No description available.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p.156-163

Thesis (Ph. D.)--State University of New York at Binghamton, Department of Biological Sciences, 2009. / Includes bibliographical references (leaves 128-145).

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 165-185). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Chez la levure à bourgeon, l’établissement de domaines silencieux pour la transcription nécessite le recrutement du complexe SIR (Silencing Information Regulator).Mon travail de thèse s’est attaché à étudier une nouvelle voie d’établissement de la répression transcriptionnelle par les SIRs. Des travaux récents ont montré que la répétition en tandem de protéines fortement liées à l’ADN favorise la mise en silence d’un gène rapporteur voisin (Dubarry et al. 2011).En combinant des approches génétiques et moléculaires, j’ai pu montrer qu’un locus composé de 120 répétitions du site opérateur de l'opéron lactose (lacO) liées par la protéine LacI génère un stress chromatinien local et représente une source d'instabilité génomique. Cette instabilité étant limitée par la recombinaison homologue.Dans la seconde partie de ma thèse, j'ai étudié la dynamique d’établissement de la répression par les complexes lacO/LacI et montré que la répression transcriptionnelle et le recrutement du complexe SIR s'établissent sur plusieurs cycles cellulaires. En outre, mes résultats montrent que le complexe SIR stabilise les nucléosomes au niveau des complexes ADN / protéines de forte affinité.Enfin, deux cribles génétiques m’ont permis d’identifier les complexes HIR et LSM comme des facteurs impliqués dans l’hétérochromatinisation induite par les répétitions lacO/LacI. Les connaissances actuelles de ces complexes étant restreintes à la régulation de la transcription et au post-traitement des ARN messagers, d'autres études seront nécessaires pour disséquer le lien entre ces complexes et l'inhibition transcriptionnelle déclenchée par les complexes lacO /lacI. / Silent domains in budding yeast are formed by the recruitment and spreading of the Silent Information Regulator (SIR) complex.Previous studies showed that an array of tight protein-DNA complexes has the ability to trigger SIR dependent silencing of an ectopically placed EADE2I reporter (Dubarry et al. 2011). It was proposed that replication stress arising due to difficulties to replicate the array of tight protein-DNA complexes is the source of this phenomenon. In my work I have demonstrated that an array of 120 lacO repeats tightly bound by a LacI protein is a source of genomic instability. Investigating the genetic requirements for this event, I have demonstrated that homologous recombination pathways maintain the stability of the locus. My work is consistent with previous reports in fission yeast demonstrating that lacO/LacI is a chromatin stress site (Sofueva et al. 2011). As a second part of my PhD project, using an inducible system that I have developed, I followed the dynamics of establishment of silencing of an ectopically placed reporter gene. My results demonstrate that transcriptional silencing in this system takes many cell cycles to be established. Additionally, I have identified a novel role of the SIR proteins in stabilizing nucleosomes. In an attempt to elucidate the functional link between lacO/LacI and EADE2I silencing, I have performed two SGA (synthetic genetic array) screens. I have identified the HIR and LSM complexes involved into transcriptional regulation and mRNA processing respectively, as potential candidates. Further studies will elucidate the role of these factors on lacO/LacI induced silencing.

The thesis deals with physico-chemical properties of the MADS box, binding domain of transcription factors, which are important for the formation of complexes with the DNA regulatory segment bearing the CArG box. The study was performed also on model oligopeptides, selected segments of the MADS box and their analogues with a point mutation. A wide range of spectroscopic techniques was employed, namely absorption, circular dichroism, fluorescence and Raman spectroscopies. Advanced approaches including multivariate methods were used for data processing. The three tyrosines of the MADS box located in amino-acid vicinities of different charge and hydrophobicity, were used as intrinsic spectroscopic probes. The obtained characteristics of the MADS box and its segments structural arrangement, flexibility and acid-base equilibria are the main results of the work.

The specific recognition of the DNA is crucial for the correct functioning of the cell. Although its mechanisms are extensively studied, the actual process is not yet fully understood, partly due to the variance observed in readout mechanisms so far. In this work, a particular type of specific recognition is examined: the shape readout in the DNA minor groove. Based on a sta- tistical analysis of three-dimensional structures of protein-DNA complexes acquired from the Protein Data Bank, I propose a previously unrecorded readout mechanism of widened minor grooves by hydrophobic amino acids. In addition, the effect of DNA sequence on the topography of the contacted locus, the preferred secondary structures and the interaction between the protein and DNA are explored, as well as the relative information amount of examined features concerning the DNA deformation. 1

Transcription factors play crucial regulatory role within the cell and the entire multicellular organism. The important factor is its ability to interact with other regulatory proteins and DNA. Despite the fact that a large part of the interaction network is already documented, detailed information on the structure and dynamics of protein-protein and protein-DNA complexes is still scarce. In this thesis we focused on the possibility of studying conformational changes given by the transcription factor-DNA complex formation using the methods of structural mass spectrometry: hydrogen/deuterium exchange and chemical crosslinking. As a model, we chose a transcription factor FOXO4 which DNA binding domain is structurally well characterized both in free form and in the complex with DNA.

Solid state approaches to structural properties like diffraction or microscopy techniques often cannot be applied to biomolecular systems, at least not without special postpreparation which often corrupts the desired properties of the pristine systems. In this work the capabilities of synchrotron-based, soft X-ray spectroscopies as an alternative way to unravel structural properties of such systems are tested. To this end, three exemplary systems were investigated each with the focus on another facet and characteristic length scale. The first example are DNA-alkanethiol self-assembled monolayers, also known as DNA microarrays or DNA chips, for which a way to monitor and controllably tune the structural composition on the mesoscopic scale of many thousands of molecules was sought for. The second example focuses on the single-molecule and submolecular scale in metalprotein hybrid compounds with the aim to identify the binding site of metal atoms or ions within protein molecules and the underlying interaction mechanisms. The most fundamental structural scale, the level of single bonds and molecular orbitals, is addressed in the last example where it was tried to elaborate an approach to map the topology of molecular orbitals based upon X-ray absorption properties. This approach was put to the practical test for the characteristic pi*peptide orbitals in protein backbones. For all three investigated examples, spectroscopies using soft X-ray synchrotron radiation were able to extract the desired information, thus confirming that they may grant alternative access to structural properties of soft-matter systems in cases where standard approaches fail. / Klassische Festkörpertechniken zur Strukturuntersuchung, wie Streu- oder Mikroskopiemethoden, können häufig nicht auf Biomolekülsysteme angewandt werden, zumindest nicht ohne spezielle Postpräparation, die die ursprünglichen Eigenschaften dieser Systeme oft verfälscht. In dieser Arbeit soll untersucht werden, inwieweit Röntgenspektroskopien basierend auf Synchrotronstrahlung einen alternativen Zugang zu Struktureigenschaften solcher Systeme bieten. Dazu wurden drei Systeme exemplarisch untersucht, jeweils mit Schwerpunkt auf einen anderen Aspekt und charakteristischen Längenbereich. Für selbstorganisierende DNA-Alkanthiol-Schichten, sogenannte DNA-Chips, wurde nach eine Weg gesucht, ihre strukturelle Zusammensetzung auf der mesoskopischen Ebene vieler tausend Moleküle zu bestimmen und kontrolliert zu modifizieren. Metallisierte Proteinstrukturen wurden auf Einzelmolekül- bzw. submolekularer Ebene untersucht, mit dem Ziel, die Orte der Metallanlagerung innerhalb des Proteins und die zugrundeliegenden Wechselwirkungsmechanismen zu identifizieren. Die unterste strukturelle Ebene, der Bereich einzelne Bindungen und Molekülorbitale, wurde adressiert am Beispiel der pi*peptide Orbitale des Proteinrückrats. Dafür wurde eine Methode zur Kartographierung einzelner Orbitale anhand von Röntgenabsorptionseigentschaften herausgearbeitet und praktisch getestet. In allen drei Fällen konnten Röntgenspektroskopien die nötigen Informationen liefern und damit ihr Potential für Strukturuntersuchungen in weicher Materie unter Beweis stellen.

Restriction modification (RM) systems play a crucial role in preventing the entry of foreign DNA into the bacterial cell. The best studied Type I RM system is EcoKI from Escherichia coli K12. Both bacteriophage and conjugative plasmids have developed a variety of strategies to circumvent the host RM system. One such strategy involves the production of antirestriction proteins that mimic a short segment of DNA and efficiently inhibit the RM system. The main aim of this project was to analyse the interaction of EcoKI and its cognate methylase (MTase) with the T7 antirestriction protein, known as overcome classical restriction (Ocr), and various ArdA antirestriction proteins. Currently, there is a paucity of structural data on the complex formed between the Type I system and the antirestriction proteins. The aim of this work was twofold; (i) compare the interaction of MTase with DNA and Ocr and (ii) quantify the strength of interaction between MTase and various ArdA proteins. The MTase was fused to the Green Fluorescent Protein (GFP) to facilitate determination of the orientation of interaction with DNA and Ocr. Time resolved fluorescence measurements were carried out using the GFP-MTase fusion to determine the fluorescence lifetime and anisotropy decay. These experiments were conducted using a time resolved fluorescence instrument fabricated in-house. The values determined in these experiments were then used to perform fluorescence resonance energy transfer (FRET) measurements with fluorescently labelled DNA or Ocr. These measurements gave information concerning the relative orientation of the MTase with either DNA or Ocr. The GFP-MTase fusion was also used to quantify the strength of interaction with various ArdA proteins. Previous attempts to determine the strength of interaction between MTase and ArdA proteins by employing conventional techniques have been unsuccessful. Therefore, a novel method was developed that exploits the interaction of MTase with a cation exchange medium, which can subsequently be displaced upon binding to ArdA. This method facilitated the determination, for the first time, of a set of binding affinities for the MTase and ArdA interaction.