TBP) and DNA. We isolated and characterized TBP proteins from barley first leaves, roots and coleoptiles of different age and differentiation stage. Also we isolated and characterized the DNA fragments from barley first leaves and water ripe and milky ripe grain TBP-DNA complexes.
We demonstrated that in different developmental stages of coleoptiles, first leaves and roots TBP-DNA complexes were identified as a group of 15-160 kDa proteins, most of TBPs are acidic. Some of barley TBPs (10, 25, 38, 40 and 55 kDa) exhibit phosphatase, maybe Ser/Thr activity. We have identified also that some of TBPs tyrosines were phosphorylated, this modification depends on organ and developmental stage. Identified barley TBPs were involved in fundamental genetic processes, as well as in chromatin rearrangement and regulation processes. Nuclear matrix proteins, enzymes, transcription factors, serpins, immunophilins, and transposon polypeptides were identified among TBPs. We demonstrated that expression of TBPs depends... [to full text]Žinoma, kad pastovi nehistoninių polipetidų frakcija yra išgryninama kartu su eukariotine DNR ir sudaro labai tvirtus (galbūt kovalentinius) kompleksus tarp branduolio baltymų ir DNR. Nustatyta, kad Erlicho ascito tvirtuose DNR-baltymų kompleksuose yra baltymas C1D, baltymai, pasižymintys fosfataziniu ir kinaziniu aktyvumais, kai kurie proteazių slopikliai ir kiti, dar neištirti baltymai. Nepaisant intensyvių tyrinėjimų, eukariotinių ląstelių tvirti DNR-baltymų kompleksai vis dar lieka menkai aprašyti ir yra objektas tolimesniems tyrimams. Augalų TBP-DNR kompleksai kol kas buvo tyrinėti labai mažai. Šiame darbe charakterizuojami miežių Hordeum vulgare tvirti DNR-baltymų kompleksai. Mes tyrėme TBP-DNR kompleksus iš miežių skirtingų ūglių organų ir skirtingų vystymosi stadijų ląstelių: lapų, šaknų, koleoptilės. Norint ištirti tokių nukleoproteidų funkcijas, svarbu charakterizuoti individualius komplekso komponentus: polipeptidus ir DNR. Taigi, išskyrėme tvirtai su DNR sąveikaujančius baltymus iš miežių skirtingos diferenciacijos bei skirtingo amžiaus ląstelių: pirminių lapelių, šaknų, koleoptilės ir juos charakterizavome. Taip pat išskyrėme ir charakterizavome DNR fragmentus iš miežių pirminių lapelių bei vandeninės brandos ir pieninės brandos grūdų TBP-DNR kompleksų. Parodėme, kad miežių TBP baltymai yra 15-160 kDa, dauguma baltymų yra rūgštiniai. Kai kurie iš miežių TBP baltymų (10, 25, 38, 40 ir 55 kDa) pasižymi fosfataziniu, galbūt, Ser/Thr aktyvumu. Nustatėme, kad tam... [toliau žr. visą tekstą]
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Luo, Dan. "Novel cross-linking technologies to assess protein-DNA binding and DNA-DNA complexes for gene delivery and expression /." The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487946776022443.
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Fischhaber, Paula L. "Investigations at the molecular interfaces of complexes formed by the proteins ADR1 and xUBF1 with DNA /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/8648.
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Yan, Junhong. "DNA-Assisted Immunoassays for High-Performance Protein Analyses." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-236591.
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Proteins play important roles in most cellular functions, such as, replication, transcription regulation, signal transduction, for catalyzing chemical reaction, etc. Technologies developed to identify proteins rely either on observing their own properties such as charge, size, mass to charge ratio or sequence composition; or on using affinity reagents that recognize specific protein targets. Immunoassays utilizing functionalized affinity reagents are powerful for targeted proteomics. Among them, DNA-assisted immunoassays in which affinity reagents are labeled with DNA molecules, offer some unique advantages. In this thesis, I will present works to improve current DNA-assisted immunoassays such as proximity ligation assays (PLA), as well as to take advantage of DNA reactions to adress other problems. In paper I, a new solid support (MBC-Ts) was functionalized with antibodies and used in the solid-phase PLA for detection of VEGF. The assay using MBC-Ts was compared among the commercially available solid supports in different matrices and it was shown to exhibit enhanced limit of detection in complex matrices. In paper II, a two-step protocol was described to prepare high-quality probes used in homogeneous and in situ PLA by purifying DNA-labeled affinity reagents from unconjugated affinity reagents and excess oligonucleotides. In paper III, PLA was applied on a capillary western blotting instrument so that both the sensitivity and specificity of the original assay were improved. In paper IV, a new method was introduced to profile protein components in individual protein complexes by DNA-barcoded antibodies. This method has been used to profile protein complexes such as surface proteins on individual secreted vesicles.
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Toma, Adriana Cristina. "DNA condensation by a basic protein, the salmon protamine." Paris 11, 2008. http://www.theses.fr/2008PA114828.
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In vivo les protamines condensent l'ADN dans la tête des spermatozoïdes, mais le mécanisme impliqué n'est pas complètement compris. Nous avons étudié la condensation de l'ADN par des protamines de saumon in vitro en utilisant différentes approches expérimentales. Les transitions d'états, la solubilité de ces complexes et la densité des précipités sont trouvés dépendants des concentrations ioniquesIn vivo protamine condense DNA in the sperm head but the mechanism of this process is not completely understood. We examine the efficiency of salmon protamine to condense DNA in vitro using different experimental approaches. The condensation transition, the solubility of these complexes and the density of the condensate are found sensitive to the ionic concentrations
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Gilljam, Karin Margaretha. "DNA repair protein complexes, functionality and significance for repair efficiency and cell survival." Doctoral thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for kreftforskning og molekylær medisin, 2010. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-12246.
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DNA‐reparasjons‐protein‐komplekser, funksjonalitet og signifikans for reparasjonseffektivitet og skadetoleranse All informasjon om en organisme er lagret i vår arvestoff, DNA. DNA er et relativt ustabilt makromolekyl som konstant blir utsatt for farer som truer dets integritet, både fra omgivelsene og fra kjemiske prosesser inne i selve cellen. I tillegg kan baser spontant bli mistet uten noe form for påvirkning. Selve kopieringen av DNA, den såkalte DNA-replikasjonen er svært rask og er en kritisk prosess i cellen hvor mye kan gå galt. I tillegg kan ureparerte DNA-skader ved replikasjonen foreviges i form av mutasjoner. Mutasjoner i gener som koder for proteiner som regulerer cellens vekst og død kan resultere i ukontrollert cellevekst og dermed kreft. En av cellens strategier for å sikre effektiv og trygg replikasjon og reparasjon av DNA’et er å samarbeide ved å danne proteinkomplekser, hvorav PCNA ofte spiller en sentral rolle. PCNA sitter som en homotrimerisk ring rundt DNA-tråden som replikeres, og fungerer som en plattform for binding av mange proteiner. I tillegg til binding av DNA-replikasjonsproteiner, bindes også mange DNA-reparasjonsproteiner til PCNA, og sørger for effektiv reparasjon av skadet DNA både før og etter selve replikasjonen. I tillegg er PCNA involvert i DNA-syntese ved reparasjon som ikke er assosiert med replikasjon. I 1998 ble det funnet et motiv (en peptid-sekvens) som er ansvarlig for at mange proteiner bindes til PCNA, kalt PCNA Interacting Peptide (PIP). I artikkel 1 fant vi ved hjelp av blant annet fluorescerende proteiner og konfokal mikroskopi et nytt motiv som er viktig for proteiners binding til PCNA. Dette motivet fant vi først i det direkte alkyleringsreparasjons-proteinet; human AlkB homologue 2 og derfor kalte vi motivet AlkB homologue 2 PCNA Interacting Motif (APIM). I denne artikkelen verifiserer vi et funksjonelt APIM motiv i fem proteiner og viser at over-uttrykk av dette motivet gjør celler mer sensitive for alkylerende skade. Dette tyder på at overuttykk av APIM hemmer bindingen mellom APIM-inneholdende DNA reparasjons-proteiner og PCNA slik at de ikke reparerer DNA-skadene optimalt. I samme artikkel viser vi også at APIM er konservert i mer enn 200 proteiner, blant annet i nukleotideeksisjonsreparasjons (NER) proteinet Xeroderma Pigmentosum group A (XPA), og i artikkel 2 verifiserer vi at APIM også er et funksjonelt PCNA bindende motiv i XPA. Vi viser og at overuttrykk av APIM-peptidet gjør celler mer sensitive for skade fra UV-lys, en type DNA-skade som hovedsakelig blir reparert av NER. I tillegg finner vi bevis som støtter at det er redusert funksjon av XPA som er årsak til at cellene er mer UV-sensitive ved overuttrykk av APIM, antagelig pga. svekket binding til PCNA. I artikkel 3 ser vi nærmere på baseeksisjonsreparasjons- og singeltrådbruddsreparasjons-proteinet XRCC1. Dette er i likhet med PCNA og XPA et protein uten enzymatisk funksjon, men med mange bindingspartnere, blant annet PCNA. Hvilken del av XRCC1 som er viktig for dens funksjon i cella er derimot ikke helt klarlagt, noe vi undersøker nærmere i denne artikkelen. Det viser seg at den delen av XRCC1 som har evnen til å binde PCNA og alkyleringsreparasjons-proteinet MPG er den eneste XRCC1 mutanten som kan stimulere reparasjon av alkyleringsskader, noe som igjen bekrefter viktigheten av å binde seg til PCNA. Oppsummert tar dette arbeidet for seg hvordan DNA-reparasjonsproteiner binder seg til hverandre og PCNA, og hvordan dette påvirker evnen til å reparere DNA og dermed tåle DNA-skade.
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Tomãs, de Oliveira Isabel. "Packing and distribution of empty space in liquids, proteins and protein-DNA complexes." Doctoral thesis, Universite Libre de Bruxelles, 2001. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/211651.
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Saurabh, Suman. "Nature of Inter-biomolecular interaction and its consequences : protein, DNA and their Complexes." Thesis, Tours, 2017. http://www.theses.fr/2017TOUR4052/document.
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Le monde biologique est rempli de mystères. La compréhension de nombreux processus biologiques extrêmement complexes est grandement améliorée par la combinaison d’approches empruntées à différentes disciplines telles que la chimie et plus récemment la physique. La physique utilise des outils expérimentaux tels que les pinces optiques et les microscopies optique et électronique pour explorer les mécanismes à l’échelle microscopique se déroulant dans la cellule. La connaissance de la nature des interactions entre biomolécules et la possibilité de traduire ces interactions en équations ont permis à la physique de construire des modèles simples mais contenant les ingrédients suffisants à la description d’un mécanisme spécifique. La simulation numérique de tels modèles permet d’améliorer notre compréhension de la relation entre les mécanismes pertinents à l’échelle moléculaire et les observations expérimentales de phénomènes biologiquesThe biological world is full of mysteries. The understanding of many extremely complex biological processes is greatly improved by the combination of approaches borrowed from different disciplines such as chemistry and more recently physics. Physics uses experimental tools such as optical tweezers and optical and electron microscopes to explore the microscopic mechanisms taking place in the cell. Knowledge of the nature of the interactions between biomolecules and the possibility of translating these interactions into equations allowed physics to construct models that are simple, but contain the ingredients sufficient to describe a specific mechanism. The numerical simulation of such models improves our understanding of the relationship between relevant molecular-scale mechanisms and experimental observations of biological phenomena. The structural organization of biomolecular complexes is a process that involves various scales of length and time
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Kim, Soojeong. "A 4-string tangle analysis of DNA-protein complexes based on difference topology." Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/528.
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An n-string tangle is a three dimensional ball with n-strings properly embedded in it. In late the 80's, C. Ernst and D. Sumners introduced a tangle model of protein-DNA complexes. This model assumes that the protein is a 3-dimensional ball and the protein-bound DNA are strings embedded inside the ball.
Originally the tangle model was applied to proteins such as Cre recombinate which binds two DNA segments. The protein breaks and rejoins the DNA segments and then creatss knotted DNA. When this kind of protein complex bounds circular DNA, there will be two DNA loops outside of the DNA-protein complex. Hence we can use a 2-string tangle model for this complex. More recently, Pathania, Jayaram and Harshey predicted that the topological structure within the Mu protein complex consists of three DNA segments containing five crossigs. Since Mu binds DNA sequences at 3 sites, the Mu protein-DNA complex can be modeled by a 3-string tangle. Darcy, Leucke and Vazquez analyzed Pathania et al's experimental results by using 3-tangle analysis.
Based on the 3-string tangle analysis of Mu protein-DNA complex, we addressed the possibility that a protein binds DNA sequences at four sites. Such a protein complex bound to a circular DNA molecule is modeled by a 4-string tangle with four loops outside of the tangle. In this thesis, we decided a biologically relevant 4-string tangle model. We also developed mathematics for solving tangle equations to predict the topology of DNA within the protein complex.
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Watkins, Jason Derrick. "X-ray structures of P22 c2 repressor-DNA complexes the mechansism of direct and indirect readout /." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/26709.
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Thesis (Ph.D)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009.Committee Chair: Loren D. Williams; Committee Member: Donald Doyle; Committee Member: Nicholas V. Hud; Committee Member: Roger Wartell; Committee Member: Stephen Harvey. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Fischer, Nina M. [Verfasser], and Oliver [Akademischer Betreuer] Kohlbacher. "Modeling Flexibility of Protein-DNA and Protein-Ligand Complexes using Molecular Dynamics / Nina M. Fischer ; Betreuer: Oliver Kohlbacher." Tübingen : Universitätsbibliothek Tübingen, 2013. http://d-nb.info/1162896728/34.
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Kolli, Ramya. "Effect of Leaving Ligands of Platinum(II) Diamine Complexes on DNA and Protein Residues." TopSCHOLAR®, 2013. http://digitalcommons.wku.edu/theses/1268.
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Platinum compounds are widely used drugs in cancer treatments. Although DNA is the biological target, reaction of platinum compounds with proteins is also potentially significant. Our objective is to study the effects of leaving ligands on the relative reactivity between 5'-GMP (guanosine 5' phosphate), a key DNA target, and N-Acetyl - L-Methionine (N-AcMet), a key protein target. We have used NMR spectroscopy to monitor reactions with N-AcMet and 5'-GMP added to a platinum complex to see which products are formed preferentially. Previous research showed that both a non-bulky complex such as [Pt(en)(D2O)2]2+ [en=ethylenediamine], and a bulky complex such as [Pt(Me4en)(D2O)2]2+ [Me4en= N, N, N', N'-tetramethylethylenediamine] react more quickly with 5'-GMP than with N-AcMet. To improve the activity of platinum compounds in our current research, oxalates as leaving ligands are used. The results suggest that [Pt(en)(Ox)] [Ox= oxalate] reacts faster with N-AcMet than with 5'-GMP. Also, [Pt(Me4en)(Ox)] reacts slowly with 5'-GMP without N-AcMet and the reaction favors N-AcMet when both ligands are added simultaneously. Interestingly, the formation of the sulfur-oxygen chelate is slow enough to be observable in the oxalate reaction; but the mono product is not independently observed in the dinitrate complex.
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Davies, Emma. "High-resolution atomic force microscopy and current-voltage characterisation of DNA and protein complexes." Thesis, Swansea University, 2006. https://cronfa.swan.ac.uk/Record/cronfa42320.
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Using the technique of non-contact atomic force microscopy (NC-AFM), the structural properties of DNA and protein complexes were studied at the singlemolecule level and at high-resolution. The electrical properties of these biomolecules were then investigated using an electrode setup. This work focussed on the visualisation of DNA strands, nucleosomes, PUT3 protein and DNA-PUT3 complexes and the effect of protein on the conductivity of DNA. NC-AFM experiments were performed in vacuum and results were compared to tappingmode AFM (TM-AFM) experiments performed in air. The detailed structure of DNA strands 2-kilobase-pairs (kbp) in length, deposited on gold substrates, was observed for the first time using NC-AFM without the need for chemical- anchoring techniques. Measurements made on individual DNA molecules revealed a strand length of 700 nm which was in good agreement with the calculated length of 680 nm for a liner 2-kbp DNA molecule. The average height of the DNA was 1.37 nm compared to 2 nm as determined with X-ray crystallography. Images of nucleosomes on mica revealed unprecedented detail with line profiles indicating peaks of 3-4 nm corresponding to DNA wrapped twice around the nucleosome core. DNA-PUT3 complexes were observed using a very high spring constant cantilever for NC-AFM. The measured diameter of PUT3 was 70 nm with corresponding height ~11 nm. Similar diameters were recorded using TM-AFM but with lower height ~2-2.5 nm. The difference in height is possibly attributed to the non-invasive nature of NC-AFM compared to tapping-mode which may have compressed soft samples into the surface. Current- voltage (I-V) measurements were performed on DNA-PUT3 samples and those prepared in binding buffer gave elevated currents at +5 V (I = 73-115 nA) compared to samples containing an equivalent concentration of DNA prepared with water (I = 0.4-3.6 nA). The presence of binding buffer appeared to improve current readings possibly by interacting with molecules via 'doping' to give to metallic-DNA (M-DNA) or by promoting DNA-PUT3 complex formation. The preferential bonding of DNA-PUT3 complexes to gold electrodes is suggested as a possible interpretation.
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Anderson, Robert James. "Development of an affinity partitioning method for DNA/protein complexes and its application to interactions of topoisomerase II with DNA." Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386626.
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Ma, Xin. "MASS SPECTROMETRY DISSOCIATION STUDIES OF PROTEIN-PROTEIN AND PROTEIN-NUCLEIC ACID COMPLEXES AND 13C FLUX OF AMINO ACIDS." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397773864.
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Grippon, Ayse Seden. "Protein complexes in base excision repair : biochemical and kinetic analysis of mismatch uracil DNA glycosylase." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/5666.
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Mismatch uracil DNA glycosylase (MUG) is an E. coli enzyme involved in the repair of ethenocytosine and uracil through the base excision repair pathway. MUG is known to bind the abasic site tightly. This may act to protect the abasic lesion, but the question then is how is the site handed over to the AP Endonuclease? Much has been made of the increase in turnover of some DNA glycosylases by AP endonucleases, but it is not clear whether this occurs via an active displacement mechanism or by passive diffusion. We are addressing these questions by studying the kinetics of MUG interactions with its product and Exonuclease III, the main AP Endonuclease in E. coli. We used fluorescence anisotropy and fluorescence resonance energy transfer assays to investigate MUG & DNA interactions. These revealed that MUG binds abasic DNA in a cooperative manner and that binding of two MUG‘s is needed for efficient DNA repair. Higher salt concentrations reduced cooperativity leading to a 1:1 binding and reduced MUG‘s activity. We also used these assays to investigate whether ExoIII displaces MUG from the DNA through an active or passive mechanism. MUG‘s role in the in vivo repair of etheno lesions has been explored by treating E. coli cells with urethane, a chemical, known to introduce etheno lesions in the DNA. A eukaryotic DNA glycosylase, hSMUG was shown to repair etheno lesions; hence we investigated hSMUG‘s ability to complement mug deficiency in E. coli cells. hSMUG was found to reduce cell viability, increase mutation rates and provided a boost in cell divisions in mug deficient cells whereas it didn‘t affect wild type cells. We then asked the question whether hAPE1, the AP endonuclease believed to increase the turnover rate of hSMUG, can reverse the effects of hSMUG expression in E. coli.
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Sachsenberg, Timo [Verfasser], and Oliver [Akademischer Betreuer] Kohlbacher. "Computational Methods for Mass Spectrometry-based Study of Protein-RNA or Protein-DNA Complexes and Quantitative Metaproteomics / Timo Sachsenberg ; Betreuer: Oliver Kohlbacher." Tübingen : Universitätsbibliothek Tübingen, 2018. http://d-nb.info/116863430X/34.
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Beamish, Eric. "Precise Size Control and Noise Reduction of Solid-state Nanopores for the Detection of DNA-protein Complexes." Thesis, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23569.
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Over the past decade, solid-state nanopores have emerged as a versatile tool for the detection and characterization of single molecules, showing great promise in the field of personalized medicine as diagnostic and genotyping platforms. While solid-state nanopores offer increased durability and functionality over a wider range of experimental conditions compared to their biological counterparts, reliable fabrication of low-noise solid-state nanopores remains a challenge. In this thesis, a methodology for treating nanopores using high electric fields in an automated fashion by applying short (0.1-2 s) pulses of 6-10 V is presented which drastically improves the yield of nanopores that can be used for molecular recognition studies. In particular, this technique allows for sub-nanometer control over nanopore size under experimental conditions, facilitates complete wetting of nanopores, reduces noise by up to three orders of magnitude and rejuvenates used pores for further experimentation. This improvement in fabrication yield (over 90%) ultimately makes nanopore-based sensing more efficient, cost-effective and accessible.
Tuning size using high electric fields facilitates nanopore fabrication and improves functionality for single-molecule experiments. Here, the use of nanopores for the detection of DNA-protein complexes is examined. As proof-of-concept, neutravidin bound to double-stranded DNA is used as a model complex. The creation of the DNA-neutravidin complex using polymerase chain reaction with biotinylated primers and subsequent purification and multiplex creation is discussed. Finally, an outlook for extending this scheme for the identification of proteins in a sample based on translocation signatures is presented which could be implemented in a portable lab-on-a-chip device for the rapid detection of disease biomarkers.
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Chichetu, Karen. "Characterization, DNA Binding and Cleavage Activities of New Prodigiosin and Tambjamine Analogues and Their Cu²⁺ and Zn²⁺ Complexes." PDXScholar, 2015. http://pdxscholar.library.pdx.edu/open_access_etds/2467.
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Prodigiosins and tambjamines are natural compounds from bacterial and marine sources belonging to a family containing a common 4-methoxy-2,2'-bipyrrole core. These compounds have received a lot of interest due to their promising biological activities. Studies have suggested DNA as a potential therapeutic target for the natural prodigiosin and tambjamine due to their ability to facilitate oxidative DNA cleavage in the presence of Cu2+. Based on this we sought to study the metal binding activity of new prodigiosin and tambjamine analogues. A new prodigiosin analogue was synthesized and complexed with Cu2+. This revealed 1:1 complex formation between the tripyrrole and Cu2+ that was confirmed by mass spectra and NMR spectra analysis. In addition in situ studies also revealed that our new analogues of prodigiosin cannot bind Zn2+ when the methoxy group on ring B is replaced by an alkyl group or when one of the ring nitrogens is methylated.
Our UV-Vis experiments with calf thymus DNA showed that prodigiosins and tambjamines bind DNA mainly through an external mode, suggesting that hydrogen bonding between the pyrrole ring nitrogens and the bases of DNA takes precedence over stacking interactions. For the new Cu2+ complex synthesized however, we observed spectral changes that suggest intercalation into DNA.
DNA cleavage experiments revealed that the prodigiosin-Cu complex is able to convert supercoiled DNA into its linear form. The data from the gel shift assays fit well to a first-order consecutive reaction model. In addition to preformed metal complexes, we showed DNA cleavage by in situ complexation of the ligands in the presence of Cu2+. However, although we showed Zn2+ complex formation with prodigiosin analogues, in situ studies did not show induction of DNA cleavage by Zn2+ complexes under our experimental conditions.
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Shajani, Zahra. "Characterizing internal dynamics in nucleic acids by nuclear magnetic resonance spectroscopy : a study of RNA, DNA, and RNA-protein complexes /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/8587.
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Nguyen, Huynh Nha Thi. "Développements en spectrométrie de masse pour l’étude des complexes biologiques." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAF045/document.
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L’élucidation des interactions non-covalentes des complexes biologiques revêt d’une importance majeure dans la compréhension du fonctionnement cellulaire. L’objectif de ce travail de thèse est d’approfondir les développements de la spectrométrie de masse (MS) pour l’étude de ces complexes, que ce soit par MALDI-MS (la désorption-ionisation laser assistée par matrice) ou par ESI-MS (l’ionisation électrospray). Ce travail s’est articulé autour de trois axes : i) étude de la stœchiométrie et de la topologie du complexe SAGA HAT (Spt-Ada-Gcn5 Acétyltransferase, module Histone Acétyl Transferase) par pontage chimique couplé à la MS ; ii) suivi de la dimérisation des complexes formés par RAR-RXR (récepteur de l’acide rétinoïque - récepteur X des rétinoïdes) avec différents ADNs ; iii) mesure de la constante de dissociation des complexes RXR-ligand. Les méthodologies développées ont permis de repousser le potentiel de la MS et d’obtenir des informations structurales des complexes biologiquesElucidation of non-covalent interactions of biological complexes takes on great importance for the understanding of cellular function. The purpose of this thesis is a further development of mass spectrometry (MS) for the study of these complexes, either by MALDI-MS (matrix-assisted laser desorption-ionization) or by ESI-MS (electrospray ionization). This work was focused on three main lines: i) study of the stoichiometry and the topology of SAGA HAT (Spt-Ada-Gcn5 Acetyltransferase, Histone Acetyl Transferase module) complex by chemical cross-linking coupled to MS; ii) monitoring the dimerization of the complexes formed by RAR-RXR (retinoic acid receptor - retinoid X receptor) with different DNAs; iii) measuring the dissociation constant of RXR-ligand complexes. The developed methodologies made it possible to expand the potential of MS and get insight into structure of biological complexes
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Quebre, Valentin. "Etude des complexes ADN-protéines impliqués dans la ségrégation de l'ADN bactérien." Thesis, Toulouse 3, 2022. http://www.theses.fr/2022TOU30072.
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La ségrégation des chromosomes et des plasmides à bas nombre de copies chez les bactéries est basée sur un mécanisme actif de positionnement. Il repose sur des systèmes de partition qui assurent la répartition intracellulaire des réplicons afin qu'ils soient transmis de façon fidèle à la descendance. Ils font intervenir trois partenaires encodés par la molécule à ségréger. Une protéine de liaison à l'ADN (ParB) s'assemble en un complexe de partition autour d'une séquence centromérique (parS). Une protéine NTPase, interagissant avec le complexe de partition, est responsable de l'activité de ségrégation et du positionnement du complexe de partition et ainsi du plasmide. Mon projet de thèse vise d'abord à approfondir le mécanisme d'assemblage du complexe de partition du système majoritaire de type I des plasmides F et pESBL et ensuite, à déchiffrer le mécanisme global de partition du système atypique de R388 récemment découvert, n'utilisant pas de NTPase codée par le plasmide pour assurer son positionnement. Ainsi, mon projet s'articule en trois parties. (i) Comprendre par une approche mutationnelle le mécanisme d'initiation de l'auto assemblage de l'ensemble des ParB du plasmide F en un complexe de haut poids moléculaire dynamique, autour de parS. (ii) Identifier les partenaires du système de partition du plasmide pESBL, caractériser in vitro l'interaction de ParB avec parS et déterminer par une étude in silico, le groupe auquel il appartient. (iii) Identifier le rôle des différents domaines de la protéine de liaison à l'ADN StbA du plasmide R388 dans ses activités et caractériser les modalités d'interaction de StbA avec son site spécifique sur le plasmide, par des approches de séquençage à haut débit et de biochimie, pour comprendre l'architecture du complexe de partition. Cette étude a permis d'approfondir nos connaissances sur la biologie des plasmides portant un système de partition de type I et a mis en lumière les spécificités d'interactions ADN/protéines d'un système atypique, ouvrant la voie à la compréhension de son mécanisme de stabilisationBacterial chromosomes and low copy number plasmids segregation is based on an active positioning mechanism. It consists in the partition systems that ensures the proper intracellular positioning of replicons to be faithfully transmitted to the daughter cells. The partition systems involves three cis-encoded partners. A DNA binding protein (ParB), is assembled in partition complexes at centromeric sequences (parS). An NTPase, which interacts with the partition complex, drives the segregation process and allows the complexes, and thus the plasmids, to be properly positioned inside the cell. My Ph.D project focused first on the better understanding of the partition complex assembly of the widespread type I system of the F plasmid and pESBL. Then, to decipher the global mechanism of the partition process of the recently discovered atypical system on R388, which does not involve any plasmid encoded NTPase to ensure its intracellular positioning. Thus, my project is divided in three parts, aiming to (i) understand by an mutational approach, the initiation mechanism for the self-assembly of the majority of F plasmid ParB in a dynamic high molecular weight complex around parS, (ii) identify the pESBL partition system partners, in vitro characterize the ParB/parS interaction profile and in silico determine the group to which it belongs, (iii) identify the roles of the different domains of the R388 DNA binding protein StbA in its activities and characterize the StbA interaction modalities on its centromere by high throughput sequencing and biochemical approaches, to understand the partition complex architecture. This study allows us to improve our knowledge on the Type I partition system and to shed light on the DNA/protein interaction specificities of an atypical system, carried by broad-host-range plasmids, opening the way to a better understanding of DNA segregation mechanism
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Padilla, Roberto. "Discovering the Potential of Photoluminescent Ruthenium(II) Complexes as Photodynamic Therapy Agents." Diss., Virginia Tech, 2016. http://hdl.handle.net/10919/78190.
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Anthracene was attached to light activated, ruthenium-based DNA disruptors to probe their distribution in cancer cells. The objective of this research is to understand the photophysical properties (Chapter 2), photoreactivity toward DNA and proteins (Chapter 3), and localization within cancer cells (Chapter 4) of ruthenium complexes that demonstrate promise as photodynamic therapy (PDT) agents.
[(AnthbpyMe)(bpy)Ru(dpp)]2+ (1) and [(AnthbpyMe)2Ru(dpp)]2+ (2) absorb visible light with metal-to-ligand charge transfer (MLCT) transitions at 459 nm (16,000 M-1 cm-1 ) and 461 nm (21,000 M-1 cm-1 ), respectively. These species exhibit 3 MLCT emissions at λem = 661 nm and λem = 663 nm for 1 and 2, respectively, while the anthracene show emissions at 450 – 560 nm. The anthracene unit(s) quench the 3 MLCT to give quantum yields (lifetime) of Φem = 0.0059 [398(1) ns] and Φem = 0.0011 [414(1) ns] for 1 and 2, respectively. Voltammetry shows an irreversible anthracene oxidation at 1.23 – 1.28 V, RuIII/II oxidation at 1.53 – 1.55 V, and quasi-reversible reduction couples attributed to dpp0/-1 at 0.98 V.
DNA gel shift assays demonstrate that complexes 1 and 2 modify DNA in the presence and absence of 3 O2 upon light activation to convert supercoiled DNA to a mixture of open circular (OC) DNA and a species that exhibit sa distinctly different migration rate than either OC and linear DNA. Binding constants, Kb, for complexes 1 and 2, toward DNA are 3.50 × 105 (3.50 × 104 ) and 4.50 × 103 (4.50 × 102 ) respectively. SDS-PAGE assays show that the complexes 1 and 2 modify bovine serum albumin (BSA) through an 3 O2-dependent mechanism upon light iii activation.
The localization and PDT potency of the anthracene-Ru-dpp complexes are tested against F98 cells, which are rat glioma cells that simulate the infiltrative patterns of growth in cancer. Confocal microscopy demonstrates that complexes 1 and 2 internalize and localize primarily along the cell membrane and associate with dot-like vesicles within the cytoplasm. Complexes 1 and 2 show IC50 values of 107 µM and 85 µM, respectively, after 15 min of drug exposure and 1 h of PDT-treatment (λPDT = 455 nm).Ph. D.
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Stadler, Jens Michael [Verfasser]. "XPF-ERCC1 protein complexes regulate 5´ DNA incision and XPC deubiquitylation in the Global Genome branch of Nucleotide Excision Repair / Jens Michael Stadler." Mainz : Universitätsbibliothek Mainz, 2019. http://d-nb.info/1188428802/34.
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Taylor, James Edward Nathan. "Biochemical and biophysical characterisation of the genetically engineered Type I restriction-modification system, EcoR124I NT." Thesis, University of Portsmouth, 2005. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424193.
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The EcoR124INT restriction-modification (R-M) system contains the genes HsdS3, HsdM and HsdR. S3 encodes the N-terminal domain of the wild-type S subunit and has been shown to dimerise in solution (Smith et al., 1998). Following purification of the subunits of the EcoR124INT R-M system, complexes of the methyltransferase S3/M and restriction endonuclease S3/M/R were formed and shown to have activity in vitro, methylating and hydrolysing a symmetrical DNA recognition sequence, respectively. The DNA mimic OCR (overcome classical restriction) protein inhibited the methyltransferase activity in vitro, with maximum inhibition at a 1: 2 molar ratio of (S3/M)2 to an ocr dimer. Dynamic light scattering (DLS), sedimentation equilibrium (SE) and sedimentation velocity (SV) experiments showed S3 to exist as a dimer and S11 (the central conserved domain of S) to exist as a tetramer in solution. M was found to be dimeric in solution, whilst the R protein was monomeric. A complex of S3/M was found to have a stoichiometry (S3/M)2 and a complex of S3/M/R had a stoichiometry of S3/M/R1, even when a 2: 1 molar ratio of R to S3/M, was added. Small angle neutron scattering (SANS) experiments provided values for the radius of gyration (Rg), which for S3 was comparable to that calculated for the recently published crystal structure of the S subunit from Methanococcus jannaschii (Kim et al., 2005). These experiments also showed a decrease in the Dmax in the presence of the 30 bp DNA recognition sequence from 200A to 140A, suggesting a similar conformational change in the positioning of the subunits as has been detected for the wild-type M. EcoR124I and a related type 1 1/2 system AhdI. This change following DNA binding was also observed by SV experiments. Furthermore ab initio modelling from the SANS data has provided a low-resolution structure for the EcoR124INT MTase and its complex with DNA.
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Heessen, Stijn. "Regulation of the ubiquitin-proteasome system : characterization of viral and cellular stabilization signals /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-600-6/.
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Xu, Guozhou. "Crystal structures of the Fanconi anemia proteins : structure of the interstrand cross-linking repair protein Fanconi anemia protein I (FANCI); structure of the human FANCF C-terminal domain; reconstitution and crystallilzation of the sub-complexes in the Fanconi anemia core complex /." Access full-text from WCMC, 2009. http://proquest.umi.com/pqdweb?did=1692100351&sid=1&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Bielskienė, Kristina. "Miežių (Hordeum vulgare) tvirtų DNR-baltymų kompleksų tyrimas." Doctoral thesis, Lithuanian Academic Libraries Network (LABT), 2009. http://vddb.library.lt/obj/LT-eLABa-0001:E.02~2009~D_20091202_112036-51456.
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Žinoma, kad pastovi nehistoninių polipetidų frakcija yra išgryninama kartu su eukariotine DNR ir sudaro labai tvirtus (galbūt kovalentinius) kompleksus tarp branduolio baltymų ir DNR. Nustatyta, kad Erlicho ascito tvirtuose DNR-baltymų kompleksuose yra baltymas C1D, baltymai, pasižymintys fosfataziniu ir kinaziniu aktyvumais, kai kurie proteazių slopikliai ir kiti, dar neištirti baltymai.
Nepaisant intensyvių tyrinėjimų, eukariotinių ląstelių tvirti DNR-baltymų kompleksai vis dar lieka menkai aprašyti ir yra objektas tolimesniems tyrimams. Augalų TBP-DNR kompleksai kol kas buvo tyrinėti labai mažai. Šiame darbe charakterizuojami miežių Hordeum vulgare tvirti DNR-baltymų kompleksai.
Mes tyrėme TBP-DNR kompleksus iš miežių skirtingų ūglių organų ir skirtingų vystymosi stadijų ląstelių: lapų, šaknų, koleoptilės. Norint ištirti tokių nukleoproteidų funkcijas, svarbu charakterizuoti individualius komplekso komponentus: polipeptidus ir DNR. Taigi, išskyrėme tvirtai su DNR sąveikaujančius baltymus iš miežių skirtingos diferenciacijos bei skirtingo amžiaus ląstelių: pirminių lapelių, šaknų, koleoptilės ir juos charakterizavome. Taip pat išskyrėme ir charakterizavome DNR fragmentus iš miežių pirminių lapelių bei vandeninės brandos ir pieninės brandos grūdų TBP-DNR kompleksų.
Parodėme, kad miežių TBP baltymai yra 15-160 kDa, dauguma baltymų yra rūgštiniai. Kai kurie iš miežių TBP baltymų (10, 25, 38, 40 ir 55 kDa) pasižymi fosfataziniu, galbūt, Ser/Thr aktyvumu. Nustatėme, kad tam... [toliau žr. visą tekstą]Despite a great deal of research, the functional significance of tightly bound DNA-protein complexes is not yet clear, therefore these complexes are perfect object for pioneering research. Very little is known about plant TBP-DNA complexes. In this work we investigated barley TBP-DNA complexes from different organs (first leaves, roots and coleoptiles) at different developmental stages. We characterized individual components of tightly bound DNA-proteins complexes: polypeptides (TBP) and DNA. We isolated and characterized TBP proteins from barley first leaves, roots and coleoptiles of different age and differentiation stage. Also we isolated and characterized the DNA fragments from barley first leaves and water ripe and milky ripe grain TBP-DNA complexes. We demonstrated that in different developmental stages of coleoptiles, first leaves and roots TBP-DNA complexes were identified as a group of 15-160 kDa proteins, most of TBPs are acidic. Some of barley TBPs (10, 25, 38, 40 and 55 kDa) exhibit phosphatase, maybe Ser/Thr activity. We have identified also that some of TBPs tyrosines were phosphorylated, this modification depends on organ and developmental stage. Identified barley TBPs were involved in fundamental genetic processes, as well as in chromatin rearrangement and regulation processes. Nuclear matrix proteins, enzymes, transcription factors, serpins, immunophilins, and transposon polypeptides were identified among TBPs. We demonstrated that expression of TBPs depends... [to full text]
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Oikonomopoulos, Spyridon. "Inferring structural properties of protein-DNA binding using high-throughput sequencing : the paradigm of GATA1, KLF1 and their complexes GATA1/FOG1 and GATA1/KLF1 : insights into the transcriptional regulation of the erythroid cell lineage." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:72b92906-4ef6-4c1d-9155-484521027e2e.
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GATA1 and KLF1 are transcription factors that regulate genes which are important for the development of erythroid cells. The GATA1 transcriptional co-factor FOG1 has been shown to be essential in a wide range of GATA1 dependent cellular functions. Here we tried to understand the diverse mechanisms by which GATA1 and KLF1 recognize their binding sites, how the GATA1 recognition mechanisms are affected by complexation with either FOG1 or KLF1 and how the GATA1 recognition mechanisms affect the transcriptional regulation of the erythroid differentiation. We profiled the DNA binding specificities/affinities of a GATA1 fragment (mGATA1NC), that contains only the two DNA binding domains (N-terminal and C-terminal Zn finger), and the DNA binding specificities/affinities of a KLF1 fragment (mKLF1257-358), that contains the three DNA binding domains, using a novel methodology that combines EMSA with high throughput sequencing (EMSA-seq (Wong et al., 2011a)). We also profiled the DNA binding specificities of the C-terminal Zn finger of GATA1 alone (mGATA1C), the wt-mGATA1, the wt-mGATA1/wt-mFOG1 complex and the mGATA1NC/mKLF1257-358 complex. At first, we confirmed that the N-terminal Zn finger of GATA1 has a strong preference for the “GATC” motif, whereas the C-terminal Zn finger of GATA1 has a strong preference for the “GATA” motif. Next, we found that in the mGATA1NC, both DNA binding domains can bind simultaneously a wide range of different positional combinations of the co-occurring “GATA” and “GATC” motifs, on the same DNA sequence. The wt-mGATA1 did not show the ability to bind in the same co-occurring motifs implying an effect of the non-DNA binding domains of the protein in the regulation of its DNA binding specificities. On the contrary, complexation of wt-mGATA1 with the wt-mFOG1 partially restored its ability to bind in a now limited range of different positional combinations of the co-occurring “GATA” and “GATC” motifs, on the same DNA sequence. Similar observations were made for other pairs of GATA1 N-terminal and C-terminal Zn finger specific motifs. We then projected the GATA1 DNA binding specificities/affinities in vivo and we classified the GATA1 ChIP-seq peaks in low, medium or high affinity based on the number of the GATA1 motifs. We noticed that high affinity GATA1 ChIP-seq peaks tend to appear in regions with low nucleosome occupancy. We also noticed that GATA1 ChIP-seq peaks in the enhancer regions are usually high affinity whereas GATA1 ChIP-seq peaks in the proximal promoter regions are usually low affinity. Additionally, we observed that high affinity GATA1 ChIP-seq peaks are usually found in regions with increased levels of H3K4me2 and are associated with a higher decrease in the H3K4me3 levels on the TSS of the nearby genes. None of these GATA1 related in vivo observations were found for the KLF1 ChIP-seq positions. These findings significantly advance our understanding of the DNA binding properties of GATA1, KLF1 and their complexes and give an insight on the importance of the GATA1 DNA binding affinities in the regulation of the erythroid transcriptional program. Overall the work establishes an experimental and analytical framework to investigate how transcriptional co-factors can change the DNA binding specificities of specific transcription factors and how integration of the transcription factor DNA binding affinities with in vivo data can give novel insights into the transcriptional regulation.
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De, Moura Miguez Araujo Sofia Jorge. "Interactions and function of nucleotide excision repair protein complexes." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322320.
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Éthève, Loic. "Étude de l’assemblage, de la mécanique et de la dynamique des complexes ADN-protéine impliquant le développement d’un modèle « gros grains »." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSE1242/document.
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Les interactions ADN-protéine sont fondamentales dans de nombreux processus biologiques tels que la régulation des gènes et la réparation de l'ADN. Cette thèse est centrée sur l'analyse des propriétés physiques et dynamiques des interfaces ADN-protéine. À partir de l'étude de quatre complexes ADN-protéine, nous avons montré que l'interface ADN-protéine est dynamique et que les ponts salins et liaisons hydrogène se forment et se rompent dans une échelle de temps de l'ordre de la centaine de picosecondes. L'oscillation des chaînes latérales des résidus est dans certains cas capable de moduler la spécificité d'interaction. Nous avons ensuite développé un modèle de protéine gros grains dans le but de décomposer les interactions ADN-protéine en identifiant les facteurs qui modulent la stabilité et la conformation de l'ADN ainsi que les facteurs responsables de la spécificité de reconnaissance ADN-protéine. Notre modèle est adaptable, allant d'un simple volume mimant une protéine à une représentation plus complexe comportant des charges formelles sur les résidus polaires, ou des chaînes latérales à l'échelle atomique dans le cas de résidus clés ayant des comportements particuliers, tels que les cycles aromatiques qui s'intercalent entre les paires de base de l'acide nucléiqueDNA-protein interactions are fundamental in many biological processes such as gene regulation and DNA repair. This thesis is focused on an analysis of the physical and dynamic properties of DNA-protein interfaces. In a study of four DNA-protein complexes, we have shown that DNA-protein interfaces are dynamic and that the salt bridges and hydrogen bonds break and reform over a time scale of hundreds of picoseconds. In certain cases, this oscillation of protein side chains is able to modulate interaction specificity. We have also developed a coarse-grain model of proteins in order to deconvolute the nature of protein-DNA interactions, identifying factors that modulate the stability and conformation of DNA and factors responsible for the protein-DNA recognition specificity. The design of our model can be changed from a simple volume mimicking the protein to a more complicated representation by the addition of formal charges on polar residues, or by adding atomic-scale side chains in the case of key residues with more precise behaviors, such as aromatic rings that intercalate between DNA base pairs
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Siu, Kit-man Phyllis. "Luminescent cyclometalated platinum(II) complexes : protein binding studies and biological applications /." View the Table of Contents & Abstract, 2005. http://sunzi.lib.hku.hk/hkuto/record/B30575357.
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Siu, Kit-man Phyllis, and 蕭潔敏. "Luminescent cyclometalated platinum(II) complexes: protein binding studies and biological applications." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B4501498X.
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Nassar, Joelle. "Caractérisation de la fonction de OBI1, une E3 ubiquitine ligase, dans la réplication de l'ADN." Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTT039.
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La division cellulaire est l’un des processus cellulaires les plus complexes. Pour que cette division se déroule correctement, la cellule doit répliquer de manière fiable l’intégralité de son génome. Durant ce processus, la réplication de l’ADN est initiée a des sites prédéfinis du génome, appelés « origines de réplication ». Vu qu’un dysfonctionnement de l'activité des origines est lié à plusieurs pathologies humaines, leur activation doit être hautement régulée. Plusieurs protéines ont été trouvées aux origines de la réplication, mais aucune n’explique comment ces origines sont reconnues et sélectionnées pour l’activation. Notre groupe de recherche vise à comprendre comment les origines de réplication sont régulées dans les cellules de métazoaires. Dans ce but, une approche protéomique a été réalisée pour définir l'interactome des origines de réplication humaine, dans l’objectif d'identifier de nouveaux facteurs qui pourraient être impliqués dans la régulation des origines. À l'aide de cette approche, une nouvelle ubiquitine ligase, nommée OBI1 (ORC-ubiquitine-ligase-1), a été identifiée avant mon arrivée au laboratoire. OBI1 se lie au complexe de reconnaissance des origines (complexe ORC) et mon projet vise à mieux caractériser le rôle de cette nouvelle protéine dans la réplication de l'ADN. Notre stratégie expérimentale est basée sur deux modèles différents: un modèle in vivo de cellules humaines en culture et un système de réplication de l'ADN in vitro dérivé d'œufs de Xénope.Nos analyses sur des cellules humaines ont d’abord révélé qu’OBI1 était crucial pour la prolifération cellulaire. Cette observation a été ensuite attribuée à son rôle dans la réplication de l’ADN et plus précisément dans l’activation des origines de réplication. En effet, la déplétion d’OBI1 a montré une diminution de recrutement à la chromatine de facteurs impliqués dans l’activation des origines. De plus, une analyse fonctionnelle a montré qu'OBI1 multiubiquitine ORC3 et ORC5, deux sous-unités du complexe ORC. Cette ubiquitination a été ensuite liée au rôle d’OBI1 dans l’activation des origines de réplication, après que la surexpression des mutants ORC3 / 5 non-ubiquitinables ait donné des résultats similaires à ceux observés lors de la déplétion d’OBI1. Dans l’ensemble, nos résultats ont démontré qu’OBI1 est une protéine essentielle à l’activation des origines et nous ont permis de mettre en place une hypothèse suggérant qu’en ubiquitinant ORC3/5, OBI1 pourrait jouer un rôle dans la sélection des origines destinées à l’activation, parmi toutes les origines définies antérieurement. Après cette étude, maintenant publiée, nous avons voulu aborder le rôle de la multiubiquitination des ORC dans l’activation des origines. Nos expériences préliminaires suggèrent un rôle de l'histone acétyl-transférase (HAT) GCN5 / KAT2A.Dans la deuxième partie de mon projet, nous avons utilisé le système in vitro, basé sur des extraits d'œufs de xénope, pour étudier le rôle de l'OBI1 et de l'ubiquitination dans l'activation des origines de réplication. Nos analyses ont confirmé la conservation d’OBI1 chez Xenopus Laevis et son recrutement a la chromatine lors de la réplication. Nous avons montré que l'ubiquitination se produit sur la chromatine lors de l'activation de l'origine. De plus, en utilisant des inhibiteurs de E1, nous avons constaté que l’ubiquitination est importante pour l’activation des origines. De façon intéressante, la déplétion de OBI1 dans ce système embryonnaire a suggéré un rôle diffèrent d’OBI1 dans l’activation des origines dans le système embryonnaire comparé aux conditions plus somatiques.Finalement, la découverte de ce nouveau facteur d'initiation a fourni des informations essentielles sur le rôle de l'ubiquitination et d’OBI1 dans l'activation et la sélection des origines de réplication. Une telle sélection pourrait également participer à la régulation du « timing » de la réplication de l'ADNCell division is one of the most complex processes a cell undergoes. For this to happen properly, the genetic material stored in a cell must be faithfully copied or replicated. During this process, DNA replication is initiated at pre-defined sites in the genome, called "origins of replication". The activation of these origins is highly regulated, as a dysfunction in origin activity is linked to several human pathologies. Several proteins have been found at replication origins, but none of them explain how to be activated origins are recognized and selected. Our research group aims to understand how DNA replication origins are regulated in metazoan cells, to this aim, a proteomic approach was performed to define the interactome of human replication origins. Our goal was to identify new factors that could be involved in replication origin regulation. Using this methodology, a novel E3 ubiquitin ligase, named OBI1 (for ORC-ubiquitin-ligase-1), was identified prior to my arrival in the laboratory. OBI1 binds the origin recognition complex (ORC complex) and my project aimed at further characterizing the role of this new protein in DNA replication. Our experimental strategy used two different model systems: an in-vivo model based on human cells in culture, and an in-vitro DNA replication system derived from Xenopus eggs.Our analyses in human cells revealed that OBI1 was a crucial gene involved in cellular proliferation, this observation was later attributed to OBI1’s role in DNA replication and more specifically, to replication origin activation. Indeed, OBI1 knockdown resulted in a deficient origin firing and a decrease in the chromatin recruitment of factors involved in origin firing. A further functional analysis showed that OBI1 multiubiquitylates two subunits of the ORC complex, ORC3 and ORC5. This ubiquitylation was directly linked to OBI1’s role in origin firing, after the over-expression of non-ubiquitylable ORC3/5 mutants yielded similar results to OBI1’s knock down. Altogether, our results demonstrated that OBI1 encoded for a protein essential for origin activation, and allowed us to propose its main role: by multiubiquitylating a subset of the ORC complex, OBI1 could select the replication origins to be activated amongst all the potential replication origins set in G1 phase of the cell cycle. After this set of experiments, now published, we wanted to address the mechanistic impact of the multiubiquitylation of ORC on origin activation. Our preliminary experiments suggest a role of the histone acetyl-transferase (HAT) GCN5/KAT2A in the “OBI1 pathway”In the second part of my project, we used the in vitro DNA replication system, based on Xenopus laevis egg extracts, to study the role of OBI1 and ubiquitylation in origin activation. Our in-vitro analyses confirmed the conservation of OBI1 in Xenopus Laevis and its recruitment to the chromatin during DNA replication. We showed that de novo ubiquitylation takes place on chromatin during origin activation. Moreover, using E1 inhibitors, we found that active ubiquitylation is important for efficient origin firing. Interestingly, our loss of function experiments suggested that OBI1’s impact on origin activation could defer in early development when compared to somatic-like conditions.Taken together, the discovery of this new replication initiation factor provided key information on the role of ubiquitylation in general and OBI1 in particular on origin activation and selection. Such selection could participate as well in the regulation of the timing of DNA replication
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Foos, Nicolas. "Etudes structurales d'un complexe HOX-PBC de drosophile. : Un exemple de régulateur transcriptionnel." Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM4075.
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Les protéines Hox sont des protéines à homéodomaine. Elles sont très conservées et impliquées dans l'identité cellulaire selon les axes antéropostérieur, dorsoventral et proximodistal. Elles reconnaissent l'ADN pour réguler l’expression des gènes. La coopérativité avec des partenaires de la famille PBC est un modèle pour l'amélioration de la spécificité. Cette étude utilise Ubx (Hox) et Exd (PBC) de D. melanogaster comme modèles. Deux modes d'interaction entre Ubx et Exd : un par le motif « hexapeptide » d'Ubx et un autre par le motif UbdA objet de ce travail. UbdA se situe en C-terminal de l'hélice de reconnaissance de l'ADN d'Ubx. Nous avons résolu différentes structures de complexes Ubx-Exd-ADN avec deux types de séquences d'ADN. Ces structures montrent que le motif UbdA peut adopter différentes conformations permettant différents rôles : surface d'interaction avec Exd ou charnière entre l'HD et les domaines C-terminaux. En plus des motifs d'Ubx important pour la fonction de régulation, Ubx et Exd comportent des motifs intrinsèquement désordonnés appelés linker et bras N-terminal de l'homéodomaine, respectivement. Ces motifs interagissent avec l'ADN au niveau du sillon mineur et ont la capacité d'explorer l’environnement. Nous avons étudié l'extrémité en C-terminale d'Exd. Ce motif forme une quatrième hélice dont le repliement peut influer sur les contacts établis avec Ubx et avec l'ADN. L'ensemble de ces observations nous a permis d'apporter des éléments supplémentaires pour la compréhension de la spécificité de fonctions et de contribuer à alimenter les modèles de scannage de l'ADN de type monkey bar et d'« interface glissante »Hox proteins are homeodomain proteins belonging to the Transcription Factors superfamilly. These proteins are necessary for the determination of the cellular identity along the anteroposterior, dorsoventral and proximodistal axes. It's essential to recognize DNA targets with high specificity. One possible mechanism to acquire specificity implies the cooperativity between Hox and their PBC partners. Ubx (Hox) and Exd (PBC) proteins from D. melanogaster are the object of this work. One mechanisme of coopertivity involves the “hexapeptide” motif in Ubx and another one that involves its UbdA motif. The UbdA motif is located C-terminal to the recognition helix. We have solved seven different structures of Ubx-Exd-DNA complexes. Thanks to these structures, we show that UbdA can have a multipurpose role : it can provide an interaction surface to contact Exd and it can also act like a hinge between the C-terminal regions of Ubx and its homeodomain. UbdA and HX motifs from Ubx are not the only regions involved in the control of these proteins functions. Ubx and Exd also contain intrinsically disordered regions which are the linker region and the homeodomain N-terminal arm, for Ubx and Exd respectively. They interact with the DNA in the minor groove and can explore the space around. We studied the Exd 's C-terminal motif and determined that it has a flexible, helical fold. The folding of this fourth helix could modify the contacts established with Ubx and with the DNA. All these observations allow us to add supplementary information for the understanding of functional specificity and provide new arguments for the monkey-bar and for the « gliding interface » DNA- scanning models
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ROBISON, JACOB. "INTERACTION OF THE Mre11/Rad50/Nbs1 (MRN) COMPLEX AND REPLICATION PROTEIN A (RPA) IN RESPONSE TO DNA DAMAGE." University of Cincinnati / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1112971385.
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Hogrel, Gaëlle. "Interactions entre composants de la maintenance génomique chez Archaea hyperthermophiles : étude des associations entre PCNA et le complexe Mre11-Rad50 et entre les hélicases MCM et XPD." Thesis, Brest, 2015. http://www.theses.fr/2015BRES0082/document.
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Vivant à des températures supérieures à 80°C, les archées hyperthermophiles ont démontré une capacité étonnante à se remettre de dommages dans leur ADN, suggérant la présence de gardiens du génome particulièrement efficaces. Ces gardiens, des protéines relativement similaires entre archées et eucaryotes, agissent et interagissent dans un ballet savamment orchestré par la cellule. Chez les archées, plusieurs protéines impliquées dans des voies essentielles à la réparation de I'ADN manquent à I'appel. Précédemment au laboratoire un réseau impliquant les protéines de la maintenance génomique de Pyrococcus abyssi a dévoilé de nouvelles interactions protéine-protéine. Décrire l'interaction pour mieux comprendre sa fonction, voici la démarche suivie et présentée dans ce manuscrit pour les duos: PCNA/Mrell-Rad50 et MCM/XPD. Pour la première fois chez Pyrococcus furiostts une interaction physique et fonctionnelle a été démontrée entre PCNA, le maestro de la réplication, et Mrell-Rad50, le complexe de la recombinaison. Pour la seconde étude, la caractérisation de l'hélicase réplicative MCM de P. abyssi a été menée via une approche biophysique basée sur des techniques de fluorescence. Les difficultés rencontrées durant la production de son partenaire potentiel XPD, n'ont toutefois pu permettre la caractérisation de leur interaction. Plus généralement ces interactions s'inscrivent dans un contexte où le couplage de la réplication avec des processus de réparation trouve son importance particulièrement chez les archées de l'extrême, archées qui se révèlent être de passionnants modèles pour l'étude des mécanismes de la maintenance génomiqueLiving at temperatures above 80°C, hyperthermophilic Archaea demonstrated amazing capacity to recover from DNA damages, suggesting they arguably have efficient genome guardians. These guardians, proteins which are relatively similar between Archaea and eukaryotes, act and interact like a ballet orchestrated by the cell. Several proteins involved in essential repair pathway in eukaryotes are missing in Archaea. To gain insights into archaeal genome maintenance processes, a previous work proposed a protein-protein interaction network based on Pyrococcus abyssi proteins. Through this network, new interactions involving proteins from DNA replication and proteins from DNA repair were highlighted. To describe interactions for a better understanding of their functions, was the aim of the work presented here for two protein interactions: PCNA/Mrell-Rad50 and MCM/XPD. For the first time in Pyrococcusfuriosus, we demonstrated both physical and functional interplay between PCNA, the replication maestro, and Mrell-Rad50, a complex involved in recombination process. For the second studied interaction, we used a biophysics approach based on fluorescent technics to characterise helicase activity of P.abyssi MCM. As several problems were encountered for XPD production, we did not characterise the helicase interaction. These two interactions are part of a more general context, where combined DNA replication and DNA repair processes could be important, especially for extremophile Archaea, Archaea which are amazing study models for understanding molecular processes ensuring genome integrity
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Zabolotnaya, Ekaterina. "DNA double-strand break repair studied by atomic force microscopy." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275890.
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DNA double-strand breaks (DSBs), where both strands of the DNA duplex are simultaneously fractured, are considered the most lethal type of DNA damage. The conserved Mre11-Rad50 DNA repair complex enables the catalytic activities of the Mre11 nuclease and the Rad50 ATPase to function together to coordinate the recognition and processing of DSBs prior to the recruitment of long-range end-resection machinery required to trigger the DSB repair by the homologous recombination (HR) pathway. Fast-scan atomic force microscopy (AFM) in fluid conditions was primarily used to explore the architectural arrangement, DNA binding and processing machinery of the Mre11-Rad50 complex from the thermophilic crenarchaeote Sulfolobus acidocaldarius. The structural analysis identified four distinct architectural arrangements and demonstrates the key role of the Rad50 zinc hooks in the oligomerisation of the complex. AFM imaging showed a dynamic and Velcro-like interplay between Mre11-Rad50 protein complexes and the DNA double-helix using the Rad50 coiled-coils in a novel mode of DNA binding. The complex appears to use the Rad50 zinc hook region to bind to and track along dsDNA for broken DNA-terminals. Furthermore, the present study shows that this archaeal complex can drive extensive ATP-dependent unwinding of DNA templates. It is the first time that such unwinding has been observed in a single molecule study. These observations reveal novel activities leading to the proposal of a new model for Mre11-Rad50 action during DSB repair. AFM was also used to visualise the structure and activity of the HerA-NurA protein complex, which has been predicted to combine the activity of the NurA nuclease and hexameric HerA-translocase to generate long single-stranded DNA overhangs essential for DSB repair by HR in archaea. The present data verify and clarify the presumed biological role of this complex. Overall, the present study provides new insights into the initial steps of DNA DSB repair by the HR pathway and, most importantly, the detection of the broken ends.
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Camacho, Inês Sofia Cortes Eusébio. "Effects of UV radiation exposure on DNA and DNA repair enzymes." Master's thesis, Faculdade de Ciências e Tecnologia, 2012. http://hdl.handle.net/10362/8263.
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Dissertação para obtenção do Grau de Mestre em
BiotecnologiaDNA integrity in the cell is under constant threat from damaging agents of endogenous or exogenous origin, such as UV light, ionizing radiation and oxidative stress. Although the effects of these carcinogens on DNA have been extensively studied, very little is known about their effect on DNA repair enzymes. The aim of the present work was the study of the effect of UV radiation on E. coli Endonuclease III, a DNA glycosylase belonging to base excision repair system. This enzyme was homologously overexpressed and then purified with a Fe/protein ratio of 3.88 ± 0.63 (fully‐loaded form). Endonuclease III exposure to UV radiation for 45 min (19.77 kJ dose) may lead to the destruction of the Fe‐S bonds of the [4Fe‐4S] cluster or to the conversion of this center into a different redox state. Electrophoretic mobility shift assays with protein‐DNA complex showed that Endonuclease III binding to plasmid DNA promotes a retardation of the free supercoiled DNA band, indicative of Endonuclease III‐DNA complex(es) formation. These assays also showed that Endonuclease III is able to bind both linear and supercoiled plasmid DNA, although with higher affinity for the linear form. Electrophoretic mobility shift assays performed after 45 min of UV irradiation (19.77 kJ) revealed that although shift occurred, the complexes formed were unstable and dissociated during electrophoresis. Moreover, the presence of aggregates suggests the unfolding of some Endonuclease III molecules. After 6 h of UV irradiation (158.18 kJ) no complexes are formed, leading to the conclusion that Endonuclease III molecules were irreversibly damaged. The electrochemical studies were performed by cyclic and differential pulse voltammetry techniques, at room temperature and anaerobic conditions; Endonuclease III and Endonuclease IIIDNA complex were adsorbed on a bare pyrolytic graphite electrode. For the first time, the direct electrochemical response of Endonuclease III unbound to DNA was observed, with a quasi‐reversible redox couple displaying a midpoint potential of 178 ± 9 mV vs. NHE. Endonuclease III binding to plasmid DNA promotes a positive shift (19 mV vs. NHE) in the characteristic redox couple of Endo III. Protein‐DNA complex UV irradiation promotes a negative shift in its redox potential of 25 mV vs. NHE.
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Schnick, Jasmin. "Structural and functional characterisation of Mcb1 and the MCMᴹᶜᵇ¹ complex in Schizosaccharomyces pombe." Thesis, University of St Andrews, 2014. http://hdl.handle.net/10023/4422.
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The MCM helicase plays an important role in eukaryotic DNA replication, unwinding double stranded DNA ahead of the replication fork. MCM is a hetero-hexamer consisting of the six related proteins, Mcm2-Mcm7. The distantly related MCM-binding protein (MCM-BP) was first identified in a screen for proteins interacting with MCM2-7 in human cells and was found to specifically interact with Mcm3-7 but not Mcm2. It is conserved in most eukaryotes and seems to play an important role in DNA replication but its exact function is not clear yet. This study contributes to the understanding of the fission yeast homologue of MCM-BP, named Mcb1, but also of MCM-BP in general. Results presented in this thesis document the initial biochemical characterisation of the complex Mcb1 forms with Mcm proteins, the MCMᴹᶜᵇ¹ complex. Interactions of Mcb1 with Mcm proteins, potential interaction sites between the proteins and the size of the complex were analysed using a variety of methods, including tandem affinity purification, co-immunoprecipitation, sucrose gradients and in vitro pull-down assays. Sequence analysis and structure prediction were utilised to gain some insight into Mcb1 and MCM-BP ancestry and structure. Results presented here indicate that fission yeast Mcb1 shares homology with Mcm proteins and forms a complex with Mcm3-Mcm7 but not Mcm2 and thus replaces the latter in an alternative high molecular weight complex that is likely to have an MCM-like appearance. Deletion of mcb1⁺ showed that Mcb1 is essential in fission yeast. To examine the cellular function of the protein, temperature-sensitive mutants were generated. Inactivation of Mcb1 leads to an increase in DNA damage and cell cycle arrest in G2-phase depending on the activation of the Chk1 dependent DNA damage checkpoint. Similar observations were made when Mcb1 was overexpressed, indicating that certain levels of the protein are important for accurate DNA replication. Construction of truncated versions of Mcb1 suggested that almost the full-length protein is needed for proper function.
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Ma, Xin. "Ion Mobility Mass Spectrometry of DNA/SgrAI Nuclease Oligomers." Thesis, The University of Arizona, 2012. http://hdl.handle.net/10150/247282.
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SgrAI is a restriction endonuclease (ENase) that cuts a long recognition sequence and exhibits self-modulation of cleavage activity and sequence specificity. Previous research has shown that SgrAI forms large oligomers when bound to particular DNA sequences and under the same conditions where SgrAI exhibits accelerated DNA cleavage kinetics. However, the detailed structure and stoichiometry of SgrAI:DNA as well as the basic building block of the oligomers, has not been fully characterized. Ion mobility mass spectrometry (IM-MS) was employed to analyze SgrAI/DNA complexes and show that the basic building block of the oligomers is the DNA-bound SgrAI dimer (DBD). The oligomers are heterogeneous containing a mixture of species with variable numbers of DBD. The collision cross sections (CCS) of the oligomers were found to have a linear relationship with the number of DBD. Models of the SgrAI/DNA oligomers were constructed and a head-to-tail arrangement was most consistent with the experimental CCS.
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Horwich, Michael D. "Small RNA Sorting in Drosophila Produces Chemically Distinct Functional RNA-Protein Complexes: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/384.
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Small interfering RNAs (siRNAs), microRNAs (miRNAs), and piRNAs (piRNA) are conserved classes of small single-stranded ~21-30 nucleotide (nt) RNA guides that repress eukaryotic gene expression using distinct RNA Induced Silencing Complexes (RISCs). At its core, RISC is composed of a single-stranded small RNA guide bound to a member of the Argonaute protein family, which together bind and repress complementary target RNA. miRNAs target protein coding mRNAs—a function essential for normal development and broadly involved in pathways of human disease; small interfering RNAs (siRNA) defend against viruses, but can also be engineered to direct experimental or therapeutic gene silencing; piwi associated RNAs (piRNAs) protect germline genomes from expansion of parasitic nucleic acids such as transposons. Using the fruit fly, Drosophila melanogaster, as a model organism we seek to understand how small silencing RNAs are made and how they function.
In Drosophila, miRNAs and siRNAs are proposed to have parallel, but separate biogenesis and effector machinery. miRNA duplexes are excised from imperfectly paired hairpin precursors by Dicer1 and loaded into Ago1; siRNA duplexes are hewn from perfectly paired long dsRNA by Dicer2 and loaded into Ago2. Contrary to this model we found one miRNA, miR-277, is made by Dicer1, but partitions between Ago1 and Ago2 RISCs. These two RISCs are functionally distinct—Ago2 could silence a perfectly paired target, but not a centrally bulged target; Ago1 could silence a bulged target, but not a perfect target. This was surprising since both Ago1 and Ago2 have endonucleolytic cleavage activity necessary for perfect target cleavage in vitro. Our detailed kinetic studies suggested why—Ago2 is a robust multiple turnover enzyme, but Ago1 is not. Along with a complementary in vitro study our data supports a duplex sorting mechanism in which Diced duplexes are released, and rebind to Ago1 or Ago2 loading machinery, regardless of which Dicer produced them. This allows structural information embedded in small RNA duplexes to direct small RNA loading into Ago1 and/or Ago2, resulting in distinct regulatory outputs.
Small RNA sorting also has chemical consequences for the small RNA guide. Although siRNAs were presumed to have the signature 2′, 3′ hydroxyl ends left by Dicer, we found that small RNAs loaded into Ago2 or Piwi proteins, but not Ago1, are modified at their 3´ ends by the RNA 2´-O-methyltransferase DmHen1. In plants Hen1 modifies the 3´ ends all small RNAs duplexs, protecting and stabilizing them. Implying a similar function in flies, piRNAs are smaller, less abundant, and their function is perturbed in hen1 mutants. But unlike plants, small RNAs are modified as single-strands in RISC rather than as duplexes. This nicely explains why the dsRNA binding domain in plant Hen1 was discarded in animals, and why both dsRNA derived siRNAs and ssRNA derived piRNAs are modified. The recent discovery that both piRNAs and siRNAs target transposons links terminal modification and transposon silencing, suggesting that it is specialized for this purpose.
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Sadji-Ouatas, Zahia. "Mécanismes de signalisation de l'analogue de la somatostatine, octréotide : rôle dans la régulation de l'activité du complexe Ku86/DNA-PKcs, de la prolifération cellulaire et des modifications post-traductionnelles de la protéine p53." Paris 7, 2001. http://www.theses.fr/2001PA077243.
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Bailly, Rémy. "Application de la dynamique moléculaire à plusieurs échelles au complexe hélicase : pontine/reptine." Thesis, Bordeaux, 2016. http://www.theses.fr/2016BORD0415/document.
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Pontine et Reptine constituent de nouvelles cibles thérapeutiques encore très méconnues à ce jour. Outre leur activité ATPase, les complexes multimériques de Pontine et Reptine ont été décrits comme des hélicases capables d’ouvrir les acides nucléiques. La modélisation moléculaire constitue un outil puissant pour l’étude des systèmes protéiques et c’est pourquoi une approche par docking et dynamique a été envisagée. Au vue de la taille d’un complexe à douze sous-unités, les simulations prenant en compte tous les atomes se sont avérées trop coûteuses en termes de puissance de calcul. Une approche mésoscopique,appelée gros-grains, a donc été utilisée pour réduire le nombre de particules à traiter. Legain de temps de calcul offert par ce modèle nous a permis d’étudier les complexes de Pontine et Reptine en présence de partenaires de type ligands, l’ATP et l’ADP, et de type acide nucléique. Par le biais d’un retour au niveau atomique, une ouverture de la double hélice d’ADN a pu être observée ainsi qu’une orientation préférentielle des brins. Des hypothèses mécanistiques de l'activité hélicase du complexe ont alors pu être formulées sur la base de ces résultatsPontin/Reptin complexes offer new therapeutic opportunities despite the fact they are still notwell known. In addition to their ATPase activity, multimeric complexes of Pontin/Reptin were reported as hélicases able to unwind nucleic acids. Molecular modeling techniques are a powerful tool to study proteins, both a docking and molecular dynamics were applied.Considering the size of a twelve sub-units complex, simulations taking into account all atoms were too expensive in terms of computational costs. A mesoscopic approach, called coarse grain,was used to reduce the number of particles. The calculation time saved with this model allowed the study of Pontin/Reptin complexes in the presence of diverse partners like small ligands (ATP or ADP) and/or nucleic acids. Reverse transformation from coarse-grain to the atomic level led to a DNA double helix opening along to the single strands rearrangement.Several mechanistic hypotheses for the complex helicase activity were formulated from these results