Functional relevance of spontaneous alternative splice variants of xeroderma pigmentosum genes: Prognostic marker for skin cancer risk and disease outcome?

Functional relevance of spontaneous alternative splice variants of xeroderma pigmentosum genes: Prognostic marker for skin cancer risk and disease outcome?

Lehmann, Janin

Doctoral thesis

Date of Examination: 2017-05-04

Date of issue: 2017-05-05

Advisor: Emmert, Steffen Prof. Dr.

Referee: Schön, Michael P. Prof. Dr.

Referee: Johnsen, Steven A. Prof. Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0023-3E36-8

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Abstract

English

The nucleotide excision repair (NER) pathway is a central DNA repair mechanism to repair a variety of bulky DNA lesions. Accumulation of these types of damage all over the genome results in the development of a cancer prone mutator phenotype, as it can be seen in patients with the autosomal recessive disease xeroderma pigmentosum, and their high frequency of ultraviolet induced skin tumors. It is known that decreased NER level are a risk factor for several cancer entities. Several components of the NER pathway already serve as biomarkers for cancer risk and treatment success. The endonucleases XPF/ERCC1 and XPG are the core components of the incision complex of NER. XPF/ERCC1 is also involved in the repair of DNA interstrand crosslinks (ICL). Due to the essential roles of this complex, patient cells retain at least one full-length allele and residual repair capabilities, rendering them unsuitable for XPF variant analyses. In the course of this thesis, the CRISPR/Cas9 technology was established in the laboratory and applied to generate an XPF knockout in a fetal lung fibroblast cell line (MRC5Vi cells), to analyze the unknown functional relevance of physiologically occurring, spontaneous XPF mRNA splice variants. Furthermore, functional roles of XPF point mutants in NER and ICL repair were investigated. The successfully generated XPF knockout cells were markedly sensitive to UVC, cisplatin, and PUVA (psoralen activated by UVA) and had reduced repair capabilities for NER and ICL repair as assessed by reporter gene assays. Using the knockout cells it was shown that human XPF is predominately involved in homologous recombination repair but dispensable for non-homologous end-joining. Notably, while ERCC1 was stably expressed in the cytosol, it was not detectable in the nucleus without its heterodimeric partner XPF, implicating the necessity of functional XPF to retain ERCC1 in the nucleus. Overexpression of wildtype XPF reversed these effects. Functional analyses revealed two XPF splice variants with residual repair capabilities (XPF-201 and XPF-003) in NER, as well as ICL repair. XPF-201 lacks the first 12 amino acids of the protein, while XPF-003 is severely C-terminally truncated. Interestingly, another variant, XPF-202, which differs to XPF-003 in the first 12 amino acids only, had no repair capability whatsoever, suggesting an important role of this protein region. It might be involved in interacting with other proteins of the DNA repair machinery. Splice variants of XPF and XPG, already characterized during my master thesis, were identified to exert dominant negative effects on NER, when stably overexpressed in wildtype cells. Additionally, the newly generated KO cells represent a highly promising tool for mechanistic studies. In this cellular background without XPF expression, point mutants showed different catalytic activities compared to reconstituted in vitro systems, which are limited by the artificial combination of recombinant proteins, or patient cell lines retaining at least one full-length allele. Finally, it was shown that the XPF and XPG splice variants varied in their inter-individual expression in healthy donors, as well as in various tissues. Together with their residual repair capability, dominant-negative effects, and different expression levels, functionally relevant spontaneous splice variants of XPF and XPG present promising prognostic marker candidates for individual cancer risk, disease outcome, and therapeutic success. Association studies and translational research within clinical trials will have to confirm this assumption in the future.

Keywords: Xeroderma pigmentosum; nucleotide excision repair; interstrand crosslink repair; homologous recombination repair; CRISPR/Cas9