The DNA contained in every living cell not only stores the genetic information; it functions in a complex molecular network that can condense, transcribe, replicate and repair genes. The essential role played by the sequence dependent structure and deformability of DNA in these basic processes of life, has received increasing attention over the past years. The present work aims at better understanding sequence dependent elasticity of double stranded DNA elasticity, across biologically relevant length scales. A theoretical description is developed that makes is possible to relate structural, biochemical and biophysical experiments and simulation. It is based on the rigid base–pair chain (rbc) model which captures all basic deformation modes on the scale of individual base–pair (bp) steps. Existing microscopic parametrizations of the rbc model rely on indirect methods. A way to relate them to biochemical experiments is provided by the indirect readout mechanism, where DNA elasticity determines protein–DNA complexation affinities. By correlating theoretical affinity predictions with in vitro measurements in a well–studied test case, different parameter sets were evaluated. As a result a new, hybrid parameter set is proposed which greatly reduces prediction errors. Indirect readout occurs mostly at particular binding subsites in a complex. A statistical marker is developed which localizes indirect readout subsites, by detecting elastically optimized sub-sequences. By a systematic coarse–graining of the rbc to the well–characterized worm–like chain (wlc) model, a quantitative connection between microscopic and kbp scale elasticity is established. The general helical rbc geometry is mapped to an effective, linear ‘on-axis’ version, yielding the full set of wlc elastic parameters for any given sequence repeat. In the random sequence case, structural variability adds conformational fluctuations which are correlated by sequence continuity. The sequence disorder correction to entropic elasticity in the rbc model is shown to coincide with the conformational correction. The results show remarkable overall agree- ment of the coarse–grained with the mesoscale wlc parameters, lending support to the model and to the microscopic parameter sets. A continuum version of the rbc is formulated as Brownian motion on the rigid motion group. Analytic expressions for angular correlation functions and moments of the end–to–end distance distribution are given. In an equivalent Lagrangian approach, conserved quantities along, and the linear response around, a general equilibrium shape are explored. / Die in jeder lebenden Zelle enthaltene DNS speichert nicht nur die genetische Information; Sie funktioniert innerhalb eines komplexen molekularen Netzwerks, das in der Lage ist, Gene zu kondensieren, transkribieren, replizieren und reparieren. Die zentrale Rolle, welche der sequenzabhängigen Struktur und Deformierbarkeit von DNS in diesen grundlegenden Lebensprozessen zukommt, erregte in den letzten Jahren zunehmendes Interesse. Die vorliegende Arbeit hat ein besseres Verständnis der sequenzabhängigen elastischen Eigenschaften von DNS auf biologisch relevanten Längenskalen zum Ziel. Es wird eine theoretische Beschreibung entwickelt, die es ermöglicht, strukturbiologische, biochemische und biophysikalische Experimente und Simulationen in Beziehung zu setzen. Diese baut auf dem Modell einer Kette aus starren Basenpaaren (rbc) auf, das alle wichtigen Deformationsmoden von DNS auf der Ebene von einzelnen Basenpaar (bp)–Schritten abbildet. Bestehende Parametersätze des rbc-Modells beruhen auf indirekten Methoden. Eine direkte Beziehung zu biochemischen Experimenten kann mithilfe des indirekten Auslese-Mechanismus hergestellt werden. Hierbei bestimmt die DNS– Elastizität Komplexierungsaffinitäten von Protein–DNS–Komplexen. Durch eine Korrelation von theoretischen Vorhersagen mit in vitro Messungen in einem gut untersuchten Beispielfall werden verschiedene Parametersätze bewertet. Als Resultat wird ein neuer Hybrid–Parametersatz vorgeschlagen, der die Vorhersagefehler stark reduziert. Indirektes Auslesen tritt meistens an speziellen Teilbindungsstellen innerhalb eines Komplexes auf. Es wird eine statistische Kenngröße entwickelt, die indirektes Auslesen durch Detektion elastisch optimierter Subsequenzen erkennt. Durch ein systematisches Coarse–Graining des rbc-Modells auf das gut charakterisierte Modell der wurmartigen Kette (wlc) wird eine quantitative Beziehung zwischen der mikroskopischen und der Elastizität auf einer kbp-Skala hergestellt. Die allgemeine helikale Geometrie wird auf eine effektive, lineare Version der Kette ‘auf der Achse’ abgebildet. Dies führt zur Berechnung des vollen Satzes von wlc-elastischen Parameters für eine beliebig vorgegebene periodische Sequenz. Im Fall zufälliger Sequenz führt die Strukturvariabilität zu zusätzlichen Konformationsfluktuationen, die durch die Kontinuität der Sequenz kurzreichweitig korreliert sind. Es wird gezeigt, daß die Sequenzunordnungs-Korrektur zur entropischen Elastizität im rbc-Modell identisch ist zur Korrektur der Konformationsstatistik. Die Ergebnisse zeigen eine bemerkenswerte Übereinstimmung der hochskalierten mikroskopischen mit den mesoskopischen wlc-Parameter und bestätigen so die Wahl des Modells und seiner mikroskopischen Parametrisierung. Eine Kontinuumsversion des rbc-Modells wird formuliert als Brownsche Bewegung auf der Gruppe der Starrkörpertransformationen. Analytische Ausdrücke für Winkelkorrelationsfunktionen und Momente der Verteilung des End-zu-End–Vektors werden angegeben. In einem äquivalenten Lagrange-Formalismus werden Erhaltungsgrößen entlang von Gleichgewichtskonformationen und die lineare Antwort in ihrer Umgebung untersucht.

Nucleic acids and proteins, some of the building blocks of life, are not static structures but highly dynamic entities that need to interact with one another to meet cellular demands. The work presented in this thesis focuses on the application of highly sensitive fluorescence methods, both at ensemble and single-molecule level, to determine the dynamics and structure of specific biomolecular interactions with nanometer resolution and in temporal scales from nanoseconds to minutes, which includes most biologically relevant processes. The main aims of my PhD can be classified in three areas: i) exploring new fluorescent sensors with increased specificity for certain nucleic acid structures; ii) understanding how some of these nucleic acids sense the presence of small molecules in the cellular environment and trigger gene regulation by altering their structure; and iii) understanding how certain molecular machines, such as helicase proteins, are able to unwind the DNA double helix by using chemical energy in the form of ATP hydrolysis.

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 résultats. / Pontin/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.

No description available.

The DNA contained in every living cell not only stores the genetic information; it functions in a complex molecular network that can condense, transcribe, replicate and repair genes. The essential role played by the sequence dependent structure and deformability of DNA in these basic processes of life, has received increasing attention over the past years. The present work aims at better understanding sequence dependent elasticity of double stranded DNA elasticity, across biologically relevant length scales. A theoretical description is developed that makes is possible to relate structural, biochemical and biophysical experiments and simulation. It is based on the rigid base–pair chain (rbc) model which captures all basic deformation modes on the scale of individual base–pair (bp) steps. Existing microscopic parametrizations of the rbc model rely on indirect methods. A way to relate them to biochemical experiments is provided by the indirect readout mechanism, where DNA elasticity determines protein–DNA complexation affinities. By correlating theoretical affinity predictions with in vitro measurements in a well–studied test case, different parameter sets were evaluated. As a result a new, hybrid parameter set is proposed which greatly reduces prediction errors. Indirect readout occurs mostly at particular binding subsites in a complex. A statistical marker is developed which localizes indirect readout subsites, by detecting elastically optimized sub-sequences. By a systematic coarse–graining of the rbc to the well–characterized worm–like chain (wlc) model, a quantitative connection between microscopic and kbp scale elasticity is established. The general helical rbc geometry is mapped to an effective, linear ‘on-axis’ version, yielding the full set of wlc elastic parameters for any given sequence repeat. In the random sequence case, structural variability adds conformational fluctuations which are correlated by sequence continuity. The sequence disorder correction to entropic elasticity in the rbc model is shown to coincide with the conformational correction. The results show remarkable overall agree- ment of the coarse–grained with the mesoscale wlc parameters, lending support to the model and to the microscopic parameter sets. A continuum version of the rbc is formulated as Brownian motion on the rigid motion group. Analytic expressions for angular correlation functions and moments of the end–to–end distance distribution are given. In an equivalent Lagrangian approach, conserved quantities along, and the linear response around, a general equilibrium shape are explored. / Die in jeder lebenden Zelle enthaltene DNS speichert nicht nur die genetische Information; Sie funktioniert innerhalb eines komplexen molekularen Netzwerks, das in der Lage ist, Gene zu kondensieren, transkribieren, replizieren und reparieren. Die zentrale Rolle, welche der sequenzabhängigen Struktur und Deformierbarkeit von DNS in diesen grundlegenden Lebensprozessen zukommt, erregte in den letzten Jahren zunehmendes Interesse. Die vorliegende Arbeit hat ein besseres Verständnis der sequenzabhängigen elastischen Eigenschaften von DNS auf biologisch relevanten Längenskalen zum Ziel. Es wird eine theoretische Beschreibung entwickelt, die es ermöglicht, strukturbiologische, biochemische und biophysikalische Experimente und Simulationen in Beziehung zu setzen. Diese baut auf dem Modell einer Kette aus starren Basenpaaren (rbc) auf, das alle wichtigen Deformationsmoden von DNS auf der Ebene von einzelnen Basenpaar (bp)–Schritten abbildet. Bestehende Parametersätze des rbc-Modells beruhen auf indirekten Methoden. Eine direkte Beziehung zu biochemischen Experimenten kann mithilfe des indirekten Auslese-Mechanismus hergestellt werden. Hierbei bestimmt die DNS– Elastizität Komplexierungsaffinitäten von Protein–DNS–Komplexen. Durch eine Korrelation von theoretischen Vorhersagen mit in vitro Messungen in einem gut untersuchten Beispielfall werden verschiedene Parametersätze bewertet. Als Resultat wird ein neuer Hybrid–Parametersatz vorgeschlagen, der die Vorhersagefehler stark reduziert. Indirektes Auslesen tritt meistens an speziellen Teilbindungsstellen innerhalb eines Komplexes auf. Es wird eine statistische Kenngröße entwickelt, die indirektes Auslesen durch Detektion elastisch optimierter Subsequenzen erkennt. Durch ein systematisches Coarse–Graining des rbc-Modells auf das gut charakterisierte Modell der wurmartigen Kette (wlc) wird eine quantitative Beziehung zwischen der mikroskopischen und der Elastizität auf einer kbp-Skala hergestellt. Die allgemeine helikale Geometrie wird auf eine effektive, lineare Version der Kette ‘auf der Achse’ abgebildet. Dies führt zur Berechnung des vollen Satzes von wlc-elastischen Parameters für eine beliebig vorgegebene periodische Sequenz. Im Fall zufälliger Sequenz führt die Strukturvariabilität zu zusätzlichen Konformationsfluktuationen, die durch die Kontinuität der Sequenz kurzreichweitig korreliert sind. Es wird gezeigt, daß die Sequenzunordnungs-Korrektur zur entropischen Elastizität im rbc-Modell identisch ist zur Korrektur der Konformationsstatistik. Die Ergebnisse zeigen eine bemerkenswerte Übereinstimmung der hochskalierten mikroskopischen mit den mesoskopischen wlc-Parameter und bestätigen so die Wahl des Modells und seiner mikroskopischen Parametrisierung. Eine Kontinuumsversion des rbc-Modells wird formuliert als Brownsche Bewegung auf der Gruppe der Starrkörpertransformationen. Analytische Ausdrücke für Winkelkorrelationsfunktionen und Momente der Verteilung des End-zu-End–Vektors werden angegeben. In einem äquivalenten Lagrange-Formalismus werden Erhaltungsgrößen entlang von Gleichgewichtskonformationen und die lineare Antwort in ihrer Umgebung untersucht.

Chapter 1: Antarctic fishes are extremely cold adapted. Despite their inability to upregulate heat shock proteins, recent studies have demonstrated a capacity for heat response in these animals. A cDNA microarray study looked at the Notothenioid fish Trematomus bernacchii and revealed heat sensitivities for hundreds of genes, two of which code for members of the CCAAT/Enhancer-binding protein (C/EBP) family of transcription factors. These molecular switches are best known for their roles in apoptosis, inflammation and cell cycle arrest. This dissertation further elucidates the role of C/EBP-delta in the Antarctic fishes T. bernacchii and Pagothenia borchgrevinki. Chapter 2: C/EBP-delta is constitutively expressed in unstressed, field-acclimated (ca. -1.86°C) animals in a highly tissue-specific manner. White muscle tissue contains the highest C/EBP-delta concentration, which is further increased in response to sublethal heat stress at 2.0 or 4.0°C. This response is mostly acute and transitory, but a lesser upregulation was observed in fishes held for one month at 4.0°C. Chapter 3: The heat-induced nuclear translocation of C/EBP-delta--as determined by immunohistochemistry--appears to be time, tissue and species specific with spleen, heart and retinae being particularly responsive in certain situations. Chapter 4: Protein concentrations of proliferating cell nuclear antigen are tissue specific and variably heat responsive. Surprisingly, levels appear to be positively correlated with C/EBP-delta. Chapter 5: Flow cytometry revealed increasingly high temperatures reduce the proportion of G1 cells while increasing the abundance of apoptotic cells. Chapter 6: These findings are discussed in the context of global climate change and the cellular stress response.

Indiana University-Purdue University Indianapolis (IUPUI) / CXXC finger protein 1 (Cfp1), a component of the Set1 histone methyltransferase complex, is a critical epigenetic regulator of both histone and cytosine methylation. Murine embryos lacking Cfp1 are unable to gastrulate and Cfp1-null embryonic stem (ES) cells fail to undergo cellular differentiation in vitro. However, expression of wild type Cfp1 in Cfp1-null ES cells rescues differentiation capacity, suggesting that dynamic epigenetic changes occurring during lineage specification require Cfp1. The domain structure of Cfp1 consists of a DNA binding CXXC domain and an N-terminal plant homeodomain (PHD). PHDs are frequently observed in chromatin remodeling proteins, functioning as reader modules for histone marks. However, the histone binding properties and underlying functional significance of Cfp1 PHD are largely unknown. My research revealed that Cfp1 PHD directly and specifically binds to histone H3K4me1/me2/me3 marks. A point mutation that abolishes binding to methylated H3K4 (W49A) does not affect rescue of cellular differentiation, but, point mutations that abolish both methylated H3K4 (W49A) and DNA (C169A) binding result in defective in vitro differentiation, indicating that PHD and CXXC exhibit redundant functions. The mammalian liver has the unique ability to regenerate following injury. Previous studies indicated that Cfp1 is essential for hematopoiesis in zebrafish and mice. I hypothesized that Cfp1 additionally plays a role in liver development and regeneration. To understand the importance of Cfp1 in liver development and regeneration, I generated a mouse line lacking Cfp1 specifically in the liver (Cfp1fl/fl Alb-Cre+). Around 40% of these mice display a wasting phenotype and die within a year. Livers of these mice have altered global H3K4me3 levels and often exhibit regenerative nodules. Most importantly, livers of these mice display an impaired regenerative response following partial hepatectomy. Collectively, these findings establish Cfp1 as an epigenetic regulator essential for ES cell function and liver homeostasis and regeneration.

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 nucléique / DNA-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

Indiana University-Purdue University Indianapolis (IUPUI) / This dissertation describes structure-function studies of CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, in order to determine the functional significance of Cfp1 protein domains and properties. Cfp1 is an important regulator of chromatin structure and is essential for mammalian development. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1-/-) are viable but demonstrate a variety of defects, including hypersensitivity to DNA damaging agents, reduced plating efficiency and growth, decreased global and gene-specific cytosine methylation, failure to achieve in vitro differentiation, aberrant histone methylation, and subnuclear mis-localization of Setd1A, the catalytic component of a histone H3K4 methyltransferase complex, and tri-methylated histone H3K4 (H3K4me3) with regions of heterochromatin. Expression of wild-type Cfp1 in CXXC1-/- ES cells rescues the observed defects, thereby providing a convenient method to assess structure-function relationships of Cfp1. Cfp1 cDNA expression constructs were stably transfected into CXXC1-/- ES cells to evaluate the ability of various Cfp1 fragments and mutations to rescue the CXXC1-/- ES cell phenotype. These experiments revealed that expression of either the amino half of Cfp1 (amino acids 1-367) or the carboxyl half of Cfp1 (amino acids 361-656) is sufficient to rescue the hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and differentiation defects. These results reveal that Cfp1 contains redundant functional domains for appropriate regulation of cytosine methylation, histone methylation, and in vitro differentiation. Additional studies revealed that a point mutation (C169A) that abolishes DNA-binding activity of Cfp1 ablates the rescue activity of the 1-367 fragment, and a point mutation (C375A) that abolishes the interaction of Cfp1 with the Setd1A and Setd1B histone H3K4 methyltransferase complexes ablates the rescue activity of the 361-656 Cfp1 fragment. In addition, introduction of both point mutations (C169A and C375A) ablates the rescue activity of the full-length Cfp1 protein. These results indicate that retention of either DNA-binding or Setd1 association of Cfp1 is required to rescue hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and in vitro differentiation. In contrast, confocal immunofluorescence analysis revealed that full-length Cfp1 is required to restrict Setd1A and histone H3K4me3 to euchromatic regions.

Indiana University-Purdue University Indianapolis (IUPUI) / Proteins are nano-machines that work inside every living organism. Functional disruption of one or several proteins is the cause for many diseases. However, the functions for most proteins are yet to be annotated because inexpensive sequencing techniques dramatically speed up discovery of new protein sequences (265 million and counting) and experimental examinations of every protein in all its possible functional categories are simply impractical. Thus, it is necessary to develop computational function-prediction tools that complement and guide experimental studies. In this study, we developed a series of predictors for highly accurate prediction of proteins with DNA-binding, RNA-binding and carbohydrate-binding capability. These predictors are a template-based technique that combines sequence and structural information with predicted binding affinity. Both sequence and structure-based approaches were developed. Results indicate the importance of binding affinity prediction for improving sensitivity and precision of function prediction. Application of these methods to the human genome and structure genome targets demonstrated its usefulness in annotating proteins of unknown functions and discovering moon-lighting proteins with DNA,RNA, or carbohydrate binding function. In addition, we also investigated disruption of protein functions by naturally occurring genetic variations due to insertions and deletions (INDELS). We found that protein structures are the most critical features in recognising disease-causing non-frame shifting INDELs. The predictors for function predictions are available at http://sparks-lab.org/spot, and the predictor for classification of non-frame shifting INDELs is available at http://sparks-lab.org/ddig.