@phdthesis{Wolski2011,
  author    = {Wolski, Stefanie Carola},
  title     = {Structural and functional characterization of nucleotide excision repair proteins},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-67183},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2011},
  abstract  = {XPD is a 5'-3' helicase of the superfamily 2. As part of the transcription factor IIH it functions in transcription initiation and nucleotide excision repair. This work focus on the role of XPD in nucleotide excision repair. NER is a DNA repair pathway unique for its broad substrate range. In placental mammals NER is the only repair mechanism able to remove lesions induced by UV-light. NER can be divided into four different steps that are conserved between pro- and eukaryotes. Step 1 consists of the initial damage recognition, during step 2 the putative damage is verified, in step 3 the verified damage is excised and in the 4th and final step the resulting gap in the DNA is refilled. XPD was shown to be involved in the damage verification step. It was possible to solve the first apo XPD structure by a MAD approach using only the endogenous iron from the iron sulfur cluster. Based on the apo XPD structure several questions arise: where is DNA bound? Where is DNA separated? How is damage verification achieved? What is the role of the FeS cluster? These questions were addressed in this work. Hypothesis driven structure based functional mutagenesis was employed and combined with detailed biochemical characterization of the variants. The variants were analyzed by thermal unfolding studies to exclude the possibility that the overall stability could be affected by the point mutation. DNA binding assays, ATPase assays and helicase assays were performed to delineate amino acid residues important for DNA binding, helicase activity and damage recognition. A structure of XPD containing a four base pair DNA fragment was solved by molecular replacement. This structure displays the polarity of the translocated strand with respect to the helicase framework. Moreover the properties of the FeS cluster were studied by electron paramagnetic resonance to get insights into the role of the FeS cluster. Furthermore XPD from Ferroplasma acidarmanus was investigated since it was shown that it is stalled at CPD containing lesions. The data provide the first detailed insight into the translocation mechanism of a SF2B helicase and reveal how polarity is achieved. This provides a basis for further anlayses understanding the combined action of the helicase and the 4Fe4S cluster to accomplish damage verification within the NER cascade.},
  subject      = {DNS-Reparatur},
  language  = {en}
}
@phdthesis{Roth2011,
  author    = {Roth, Heide Marie},
  title     = {Nucleotide Excision Repair: From Recognition to Incision of damaged DNA},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-57098},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2011},
  abstract  = {The Nucleotide Excision Repair (NER) pathway is able to remove a vast diversity of structurally unrelated DNA lesions and is the only repair mechanism in humans responsible for the excision of UV induced DNA damages. The NER mechanism raises two fundamental questions: 1) How is DNA damage recognition achieved discriminating damaged from non damaged DNA? 2) How is DNA incision regulated preventing endonucleases to cleave DNA non specifically but induce and ensure dual incision of damaged DNA? Thus, the aim of this work was to investigate the mechanisms leading from recognition to incision of damaged DNA. To decipher the underlying process of damage recognition in a prokaryotic model system, the intention of the first part of this work was to co crystallize the helicase UvrB form Bacillus caldotenax together with a DNA substrate comprising a fluorescein adducted thymine as an NER substrate. Incision assays were performed to address the question whether UvrB in complex with the endonuclease UvrC is able to specifically incise damaged DNA employing DNA substrates with unpaired regions at different positions with respect to the DNA lesion. The results presented here indicate that the formation of a specific pre incision complex is independent of the damage sensor UvrA. The preference for 5' bubble substrate suggests that UvrB is able to slide along the DNA favorably in a 5' → 3' direction until it directly encounters a DNA damage on the translocating strand to then recruit the endonuclease UvrC. In the second part of this work, the novel endonuclease Bax1 from Thermoplasma acidophilum was characterized. Due to its close association to archaeal XPB, a potential involvement of Bax1 in archaeal NER has been postulated. Bax1 was shown to be a Mg2+ dependent, structure specific endonuclease incising 3' overhang substrates in the single stranded region close to the ssDNA/dsDNA junction. Site directed mutagenesis of conserved amino acids was employed to identify putative active site residues of Bax1. In complex with the helicase XPB, however, incision activity of Bax1 is altered regarding substrate specificity. The presence of two distinct XPB/Bax1 complexes with different endonuclease activities indicates that XPB regulates Bax1 incision activity providing insights into the physical and functional interactions of XPB and Bax1.},
  subject      = {DNS-Reparatur},
  language  = {en}
}
@phdthesis{Mehringer2021,
  author    = {Mehringer, Christian Felix},
  title     = {Optimierung und Objektivierung der DNA-Biegewinkelmessung zur Untersuchung der initialen Schadenserkennung von Glykosylasen im Rahmen der Basen-Exzisions-Reparatur},
  doi       = {10.25972/OPUS-23084},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-230847},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Im Rahmen dieser Doktorarbeit sollte ankn{\"u}pfend an die Ergebnisse aus vo-rangegangenen Untersuchungen der AG Tessmer, das von B{\"u}chner et al. [1] vorgestellte Modell zur DNA-Schadenserkennung, welches im Speziellen auf Daten zu den Glykosylasen hTDG und hOGG1 basierte, auf seine Allgemein-g{\"u}ltigkeit f{\"u}r DNA-Glykosylasen untersucht werden. Das Modell beschreibt den Prozess der Schadenserkennung als eine notwendige {\"U}bereinstimmung der passiven Biegung am Schadensort mit dem aktiven BiegungswinkeI der scha-densspezifischen Glykosylase. Ein wesentlicher Bestandteil dieser Arbeit war zudem die Etablierung einer automatisierten Messsoftware zur objektiven Biegewinkelmessung an DNA-Str{\"a}ngen in rasterkraftmikroskopischen Aufnah-men. Dies wurde mit verschiedenen Bildverarbeitungsprogrammen sowie einer in MATLAB implementierten Messsoftware erreicht und das Programm zudem auf die Biegewinkelmessung von proteininduzierten Biegewinkeln erweitert. Zur Anwendung kam die Methode der automatisierten Biegewinkelmessung sowohl an rasterkraftmikroskopischen Aufnahmen der Glykosylase MutY gebunden an ungesch{\"a}digter DNA als auch an Aufnahmen von DNA mit und ohne Basen-schaden. Neben oxoG:A und G:A, den spezifischen MutY-Zielsch{\"a}den, wurden auch andere Basensch{\"a}den wie beispielsweise oxoG:C und ethenoA:T vermes-sen und zudem die von der Glykosylase MutY an ungesch{\"a}digter DNA induzier-te Biegung mit den Biegewinkeln der jeweiligen Zielsch{\"a}den verglichen. Die {\"U}bereinstimmung in den Konformationen der Zielsch{\"a}den und der Reparatur-komplexe auch f{\"u}r die Glykosylase MutY (wie bereits f{\"u}r hTDG und hOGG1 in oben genannter Arbeit gezeigt) erlauben ein verbessertes Verst{\"a}ndnis der Schadenssuche und -erkennung durch DNA-Glykosylasen, indem sie die All-gemeing{\"u}ltigkeit einer Biegungsenergie-basierten initialen Schadenserkennung durch DNA-Glykosylasen unterst{\"u}tzen. Die etablierte Messsoftware kann zu-k{\"u}nftig an weiteren DNA-Sch{\"a}den und den entsprechenden Protein-DNA-Komplexen ihre Anwendung finden und kann somit durch die effektive Gewin-nung objektiver Daten in großer Menge zur St{\"u}tzung des Modells beitragen.},
  subject      = {DNS-Reparatur},
  language  = {de}
}
@misc{Fronczek2009,
  type      = {Master Thesis},
  author    = {Fronczek, David Norman},
  title     = {Integration of fluorescence and atomic force microscopy for single molecule studies of protein complexes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70731},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2009},
  abstract  = {The scope of this work is to develop a novel single-molecule imaging technique by combining atomic force microscopy (AFM) and optical fluorescence microscopy. The technique is used for characterizing the structural properties of multi-protein complexes. The high-resolution fluorescence microscopy and AFM are combined (FIONA-AFM) to allow for the identification of individual proteins in such complexes. This is achieved by labeling single proteins with fluorescent dyes and determining the positions of these fluorophores with high precision in an optical image. The same area of the sample is subsequently scanned by AFM. Finally, the two images are aligned and the positions of the fluorophores are displayed on top of the topographical data. Using quantum dots as fiducial markers in addition to fluorescently labeled proteins, fluorescence and AFM information can be aligned with an accuracy better than 10 nm, which is sufficient to identify single fluorescently labeled proteins in most multi-protein complexes. The limitations of localization precision and accuracy in fluorescence and AFM images are investigated, including their effects on the overall registration accuracy of FIONA-AFM hybrid images. This combination of the two complementary techniques opens a wide spectrum of possible applications to the study of protein interactions, because AFM can yield high resolution (5-10 nm) information about the conformational properties of multi-protein complexes while the fluorescence can indicate spatial relationships of the proteins within the complexes. Additionally, computer simulations are performed in order to validate the accuracy of the registration algorithm.},
  subject      = {Kraftmikroskopie},
  language  = {en}
}