Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06 Archiv

The Mfd (mutation frequency decline) protein is responsible for connecting the cellular processes of transcription and DNA repair in bacteria. Mfd, also termed transcription-repair coupling factor (TRCF), recognizes arrested transcription elongation complexes and catalyzes their dissociation from damaged template DNA in an ATP-dependent manner. Subsequently, Mfd recruits the UvrABC nucleotide excision repair machinery to the damage site. The mechanistic details of this process are not fully understood. X-ray crystallography was used in order to give structural insights into the mechanism of bacterial transcription-coupled repair. During this PhD thesis, the crystal structure of the N terminus (residues 1-333) of Escherichia coli Mfd ("Mfd-N2") was solved. The Mfd N terminus is implicated to function in UvrA-binding. It bears a region with high homology to the nucleotide excision repair protein UvrB. Mfd-N2 is a triangularly shaped molecule of approximately 60×60×30 Å dimensions which contains three structural domains (domains 1A, 1B and 2). Interestingly, the structure of Mfd-N2 very much resembles that of the three N-terminal domains of UvrB. Mfd domain 1A adopts a typical RecA fold. However, it lacks the functional motifs of active ATPases, and we could confirm that the Mfd N-terminus does not possess any ATPase activity. Domain 1B matches the damage-binding domain of the UvrB. Interestingly, Mfd is bereft of the damage-binding motif of UvrB domain 1B, and no DNA binding is associated with this part of Mfd. Domain 2, which possesses the highest sequence homology to UvrB, closely matches the three-dimensional structure of the implicated UvrA-binding domain of UvrB. Highly conserved amino acids between Mfd and UvrB can be found on the surface of domain 2. Using site-directed mutagenesis, several of these residues could be shown to function in the UvrA-Mfd interaction. Remarkably, the corresponding residues in UvrB are required for productive interaction between UvrA and UvrB as well. Taken together, these results suggest that Mfd and UvrB interact with UvrA in a similar manner. Mfd may form an UvrA-recruitment factor at stalled transcription complexes that resembles UvrB architecturally but not catalytically. The molecular similarity between Mfd and UvrB indicates an evolutionary connection between global genome and transcription-coupled nucleotide excision repair in bacteria.

Im Rahmen dieser Dissertation wurden neue Eisen- und Nickel-katalysierte Aryl-Aryl- Kreuzkupplungen mit funktionalisierten Grignard-Verbindungen untersucht. Des Weiteren wurde eine Kupfer- katalysierte Aryl-Benzyl-, sowie eine Nickel-katalysierte Benzyl-Aryl-Kreuzkupplung zur Darstellung von Diarylmethanen entwickelt. Außerdem wurde eine Methode zur Darstellung von Alkyl-Aryl-Aminen ausgearbeitet.

This work has been focused on the use of copper(I)-mediated anti-SN2’ allylic substitution reactions with non directing leaving groups. This proved to be a general and highly reliable method for the preparation of new chiral stereogenic centers in high enantioselectivity. Moreover a particular effort was made to apply this method to more complex systems, and compounds like alcohols, amines, alkenylsilanes or unsaturated nitriles could be successfully prepared in their enantiomerically enriched form.

RNA polymerase II (RNAP II) transiently associates with many different proteins and multiprotein complexes during the mRNA transcription cycle, which includes three phases, initiation, elongation, and termination. This thesis describes structural studies of two factors that facilitate transcription through chromatin. The heterodimeric Saccharomyces cerevisiae elongation factor Spt4-Spt5 (human DSIF) has been identified by biochemical and genetic approaches to help RNAP II transcribe through chromatin. It is assumed that Spt4-Spt5 pauses RNAP II to open a time window for capping enzyme recruitment and addition of a cap to the 5'-end of the nascent RNA. The preparation of milligram quantities of soluble Spt4-Spt5 variants that are suited for structural studies has been achieved. Several strategies to resolve the structure of the RNAP II–Spt4-Spt5 complex were unsuccessful, possibly indicating an intrinsic flexibility of the complex. In addition, there is now evidence for direct links between chromatin modification and transcription elongation. A major player in this process is the histone lysine methyltransferase Set2 which has a modular structure. The catalytic activity of Set2 is mediated by the SET [Su(var)3-9, Enhancer of Zeste, Trithorax] domain. During mRNA elongation, the SRI (Set2 Rpb1-interacting) domain of Set2 binds to the phosphorylated CTD (carboxyl-terminal domain) of RNAP II. The NMR solution structure of yeast Set2 SRI domain has been determined. The structure reveals a novel CTD-binding fold consisting of a left-handed three-helix bundle. Unexpectedly, the SRI domain fold resembles the structure of an RNA polymerase-interacting domain in sigma factors that mediate transcription initiation in bacteria (domain sigma2 in sigma70). NMR titration experiments show that the SRI domain binds a Ser2/Ser5-phosphorylated CTD peptide comprising two heptapeptide repeats and three flanking NH2-terminal residues. Amino acid residues that show strong chemical shift perturbations upon CTD binding cluster in two regions on the SRI surface. The results will enable a detailed analysis of the specific CTD interactions underlying the coupling of transcription and chromatin modification by Set2.

This work focused on the copper(I)-mediated stereoselective anti-allylic substitution reactions using organozinc reagents and chiral cyclic phosphates or pentafluorobenzoates. The applications of this method to the preparation of natural products were also reported.

Die vorliegende Arbeit befasst sich mit der Identifikation und Lokalisation eines ATP-abhängigen Kaliumkanals (KATP) in isolierten humanen Granulosazellen und im humanen Ovar. In RT-PCR- und Western Blot-Experimenten zeigte sich, dass dieser in humanen GZ von IVF-Patientinnen ohne und mit PCOS aus den Untereinheiten Kir 6.1 und SUR 2B besteht. Die Proteine der KATP-Untereinheiten in kultivierten humanen GZ ließen sich in immunzytochemischen Färbungen mit Antikörpern, die gegen die Kir 6.1- und die SUR 2B-Untereinheit gerichtet waren, nachweisen. Immungoldfärbungen mit anschließender elektronenmikroskopischer Betrachtung zeigten, dass sich der KATP in der Plasmamembran humaner GZ befindet, nicht jedoch in Mitochondrien. Dies ergab sich auch in funktionellen Untersuchungen, in denen Diazoxid und Glibenclamid keine Wirkung auf das mitochondriale Membranpotential hatten. Der KATP, der aus den Untereinheiten Kir 6.1 und SUR 2B zusammengesetzt ist, entspricht dem Subtyp des KATP-Kanals, der typischerweise im glatten Muskel in Gefäßen vorkommt. Die elektrophysiologischen Eigenschaften des KATP wurden durch Einzelkanalmessungen untersucht und ergaben, dass der beschriebene Kanal alle charakteristischen Merkmale eines KATP aufweist, nämlich eine Leitfähigkeit von etwa 44 pS, Einwärtsgleichrichtung und eine Regulation durch Nukleotide. Neben der Charakterisierung des KATP auf Einzelkanalebene wurde auch das Ansprechen des Kanals auf KATP-Öffner und -Inhibitoren in Whole-Cell-Messungen und durch Fluoreszenzmikroskopie untersucht. Der KATP spricht auf den KATP-Inhibitor Glibenclamid an, was zu einer Depolarisation der Plasmamembran führt. Somit ist der KATP der erste Kanaltyp in humanen GZ, für den eine Rolle bei der Regulation des Membranpotentials gezeigt werden konnte. Es ergaben sich Hinweise darauf, dass der KATP-Inhibitor Glibenclamid Auswirkungen auf die Expression des für die Östradiolsynthese wichtigen Enzyms Aromatase hat. Des Weiteren führte das Schließen des KATP durch Glibenclamid zu einer Senkung der hCG-induzierten Progesteronproduktion, der Hauptaufgabe der Granulosalutealzellen. Zur Klärung des zugrundeliegenden Mechanismus wurde der Einfluss des KATP auf verschiedene Syntheseschritte der Progesteronproduktion und die daran beteiligten Enzyme untersucht. Es konnte jedoch weder für das Enzym StAR noch für die Enzyme SCC und 3β-HSD eine Veränderung der Expression durch die Behandlung humaner GZ mit den KATP-Modulatoren Diazoxid und Glibenclamid nachgewiesen werden. Bei der Betrachtung von Ultradünnschnitten im Elektronenmikroskop zeigten sich in humanen GZ, die mit hCG und Glibenclamid behandelt worden waren, weniger Lipidtröpfchen als nach der Behandlung mit hCG und DMSO. Zudem traten unter hCG und Glibenclamid vermehrt Mitosen und Apoptose auf, aber es bleibt unklar, wie sich dies auf die Progesteronproduktion auswirkt. Diese Effekte von Glibenclamid auf den KATP bzw. das Membranpotential einerseits und auf die hCG-induzierte Progesteronproduktion in humanen GZ andererseits legt einen Zusammenhang zwischen der KATP-Aktivität und der Steroidhormonsynthese in humanen GZ nahe. Der Mechanismus dieses Effekts bleibt aber noch zu klären. Da der KATP in humanen GZ vorkommt und einen potentiellen Angriffspunkt für pharmakologische Wirkstoffe darstellt, sollte er als möglicher Auslöser eventuell noch nicht berücksichtigter Wirkungen im Ovar weiter untersucht werden.

In this study a structure–function analysis has been employed to analyze transcriptional regulation through the Mediator subunit MED25. A relationship could be established between predicted structural domains and functional characteristics of this protein. Most critically the region responsible for interaction of MED25 with the Mediator was identified. Immunoprecipitation experiments demonstrated that the so–called VWA domain (von–Willebrand A domain, amino acids 1–290) is both sufficient and required for this contact. Site–directed mutagenesis indicates that this binding reaction involves the non–conserved loop SR2, which is protruding from this domain. Based on the results of this analysis a model was proposed, in which the primary contact is established by ionic forces and is further stabilized by hydrophobic interactions. The previously identified ACID domain was reported to bind to VP16. Targeted mutagenesis of four different motifs in this region impaired not only transcriptional activation through MED25 but also led to reduced binding to VP16. In particluar a lysine–rich motif is also present in two domains of PTOV1, a close homolog of MED25. Noteworthy, K518 is not conserved in the PTOV1_B domain, which in contrast to PTOV1_A and the ACID domain of MED25 does not bind to VP16. This led to the hypothesis that K518 is critically involved in the binding of VP16 to MED25. Furthermore it could be demonstrated that MED25 contains an intrinsic transcriptional activation capacity, which is localized in the region 290–715. This indicates additional recruitment of other factors to promoters through this region. Together with the Mediator binding VWA–domain and the VP16–interaction domain this region might facilitate transcriptional activation. A genome–wide screen showed downregulation of c–Jun and FosB following overexpression of MED25. Interestingly, expression of GSK3β, a downstream target of which is cyclin D1, seems to be stimulated by MED25. Together with the finding that overexpression of MED25 leads to activation of a p21 reporter, this raises the possibility that MED25 is involved in cell cycle control. An overlap has been discovered by comparison of MED25 target genes and genes identified previously as target for the viral activator EBNA2. The close homology between the activation domains of EBNA2 and VP16 implies a common mechanism of transcriptional activation by these two viral proteins through MED25. The involvement of MED25 in gene activation by viral activators might indicate a role for this Mediator subunit in viral transcription.

In this thesis novel carbohydrate transition metal complexes are presented. As transition metals rhenium(V), copper(II) and palladium(II) are used, and their complexation behaviour towards carbohydrates is investigated. Various carbohydrates, such as tartaric acid, xylaric acid, polyols, hydroxyaldehydes, glyceraldehyde, erythrulose, erythrose, threose and xylose act as ligands in these coordination compounds. The complexes are synthesised and characterised by NMR spectroscopy and single crystal X-ray diffraction. Heteroleptic complexes of copper(II) with xylaric acid and tartaric acid are synthesised and characterised. Different ancillary ligands such as bpma and tpa are used. In these structures the coordination number of copper(II) varies from 4 to 6, and different geometries depending on the ancillary ligand are observed. At last the reactivity of palladium(II) towards highly reducing sugars and aldehydes such as D-erythrose, D-threose, D-erythrulose, D-glyceraldehyde, glyoxal and glycolaldehyde is investigated. Complexes of the hydrated aldehydes can be observed in solution, with the hydrate coordinating up to two metal centers. For the first time complexes of monosaccharides in their open chain form can be identified and characterised in solution. These forms are stabilised and enriched in solution by palladium(II). The crystal structure of a palladium(II)-erythrose complex is presented, which is the first crystal structure of this tetrose as yet.

The preparation of polyfunctinalized arynes has been achieved by the controlled elimination of readily generated 2-magnesiated aryl arylsulfonates obtained by a low temperature halogen/Mg-exchange starting from the corresponding iodides. New types of Grignard reagents via the addition of magnesium aryl thiolates, amides and selenides to arynes and heteroarynes have been prepared. An easy access to highly functionalized arylmagnesium reagents prepared by direct magnesiation of carbocyclic aromatic rings with TMPMgClLiCl was developed.