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HOX-Gene sind essentielle Transkriptionsfaktoren (TFs), die den Körperplan, die Struktur und die Organbildung während der Entwicklung bestimmen. Diese komplexen Prozesse werden präzise von in verschachtelter Weise exprimierten HOX-Genen reguliert. In vitro Experimente zeigten jedoch, dass die HOX-DNA-Bindungsdomäne stark konserviert ist und oft ähnliche DNA-Sequenzen bindet. Die niedrige biochemische Bindungsspezifität und die hochspezifischen Funktionen stehen oft im Widerspruch und bilden das Schlussthema des so genannten Hox-Paradoxons. Das Paradox besteht aufgrund der folgenden Hindernisse: hohe Proteinhomologie, unspezifischen Antikörper sowie die verschachtelte HOX-Expressionsmuster. Das Ziel dieser Arbeit war, diese Probleme zu überwinden, die HOX-DNA-Bindung in kontrollierten und physiologischen Umstände zu untersuchen und die Bindung von neun Gliedmaßen-spezifischen posterioren HOXA und -D-TFs zu vergleichen. Zu diesem Zweck wurden neun Hühner-HOX-Gene (HOXA- und HOXD9-13) mit dem FLAG markiert und mittels Viren in Gliedmaßen-mesenchymalen Zellen exprimiert. Somit wurde der Vergleich unter identischen und kontrollierten Bedingungen ermöglicht. Im Einklang mit in vivo Funktionsdaten zeigten die HOX-Bindungsprofile, dass zwei direkte Paraloge (z. B. HOXA10 und D10) häufiger dieselben Regionen binden als zwei Nicht-Paraloge (z. B. HOXA9 und A13). Außerdem, die hier beschriebene HOX-DNA-Bindung unterscheidet sich von in vitro Bindung, was darauf hinweist, dass Kofaktoren für deren biologische Funktion wichtig sind. Zusätzlich ergab sich aus dem Bindungsvergleich, dass es zuvor unbekannte Unterschiede zwischen Bindungsweise von HOX-TFs gibt, die zumindest teilweise auf der Häufigkeit von direkter Bindung und Ko-Bindung mit anderen TFs beruhen. Schließlich wurde mit der Kombination von Genetik, Genomik und Biochemie einen neuen HOX-Kofaktor entdeckt, CTCF, der auf ein mögliches Wechselspiel zwischen der HOX-Zielregulation und der Chromatinarchitektur hindeutet. / HOX genes are essential developmental transcription factors (TFs) that pattern the animal body plan, their structures and organs. To precisely control these very diverse processes HOX genes are expressed in a nested fashion and regulate their targets in a context specific way. However, in vitro experiments indicated that HOX DNA binding domain (Homeodomain) is remarkably rigid and often binds very similar DNA sequences. This discrepancy between high functional specificity and low in vitro biochemical specificity is at the core of a problem termed Hox paradox. This paradox persists due to several biological and technical obstacles; namely high HOX protein homology and lack of sufficiently specific antibodies as well as nested HOX expression pattern. The aim of this study was to address these problems, study HOX-DNA binding in a controlled, Hox-native environment and to compare HOX-DNA binding of nine posterior vertebrate HOXA and HOXD TFs. To do this, nine chicken HOX genes (HOXA9-13 and HOXD9-13) were FLAG-tagged and virally expressed in chicken mesenchymal limb-derived cells enabling comparison of their binding in an identical setup and controlled conditions. HOX binding profiles uncovered two direct paralogues (i.e. HOXA10 and D10) bind more often same regions than two non-paralogues (i.e. HOXA9 and A13) reminiscent of in vivo functional data. Moreover, the here described in vivo HOX-DNA binding differs from in vitro binding, indicating the importance of cofactors and biological context for HOX binding and functional outcome. Additionally, binding comparison uncovered previously unknown differences between binding modes of HOX-TFs that at least partially rely on the abundance of direct binding and co-binding with other TFs. Finally, with combination of genetics, genomics and biochemistry a novel HOX cofactor, CCCTC binding factor (CTCF), was discovered suggesting potential interplay between HOX target regulation and chromatin architecture.

Der DNA-Verpackungsmechanismus des humanen Cytomegalievirus (HCMV) ist charakteristisch für große DNA-Viren wie Herpesviren und ds-Bakteriophagen. Er beruht auf der Spaltung der konkatemeren DNA durch einen viralen, hetero-oligomeren Proteinkomplex, der Terminase. In der vorliegenden Arbeit konnten die funktionellen Domänen der Terminase-Untereinheit pUL89 in vitro identifiziert und charakterisiert werden. Neben einer Nuklease-Aktivität besitzt pUL89 auch die Fähigkeit dsDNA sequenz-unabhängig zu binden. Durch Nuklease-Untersuchungen konnte gezeigt werden, dass pUL89 sowohl dsDNA, als auch lineare DNA spaltet. PUL89 weist dabei eine größere Spezifität zu dsDNA auf. Des Weiteren konnte nachgewiesen werden, dass die Aminosäure D463 eine zentrale Funktion innerhalb der Nuklease-Aktivität besitzt. Durch kolorimetrische DNA-Bindungsuntersuchungen konnte die Aminosäure R544 als essenziell für die dsDNA-Bindungsfähigkeit von pUL89 identifiziert werden. Basierend auf den in vitro Ergebnissen wurden rekombinanten TB40/E-Virusmutanten mit Mutationen im ORF UL89 durch die En Passant Mutagenese generiert. Mit Hilfe dieser Viren sollte der Einfluss der Mutationen auf die Replikation des Virus charakterisiert werden. Es war möglich nachzuweisen, dass die Aminosäuren E534 und R544 eine essenzielle Aufgabe innerhalb von HCMV erfüllen, da die Mutation einer dieser Aminosäure zu nicht wachstums-fähigen BAC-Mutanten führte. Zur Charakterisierung dieser Konstrukte wurden die Zelllinien HELF Fi301-UL89 und HELF Fi301-vProm-UL89 verwendet. Durch Untersuchungen hinsichtlich der Wachstumseigenschaften, Proteinexpression, DNA-Spaltung, DNA-Bindung sowie elektronenmikroskopischen Aufnahmen, konnte gezeigt werden, dass die wachstums-kompetenten BAC-Mutanten keinen signifikanten Unterschied zum Wildtyp-Virus TB40/E zeigten. Sodass nachgewiesen werden konnte, dass die basischen Aminosäuren H565 und H571 keine essenzielle Funktion in pUL89 erfüllen. / The human cytomegalovirus DNA packaging mechanism is characteristic for large DNA viruses like Herpes viruses and ds bacteriophages. This mechanism is based on the cleavage of concatemeric DNA by the viral heterooligomeric protein complex terminase. This dissertation includes the identification and characterization of functional domains of the HCMV terminase subunit pUL89. PUL89 contain a nuclease activity and the ability to bind dsDNA. This protein shows the property to cut as well dsDNA as linear DNA. The amino acid D463 shows a significant role in this cleavage event. Colorimetric DNA binding experiments show the central role of R544 in DNA binding by pUL89. Based of the in vitro results recombinant TB40/E viruses with mutations in the ORF UL89 were generated. These viruses allow a characterization of the impact of virus replication. It was possible to show that the amino acids E534 and R544 have a functional role in HCMV. The mutation of one of these amino acids was enough to generate a growth deficient mutant. The stable cell lines HELF Fi301-UL89 and HELF Fi301-vProm UL89 were used for the characterization of the growth deficient mutants. The growth competent mutants H656A and H571A show no significant differences in comparison with the wild type TB40/E virus. This was verified by growth kinetics, protein expression characterizations, pulse field gel electrophoresis, DNA binding assays and electron microscopy.

Self-assembly processes were used to construct supramolecular architectures based on metal-ligand interactions. The structures formed strongly depend on the used metal ion, the ligand type, the chosen counter ion and solvent as well as on the experimental conditions. The focus of the studies was the design of multifunctional N-donor ligands and the characterization of their complexing and structural properties. This work was divided into three distinct main parts: The bis(2-pyridylimine), the bis(2-hydroxyaryl) imine and the tripodal imine / amine ligand approach. In the first part a series of bis(2-pyridylimine) derivatives having different linking elements were employed as building blocks for novel supramolecular architectures. Reaction of individual d-block metal salts with these ligands has led to the isolation of coordination polymers, a metallamacrocycle, double-stranded helicates, triple-stranded helicates as well as of circular meso-helicates. The nature of the spacer in the Schiff base ligands, the noncovalent weak interactions, such as hydrogen bond, face-to-face π-π and edge-to-face CH-π interactions, are all important factors influencing the architecture of the final products. Topological control of the assembly process of the hexanuclear meso-helicates is clearly associated with the bidentate coordination of the sulfate anion which directs the formation of a double- rather than a triple-stranded helicate around the octahedrally coordinated Cu(II). Surprisingly, the variation of the linker function in the ligands, which significantly changes the linking angle of the pyridylimine strands, has only a little influence of the resulting structure. Also the use of a mixture of ligands does not influence the meso-helicate topology; the result is the symmetrically mixed meso-helicate. The new iron(II) triple helicate [Fe2(L5)3](PF6)4 14 {L5 = bis[4-(2-pyridylmethyleneimino)phenyl]-1,1-cyclohexane} in its chloride form binds strongly to DNA as confirmed by induced circular dichroism signals in both the metal-to-ligand charge transfer (MLCT) and in-ligand bands of the helicate. The induced CD spectrum gives some evidence that [Fe2(L5)3]4+ interacts with the DNA in a single binding mode, which is consistent with major groove binding. The cytotoxicity of the new iron(II) triple helicate 14 was evaluated on human lung cancer A549 cells and compared with that of cisplatin and that of the previously reported iron(II) triple helicate [Fe2(L1)3]4+{L1 = bis[4-(2-pyridylmethyleneimino)phenyl]methane}. The first results show some distinguishing features for 14 obviously caused by the existing structural differences of the complexes. In the second part of the thesis, novel uranyl complexes of the bis(2-hydroxyaryl) imine ligands have been synthesized and characterized. 1D coordination polymers and mononuclear structures were formed. In all complexes a distorted hexagonal bipyramidal coordination geometry around the uranyl centre is observed. The imine nitrogen atoms of the ligands do not bind to the metal centre but interact strongly with the hydroxy group via H-bonding. DFT calculations made with L8 ( α,α’-Bis(salicylimino)-m-xylene) are in good agreement with the X-ray crystal structure data. Liquid-liquid extraction studies involving selected ligands and Eu(III) or U(VI) indicate remarkably high selectivity for U(VI) over Eu(III) at weak acidic pH conditions. We believe that the study made opens up new possibilities for uranyl ion extraction which could be interesting in view of the treatment of nuclear waste. In the third part of the thesis, a series of multifunctional tripodal ligands with different N-donor centres were used for U(VI) and lanthanide, Nd(III), Eu(III) and Yb(III), binding and extraction. Reaction of these metal ions with selected tripodal ligands afforded complexes which were characterized by ESI mass spectroscopy. The complex composition was found to be 1:1 in all cases. The extraction behaviour of the tripodal ligands towards Eu(III) and U(VI) was studied both in the absence and presence of octanoic acid as co-ligand using the extraction system Eu(NO3)3 or UO2(NO3)2–buffer–H2O/ ligand–CHCl3. These separation systems show a remarkably high selectivity for U(VI) over Eu(III). It is interesting to note that the addition of the octanoic acid to the extraction system leads to high synergistic effects. A series of Eu(III) extraction experiments were done to clarify the composition of the extracted complexes. The results clearly point to the formation of various species with changing composition.

Self-assembly processes were used to construct supramolecular architectures based on metal-ligand interactions. The structures formed strongly depend on the used metal ion, the ligand type, the chosen counter ion and solvent as well as on the experimental conditions. The focus of the studies was the design of multifunctional N-donor ligands and the characterization of their complexing and structural properties. This work was divided into three distinct main parts: The bis(2-pyridylimine), the bis(2-hydroxyaryl) imine and the tripodal imine / amine ligand approach. In the first part a series of bis(2-pyridylimine) derivatives having different linking elements were employed as building blocks for novel supramolecular architectures. Reaction of individual d-block metal salts with these ligands has led to the isolation of coordination polymers, a metallamacrocycle, double-stranded helicates, triple-stranded helicates as well as of circular meso-helicates. The nature of the spacer in the Schiff base ligands, the noncovalent weak interactions, such as hydrogen bond, face-to-face π-π and edge-to-face CH-π interactions, are all important factors influencing the architecture of the final products. Topological control of the assembly process of the hexanuclear meso-helicates is clearly associated with the bidentate coordination of the sulfate anion which directs the formation of a double- rather than a triple-stranded helicate around the octahedrally coordinated Cu(II). Surprisingly, the variation of the linker function in the ligands, which significantly changes the linking angle of the pyridylimine strands, has only a little influence of the resulting structure. Also the use of a mixture of ligands does not influence the meso-helicate topology; the result is the symmetrically mixed meso-helicate. The new iron(II) triple helicate [Fe2(L5)3](PF6)4 14 {L5 = bis[4-(2-pyridylmethyleneimino)phenyl]-1,1-cyclohexane} in its chloride form binds strongly to DNA as confirmed by induced circular dichroism signals in both the metal-to-ligand charge transfer (MLCT) and in-ligand bands of the helicate. The induced CD spectrum gives some evidence that [Fe2(L5)3]4+ interacts with the DNA in a single binding mode, which is consistent with major groove binding. The cytotoxicity of the new iron(II) triple helicate 14 was evaluated on human lung cancer A549 cells and compared with that of cisplatin and that of the previously reported iron(II) triple helicate [Fe2(L1)3]4+{L1 = bis[4-(2-pyridylmethyleneimino)phenyl]methane}. The first results show some distinguishing features for 14 obviously caused by the existing structural differences of the complexes. In the second part of the thesis, novel uranyl complexes of the bis(2-hydroxyaryl) imine ligands have been synthesized and characterized. 1D coordination polymers and mononuclear structures were formed. In all complexes a distorted hexagonal bipyramidal coordination geometry around the uranyl centre is observed. The imine nitrogen atoms of the ligands do not bind to the metal centre but interact strongly with the hydroxy group via H-bonding. DFT calculations made with L8 ( α,α’-Bis(salicylimino)-m-xylene) are in good agreement with the X-ray crystal structure data. Liquid-liquid extraction studies involving selected ligands and Eu(III) or U(VI) indicate remarkably high selectivity for U(VI) over Eu(III) at weak acidic pH conditions. We believe that the study made opens up new possibilities for uranyl ion extraction which could be interesting in view of the treatment of nuclear waste. In the third part of the thesis, a series of multifunctional tripodal ligands with different N-donor centres were used for U(VI) and lanthanide, Nd(III), Eu(III) and Yb(III), binding and extraction. Reaction of these metal ions with selected tripodal ligands afforded complexes which were characterized by ESI mass spectroscopy. The complex composition was found to be 1:1 in all cases. The extraction behaviour of the tripodal ligands towards Eu(III) and U(VI) was studied both in the absence and presence of octanoic acid as co-ligand using the extraction system Eu(NO3)3 or UO2(NO3)2–buffer–H2O/ ligand–CHCl3. These separation systems show a remarkably high selectivity for U(VI) over Eu(III). It is interesting to note that the addition of the octanoic acid to the extraction system leads to high synergistic effects. A series of Eu(III) extraction experiments were done to clarify the composition of the extracted complexes. The results clearly point to the formation of various species with changing composition.

Das Immediate-Early Protein 2 (IE2) des humanen Zytomegalievirus ist ein essentieller Regulationsfaktor des lytischen Infektionszyklus. Es aktiviert verschiedene early Promotoren, autoreprimiert seine eigene Expression und besitzt darüber hinaus auch zellzyklusregulatorische Aktivitäten. Um einzelne Funktionen des IE2 Proteins gezielt analysieren zu können, ist eine genaue Kenntnis seiner regulatorischen Domänen unabdingbar. Im Rahmen dieser Arbeit wurde daher eine Struktur-Funktionsanalyse des IE2 Proteins durchgeführt mit dem Ziel, seine funktionellen Domänen genauer zu charakterisieren. Hierfür wurden verschiedene IE2-Mutanten hergestellt und ihre Aktivität im Hinblick auf Transaktivierung, Autorepression und DNA-Bindung sowie Zellzylusarrestinduktion bestimmt. Die Untersuchungen ergaben, dass innerhalb einer Core-Region im C-Terminus des Proteins (AS 450-544) die regulatorischen Domänen der untersuchten Funktionen überlappen und hier schon kleinere Mutationen zu einem Funktionsverlust führen. Im Gegensatz dazu ist der Bereich N-terminal des Core deutlich weniger sensitiv gegenüber Mutationen. Hier konnten Sequenzen identifiziert werden, die spezifisch für einzelne Funktionen wie die Transaktivierung oder die Zellzyklusarrestinduktion erforderlich sind. Darüber hinaus hat sich gezeigt, dass eine im bisherigen Verständnis essentielle putative Zinkfingerdomäne außerhalb des Core liegt und für die Funktionalität des Proteins, vor allem für seine DNA-Bindung, nicht benötigt wird. Somit ist der Bereich, in dem die regulatorischen Domänen der untersuchten Funktionen überlappen, deutlich kleiner, als bisher angenommen. Vor diesem Hintergrund lässt sich eine Strategie für die Erstellung von diskriminierenden Virusmutanten ableiten, bei der Einzelfunktionen von IE2 im Viruskontext eliminiert und somit im Sinne ihrer physiologischen Relevanz analysierbar werden. / The Immediate Early Protein 2 (IE2) of human cytomegalovirus is an essential regulatory factor of the viral replicative cycle. It fulfills several functions including transactivation, negative autoregulation and cell cycle regulation. In order to analyse the physiological significance of each of the IE2 functions a precise knowledge of the regulatory protein domains is needed. Therefore, a structure-function analysis of the IE2 protein was performed in this work. Different sets of IE2 mutants were tested in parallel with regard to transactivation, DNA-binding, autoregulation and cell cycle regulation. We found the IE2 protein to contain an unexpectedly clear-cut core domain (amino acids (aa) 450-544) that is defined by its absolute sensitivity to any kind of mutation. In contrast, the region adjacent to the core (aa 290-449) generally displays greater tolerance towards mutations. Although specific sequences correlate with distinct IE2 activities none of the mutations analysed completely abolished any particular function. The core is separated from the adjacent region by the putative zinc finger (428-452) which was found to be entirely dispensable for any function tested. Our work supports the view that the 100 amino acids of the core domain hold the key to most functions of IE2. A systematic, high-density mutational analysis of this region may identify informative mutants which discriminate between various IE2 functions. Such mutants could then be tested in a viral background.

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