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dc.contributor.advisor Cramer, Patrick Prof. Dr.
dc.contributor.author Kokic, Goran
dc.date.accessioned 2021-04-09T10:44:20Z
dc.date.available 2021-04-09T10:44:20Z
dc.date.issued 2021-04-09
dc.identifier.uri http://hdl.handle.net/21.11130/00-1735-0000-0008-57EA-F
dc.language.iso eng de
dc.relation.uri http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc 572 de
dc.title Structure-function analysis of human nucleotide excision DNA repair de
dc.type doctoralThesis de
dc.contributor.referee Stark, Holger Prof. Dr.
dc.date.examination 2019-04-10
dc.description.abstracteng An average human cell accumulates tens of thousands of DNA lesions per day. Therefore, DNA repair pathways are set up to continuously proofread the DNA and correct DNA lesions, thus ensuring accurate expression and propagation of genetic material. Nucleotide excision DNA repair (NER) is a major DNA repair pathway which removes UV-induced lesions and bulky DNA modifications. Defects in NER promote cancer development and cause human diseases, such as Xeroderma pigmentosum (XP), Cockayne syndrome and trichothiodystrophy. During NER, lesions are cut out of the DNA as part of a short oligonucleotide and the resulting gap is filled by DNA synthesis using the nondamaged DNA strand as a template. The NER machinery assembles around the heterodecameric transcription factor IIH (TFIIH), comprised of the core module and the kinase module. The TFIIH core module utilizes the ATP-consuming subunits XPB and XPD to open the DNA repair bubble, scan for the lesion and coordinate the excision of the damaged DNA. However, due to the complete lack of structural information on NER assembly intermediates and difficulties in preparing the NER complexes for in vitro analysis, the molecular mechanism of NER is still not well understood. Here we prepare human TFIIH and other NER factors involved in DNA excision. We reconstitute several steps of the NER pathway and analyze the trapped intermediates with biochemical assays, cross-linking mass-spectrometry and electron microscopy (EM). We systematically dissect the regulation of the TFIIH ATPases XPB and XPD and show that the additional NER factors XPA and XPG stimulate the enzymatic activities of the ATPases. We report the core TFIIH-XPA-DNA structure at 3.6 Å resolution, which represents the lesion scanning NER intermediate, and we map the position of XPG within the complex by chemical crosslinking. The structure expands the previous model for the TFIIH core and explains many disease mutations. The structure further elucidates the topology of NER factors around the 5’ edge of the repair bubble: XPB binds the DNA duplex, XPD binds the 3’ single strand extension and XPA wraps around the duplex single-strand junction and bridges the ATPases. XPA and XPB form a DNA duplex tunnel which anchors the NER machinery to the DNA. Our biochemical analysis and comparison to previous structures reveal how XPA and XPG activate TFIIH for repair. The TFIIH kinase module inhibits NER by repressing the XPD helicase activity. XPA stabilizes a completely novel TFIIH conformation in which the ATPases are dramatically reoriented, which displaces the TFIIH kinase module and removes the “plug” element from the DNA-binding pore in XPD. This allows XPD to move by ~80Å, engage the DNA and rapidly scan for the lesion. XPG facilitates lesion scanning by directly stimulating XPD migration on DNA and by sequestering the kinase module binding site on XPD. The results presented here greatly extend our understanding of human NER and provide the basis for future structure-function analysis of this repair pathway, also in the context of transcription. de
dc.contributor.coReferee Höbartner, Claudia Prof. Dr.
dc.contributor.thirdReferee Pena, Vladimir Dr.
dc.contributor.thirdReferee Rodnina, Marina Prof. Dr.
dc.contributor.thirdReferee Urlaub, Henning Dr.
dc.subject.eng cryo-EM de
dc.subject.eng transcription de
dc.subject.eng DNA repair de
dc.identifier.urn urn:nbn:de:gbv:7-21.11130/00-1735-0000-0008-57EA-F-3
dc.affiliation.institute Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) de
dc.subject.gokfull Biologie (PPN619462639) de
dc.identifier.ppn 1753599024

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