| 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.identifier.uri | http://dx.doi.org/10.53846/goediss-8540 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-8540 | |
| dc.language.iso | eng | de |
| dc.rights.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 | |