No description available.

No description available.

No description available.

No description available.

In patients with lung cancer whose tumors harbor activating mutations in the EGF receptor (EGFR), increased responses to platinum-based chemotherapies are seen compared with wild-type cancers. However, the mechanisms underlying this association have remained elusive. Here, we describe a cellular phenotype of crosslinker sensitivity in a subset of EGFR-mutant lung cancer cell lines that is reminiscent of the defects seen in cells impaired in the Fanconi anemia pathway, including a pronounced G2–M cell-cycle arrest and chromosomal radial formation. We identified a defect downstream of FANCD2 at the level of recruitment of FAN1 nuclease and DNA interstrand crosslink (ICL) unhooking. The effect of EGFR mutation was epistatic with FANCD2. Consistent with the known role of FANCD2 in promoting RAD51 foci formation and homologous recombination repair (HRR), EGFR-mutant cells also exhibited an impaired RAD51 foci response to ICLs, but not to DNA double-strand breaks. EGFR kinase inhibition affected RAD51 foci formation neither in EGFR-mutant nor wild-type cells. In contrast, EGFR depletion or overexpression of mutant EGFR in wild-type cells suppressed RAD51 foci, suggesting an EGFR kinase-independent regulation of DNA repair. Interestingly, EGFR-mutant cells treated with the PARP inhibitor olaparib also displayed decreased FAN1 foci induction, coupled with a putative block in a late HRR step. As a result, EGFR-mutant lung cancer cells exhibited olaparib sensitivity in vitro and in vivo. Our findings provide insight into the mechanisms of cisplatin and PARP inhibitor sensitivity of EGFR-mutant cells, yielding potential therapeutic opportunities for further treatment individualization in this genetically defined subset of lung cancer.

The integrity of DNA in living organisms is permanently threatened by many endogenous and exogenous factors. Damages can lead to cell death and serious diseases and have to be repaired by the organism. The focus of this thesis is the investigation of the ability of high (Bst Pol I, Klenow fragment exo-) and low fidelity polymerases (Pol kappa, Pol eta) to insert and bypass several oxidatively-derived purine-lesions, such as 8-oxopurines (8-oxodA, 8-oxodG) and formamidopyrimidines (FaPydA, FaPydG), but also guanine-derived imidazolone (dIz) and its further degradation products oxazolone and guanidinoformimine. In order to support the biochemical data and to reveal the replication mechanism of a high fidelity polymerase with FaPy-lesions we performed co-crystallization studies with Bst Pol I. After the discovery that oxidative modifications of the base methylcytosine (mC) occur naturally within the genome and play a key role during cellular development we sought to address the question of their mutagenic potential.

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Eukaryotic genomes are organized into highly condensed chromatin. This packaging obviously impedes essential DNA mediated processes. ATP-dependent chromatin remodelers are therefore required to establish a dynamic chromatin environment. The chromatin remodeler INO80 is involved in various fundamental nuclear processes such as DNA repair, DNA replication and transcription. INO80 is thought to contribute to these processes by controlling genome wide levels of the histone variant H2A.Z. The INO80 chromatin remodeler is a macro-molecular complex composed of >15 subunits and a molecular mass of ~1.3 MDa. INO80 is found in human, fly and yeast. INO80 contains core subunits, which are conserved across species, as well as species-specific proteins. Not much was known about the organization of the INO80 subunits and their contribution to chromatin remodeling. Therefore, a hybrid approach was applied on yeast INO80 combining chemical cross-linking and mass spectrometry (XL-MS) (in collaboration with Franz Herzog, Ruedi Aebersold’s group, ETH, Zurich), electron microscopy (EM) (in collaboration with Caroline Haas, Roland Beckmann’s group, Gene Center, Munich) and biochemical analysis. For this, firstly the purification of INO80 was established. In order to yield sufficient amounts of highly purified and monodisperse complex, INO80 was purified endogenously from yeast by a combination of affinity and chromatography methods. In addition, nanobodies targeting the INO80 complex were generated that could yield even larger amounts of INO80 in the future. EM analysis revealed that INO80 is an embryo-shaped particle with a dynamic head-neck-body-foot architecture that can undergo large conformational changes. XL-MS unraveled the interaction map of the INO80 complex. The analysis of INO80 deletion mutants verified the observed interactions in vivo and proved the modular architecture of INO80. Additionally, the gained knowledge allowed the design and purification of stable and novel sub-complexes that could improve crystallization behavior. An integration of the results from different techniques deepened our understanding of the molecular architecture of INO80. The enigmatic subunits Rvb1 and 2 assemble as a dodecamer composed of two hetero-hexameric rings within the head of the INO80 complex. Rvb1/2 is flanked by the Swi2/Snf2 ATPase of Ino80 and the actin related protein (Arp) 5 in the neck. The Nhp10-module localizes to the body and the Arp8-module to the foot. Biochemical analysis showed that the Nhp10-module is a high affinity DNA/nucleosome binder. The Nhp10-module might together with the Arp8-module target INO80 to chromatin. The Arp5-module is catalytically crucial for nucleosome remodeling and senses the histone entity in chromatin. In order to map interaction sites to the substrate, INO80-nucleosome complexes were analyzed by XL-MS and were visualized by EM. Two-dimensional class averages showed that the nucleosome bound to the central groove of INO80 and was flanked by the head and foot module. The nucleosome was oriented in respect to INO80 as the H2A/H2B dimer- the moiety to be exchanged- was in contact with subunits situated in the neck. All INO80 modules contribute to nucleosome binding and the observed flexibility proposes a mechanism of how INO80 may remodel its substrate. This study established a structural and functional framework of these large remodelers. The investigation of the interaction with the checkpoint kinase Mec1 will contribute to the understanding of the obscure signaling of INO80.

Current efforts in the field of plasmonics towards device integration and miniaturization require detailed knowledge about the coupling between surface plasmons and emitters. In this work coupling between surface plasmon polaritons and different emitter systems has been investigated by the technique of back focal plane imaging. To develop a deeper understanding of the interaction phenomena the studies focused on single emitters in elementary plasmonic configurations that allow for an analytical description. The first part of the thesis reports on the successful demonstration of surface plasmon polaritons launched by a single dipolar carbon nanotube emitter on a metal thin film after local optical excitation. Leakage radiation microscopy images, recorded in the back focal plane of a microscope objective, could be modeled successfully and contained the propagation length and direction of surface plasmon polaritons. Corresponding real-space images revealed plasmon propagation away from the single dipolar plasmon source. The polarization behavior of surface plasmon polaritons launched by single carbon nanotubes was found to be radial as predicted by theoretical calculations. Remote excitation of single walled carbon nanotube excitons via propagating surface plasmons is demonstrated in the second part. A scanning aperture probe was used as source for propagating surface plasmons with fine controllability over excitation position and propagation direction. It was raster scanned in close proximity over a single carbon nanotube located on a metal film while recording the emission response from the nanotube. The carbon nanotube showed an emission response while the aperture plasmon source was still far away from the nanotube position. Theoretical modeling of the excited surface plasmon fields confirmed that the nanotube maps the surface plasmons locally with sub-diffraction resolution. In the last part, radiation channels in the vicinity of a plasmonic nanowire were investigated. Radiation patterns of a coupled system of rare earth nanocrystals and silver nanowires in the back focal plane revealed that the emission in the vicinity of a nanowire can be approximately described by two emission channels that can be calculated analytically: Dipolar emission, also observed in the absence of the nanowire, and leakage radiation from the nanowire. The latter can be calculated using an antenna-resonator model that considers the air-dielectric interface on which the nanowire is deposited and the position of excitation along the nanowire. Fitting of the experimentally observed patterns provides estimates for the branching ratio between the two emission channels and further enable the determination of the plasmon wave-vector supported by the nanowires.