Structural Maintenance of Chromosomes (SMC) proteins are vital for a wide range of cellular processes including chromosome structure and dynamics, gene regulation, and DNA repair. Whereas prokaryotic genomes encode for only one SMC protein that exists as a homodimer, eukaryotes possess six different SMC proteins that form three distinct heterodimeric complexes, with the holocomplexes additionally containing several specific regulatory subunits. The prokaryotic SMC complex is required for chromosome condensation and segregation. In eukaryotes, this function is carried out by the condensin complex with SMC2 and SMC4 at its core. The complex containing SMC1 and SMC3, named cohesin, is responsible for sister chromatid cohesion during mitosis and meiosis. Cohesin is also employed in DNA double-strand break repair, whereas condensin participates in single-strand break repair. The as yet unnamed SMC5-SMC6 complex is involved in several DNA repair pathways as well as homologous recombination in meiosis.

SMC proteins consist of N and C-terminal domains that fold back onto each other to create an ATPase “head” domain, connected to a central “hinge” domain via long antiparallel coiled-coils. The hinge domain mediates dimerisation of SMC proteins and binds DNA, but it is not clear to what purpose this activity serves.

The aim of this work was therefore to characterise the structure and function of the SMC hinge domain in more detail. Specifically, the hinge domains of the Pyrococcus furiosus SMC protein and of mouse condensin were studied. Both their high-resolution crystal structures as well as low-resolution solution envelopes were determined, and their DNA-binding activity was analysed qualitatively and quantitatively.

While the SMC hinge domain fold is largely conserved from prokaryotes to eukaryotes, functionally relevant structural differences can be observed. Most importantly, the surface charge has been almost reversed throughout evolution. The data obtained confirm that of all three eukaryotic SMC complexes, condensin is most closely related to prokaryotic SMC proteins. Both the P. furiosus and the mouse condensin hinge domain preferentially bind single-stranded DNA, but the mouse condensin hinge displays a much higher affinity than its prokaryotic counterpart, suggesting that this function has been enhanced during the course of evolution. The single-stranded DNA-binding activity might be important for the function of the condensin complex in single-strand break repair, but probably plays a different role in prokaryotes, possibly in the DNA-loading process of the prokaryotic SMC complex during replication.

Cisplatin ist ein weit verbreitetes Zytostatikum, das seine Wirkung durch die Ausbildung von Quervernetzungen innerhalb eines DNA-Stranges entfaltet. Für das Cisplatin 1,2-d(GpG) Dinukleotid-Addukt konnte bereits sowohl in vitro als auch in Zellen die zentrale Bedeutung von DNA Polymerase η (Pol η) während des TLS-Prozesses nachgewiesen werden. In Zusammenarbeit mit der Gruppe Livneh wurde untersucht, ob der 1,3-d(GpTpG) Cisplatin Schaden ebenfalls in Zellen überlesen werden kann. Erstaunlicherweise sind humane Zellen in der Lage auch diesen komplexen Schaden zu überlesen, allerdings nur sehr ineffektiv. Da die Anzahl an Mutationen in Pol η defizienten Zellen deutlich erhöht war, konnte eine Beteiligung von Pol η am TLS-Prozess des 3’dGs des Schadens nachgewiesen werden. Somit konnte erneut die Initiator-Rolle von Pol η im Multi-Polymerasen Modell beim Überlesen von Cisplatin-Schäden bestätigt werden.

Um mechanistische Details des Pol η vermittelten TLS-Prozesses zu erhalten, wurde die TLS-Fähigkeit von S. cerevisiae Pol η mittels in vitro Primerverlängerungsstudien untersucht. Dazu wurde DNA, die den 1,3-d(GpTpG) Cisplatin Trinukleotid-Schaden enthielt, hergestellt und mittels enzymatischen Verdaus charakterisiert. Mit dieser Methode konnte die Anwesenheit unerwünschter Nebenprodukte ausgeschlossen und die Entstehung des 1,3 Adduktes eindeutig nachgewiesen werden. Im Gegensatz zum 1,2-d(GpG) Addukt konnte Pol η alleine nicht über den 1,3-d(GpTpG) Cisplatin Schaden replizieren. Einzelnukleotid-Insertionsstudien zeigten, dass gegenüber dem 3’dG fehlerfrei ein dC eingebaut wurde, während der nächste Insertionsschritt nur mit erhöhten Enzymkonzentrationen erfolgte und mutagen war.

Der Mechanismus des Replikationsblockes durch den 1,3-d(GpTpG) Cisplatin Schaden wurde mittels Röntgenstrukturanalyse auf atomarer Ebene untersucht. Dazu wurde Cisplatin-geschädigte DNA als Templat in Komplex mit dem katalytischen Fragment von Pol η, einem dATP in der aktiven Tasche und einem Primer mit terminaler 2’,3’-Didesoxyribose im zweiten Schritt der Verlängerung kristallisiert. Die Kristalle beugten die Röntgenstrahlung bis zu einer Auflösung von 2.5 Å. Im Kristall liegt der Komplex in zwei konformationell verschiedenen Formen vor. In beiden Formen (Komplex A und Komplex B) ist eine perfekte Watson-Crick Basenpaarung des 3’dGs (Pt-GTG) mit dem Primer zu erkennen. Dies erklärt die fehlerfreie Replikation des 3’dGs durch Pol η, welche sowohl in Zellen als auch in vitro beobachtet wurde. Das Hauptmerkmal der beschriebenen Struktur ist das zentrale Thymin des Schadens. Es befindet sich nicht zwischen den beiden Guaninen, sondern ist komplett aus dem DNA-Doppelstrang herausgedreht. Folglich ist das zentrale Thymin unfähig, den zweiten Verlängerungsschritt zu dirigieren, der nun gegenüberliegend zum 5’dG (Pt-GTG) erfolgt.

Die Struktur beschreibt, wie der 1,3-d(GpTpG) Cisplatin Schaden eine Verlängerung durch Pol η verhindert. Das zentrale Thymin, das durch die Koordination des Platinatoms aus den Trinukleotidschaden herausgedreht ist, ist direkt vor dem Protein positioniert. Es tritt dadurch in sterische Wechselwirkung mit Methionin 74 und verhindert eine weitere Bewegung der Polymerase entlang des DNA-Strangs.

Zudem ist zu sehen, warum die Inkorporation gegenüber dem 5’dG des Schadens so ineffizient verläuft. Die Rotation der DNA in die aktive Tasche des Enzyms (Komplex A → Komplex B) wird durch die Ausbildung einer Wasserstoffbrückenbindung zwischen dem dATP in der aktiven Tasche und dem 5’dG des Schadens getrieben. Dabei vergrößert sich aber der Abstand zwischen dem α-Phosphat des dATPs und der „gemodelten“ 3’OH-Gruppe des Primers. Der Abstand von ~8.5 Å ist für einen effektiven Nukleotidyltransfer eindeutig zu groß.

Quantum control spectroscopy denotes the combination of optical quantum coherent control with femtosecond spectroscopy.

The molecular response to a photo induced process,

controlled by shaped ultrashort light pulses,

carries information about the system and the induced chemical reaction

not obtainable by unshaped pulses.

In this work quantum control spectroscopy is used to investigate the photochemical process of

beta-carotene during its first few hundred femtoseconds, which are important in the photosynthesis

of light harvesting complexes.

A special class of shaped pulses, called pulse trains, are investigated.

Pulse trains are obtained from Fourier limited pulses, by modulation with a

sinusoidal phase mask $\phi(\omega) = a \sin(b\omega_0+c)$,

leading to a sequence of three or more phase stabilized Gaussian shaped pulses in the time domain.

The intensities of these pulses are defined by a, they are separated by

equal interpulse distances b and have a distinct phase relation which is defined by c.

In this work it will be shown that it is possible

to draw a very unique relation between molecular properties and

the molecular response to the electrical field in dependance of these parameters.

In terms of quantum coherent control, sinusoidal modulated pulse trains

have attracted special attention in the context of mode selectivity.

In a series of experiments it was observed that pulse train excitation can suppress spectral features in the detection signal when the

interpulse distance is adjusted to molecular characteristics like vibrational frequencies.

Furthermore, in many control experiments aiming to steer a chemical reaction,

the use of learning loops for field optimization leads to pulse shapes

that could be reduced to sequences of pulses, comparable to the pulse trains introduced.

Replacement of optimized light fields by appropriate adjusted pulse trains were successful in experiments controlling the energy flow in a light harvesting complex.

Control could be obtained by variation of the phase parameter c, suggesting that the achieved effect was of coherent origin.

The assumption that the carotene units in LH2 were responsible for the successful control,

was the motivation for the presented work of quantum control spectroscopy

of beta-carotene.

Although many efforts have been made to understand

the non-linear effects induced by pulse trains,

the underlying mechanism is not yet clear.

Neither the background of mode selectivity nor the mechanism of chemical reaction control

could be deciphered satisfactorily.

For spectroscopical investigations, however, the knowledge of the

underlying process and its connection to the molecular response is inevitable and are

analyzed in detail.

Starting with a simple model of bound states in a diatomic molecule,

the induced dynamics of the molecular system and the characteristics

of the response field are analyzed.

First phenomenological investigations of the pulse train induced wave packet dynamics

show dependancies between the populations and coherences of the generated molecular state

and the choice of the sinusoidal mask parameters.

Further investigations imply a mechanism connecting the outcome of the control experiment

with the pulse train parameters and the molecular properties which is

confirmed by derivation of a formula based on time dependent perturbation theory.

The proposed mechanism leads to results which are in accordance with

many experimentally observed effects.

It is found that pulse train excitation generates vibrational wave packets that can exhibit symmetric phase space structures.

Comparable structures appear during long time evolution after excitation with Fourier limited pulses

and are known as partial revival states.

Experimentally observed effects, like annihilation of spectral signals, are attributed to temporal interference effects between phase shifted vibrational coherences of these symmetr

In dieser Arbeit werden Ester der Borsäure und von Arylboronsäuren mit Diolen, Methylglycosiden und Glycosen vorgestellt. Die Verbindungen werden durch NMR-Spektroskopie und Einkristallröntgenstrukturanalyse charakterisiert. DFT-Rechnungen werden unterstützend eingesetzt.

MicroRNAs (miRNAs) are a conserved class of small non-coding RNA genes with 19 to 25 nucleotides in length that are found in all higher eukaryotes as well as some DNA viruses. MiRNAs have been elucidated to exert important regulatory functions in many biological and pathological processes, by imperfect base pairing to the 3’ untranslated region (UTR) of target mRNAs, leading to translational repression or mRNA degradation. In the recent years, the rapid development of next generation sequencing (NGS) technologies has tremendously enhanced the identification of novel miRNA genes as well as the profiling of miRNA expression levels. In this study, NGS method has been applied to two different gammaherpesviruses (γ-herpesviruses) associated models: Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC) in human and murine gammaherpesvirus 68 (MHV-68)-infected cell lines from

mouse. As a result, two novel miRNA precursors from EBV and six novel miRNA precursors from MHV-68 have been successfully identified and characterized. In addition, the completion of MHV-68 miRNA set has revealed a unique viral tRNA (vtRNA)-miRNA-miRNA structure in MHV-68 genome. Furthermore, expression levels of the viral miRNAs suggest a distinct pattern during different stages of the viral life

cycle. On the other hand, the profiling of cellular miRNAs also defined a number of miRNAs that have been dysregulated in EBV-positive NPC tissues compared to healthy control tissues, and in MHV-68-infected compared to non-infected NIH 3T3 murine fibroblasts. Among them, miR-15 and miR-16 were upregulated upon EBV or MHV-68 infection. The tumor suppressor gene BRCA1 has been revealed to be the repressed target protein of miR-15/16 in both models, implying an interesting role of miRNAs in the pathogenesis of γ-herpesviruses. Meanwhile, to facilitate the analysis of NGS data, an automated software was designed and developed. Additional information gained during the analysis processes revealed the possible mis-annotations existing in the miRNA registry database, suggesting that the definition and characterization of novel miRNA genes has to be performed with much more caution.