Characterization of the multifunctional XPG protein during Nucleotide-excision-repair
by Steffen Schubert
Date of Examination:2014-05-15
Date of issue:2014-06-03
Advisor:Prof. Dr. Steffen Emmert
Referee:Prof. Dr. Michael P. Schön
Referee:PD Dr. Wilfried Kramer
Persistent Address:
http://dx.doi.org/10.53846/goediss-4515
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Description:Characterization of the multifunctional XPG protein during Nucleotide-excision-repair
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Abstract
English
Xeroderma pigmentosum (XP), a cancer model disease, is the perfect proof for the existing model of carcinogenesis activated by mutations. All patients share a defect in Nucleotide excision repair (NER). The gene, which is disease-causing for XP complementation group G (XPG) patients, encodes for the multifunctional endonuclease XPG. This enzyme has many binding partners like TFIIH, RPA and PCNA, and acts at a crucial step at the very end of NER. Several functional domains of XPG were mutated to investigate the behavior of the respective mutants during NER intermediates of dual incision, using DNA repair synthesis (UDS) and Host cell reactivation (HCR) assays. Furthermore, a new XPG patient with implications for the functional XPG-TFIIH interaction has been studied. By genotype-phenotype correlation of a XPG patient (XP172MA), this study greatly suggests to narrow down the functionally important XPG interaction domain between TFIIH and XPG to the XPG amino-acids 30-85. This study demonstrates that the functional PCNA-XPG interaction is more important for NER than the endonuclease function of XPG. The C-terminally located PIP-box of XPG is required for immediate UV response but not for the functionality of XPG during NER in transiently transfected primary fibroblasts. The N-terminal PIP-UBM ubiquitin binding domain is more important for integrity of NER than the C-terminal PIP-box. I raise the model of an NER intermediate state that involves obligatory ubiquitination during NER and the blocking of error-prone translesion polymerases by XPG. This study excludes XPG as the responsible factor for PCNA recruitment and designates XPG as the factor as restrictive element for UV-damage dependent activation of translesion polymerases to S-phase. The results obtained with the endonuclease defective E791A XPG mutant confirm the actual “cut-patch-cat-patch” model of dual incision during NER. Moreover, this study clearly demonstrates the ability of endonuclease defective XPG to perform accurate NER in living cells. This accounts for the existence of a cellular backup mechanism for the XPG endonuclease function. The proposal for a nuclear backup mechanism is supported by the investigation of a physiologically relevant (evolutionary developed) XPG splicevariant with NER activity (IsoVI). The severely truncated XPG isoform is able to structurally complement a XPG defect. This complementation is dependent on the endonuclease function of Fen1. This suggests the existence of an evolutionary developed backup mechanism for XPG during NER.
Keywords: Nucleotide-Excision-Repair, NER, XPG, skin cancer, Xeroderma pigmentosum, splice variant