Splice variants of the endonucleases XPF and XPG contain residual DNA repair capabilities and could be a valuable tool for personalized medicine

Abstract

The two endonucleases XPF and XPG are essentially involved in nucleotide excision repair (NER) and interstrand crosslink (ICL) repair. Defects in these two proteins result in severe diseases like xeroderma pigmentosum (XP). We applied our newly CRISPR/Cas9 generated human XPF knockout cell line with complete loss of XPF and primary fibroblasts from an XP-G patient (XP20BE) to analyze until now uncharacterized spontaneous mRNA splice variants of these two endonucleases. Functional analyses of these variants were performed using luciferase-based reporter gene assays. Two XPF and XPG splice variants with residual repair capabilities in NER, as well as ICL repair could be identified. Almost all variants are severely C-terminally truncated and lack important protein-protein interaction domains. Interestingly, XPF-202, differing to XPF-003 in the first 12 amino acids only, had no repair capability at all, suggesting an important role of this region during DNA repair, potentially concerning protein-protein interaction. We also identified splice variants of XPF and XPG exerting inhibitory effects on NER. Moreover, we showed that the XPF and XPG splice variants presented with different inter-individual expression patterns in healthy donors, as well as in various tissues. With regard to their residual repair capability and dominant-negative effects, functionally relevant spontaneous XPF and XPG splice variants present promising prognostic marker candidates for individual cancer risk, disease outcome, or therapeutic success. This merits further investigations, large association studies, and translational research within clinical trials in the future.

Xeroderma Pigmentosum: Gene Variants and Splice Variants

“…Due to XPF-202 not displaying any significant repair capabilities, the first 12 amino acids seem to be essential for protein function. Interestingly, when overexpressed, all XPF splice variants as well as the full-length protein reduced NER capabilities [38,39].…”

RNA splicing alteration in the response to platinum chemotherapy in ovarian cancer: A possible biomarker and therapeutic target

“…88 Also XPG (ERCC5) and XPF (ERCC4) endonucleases can be transcribed in several splicing variants, differently expressed in human tissues and displaying different repair abilities following exposure to PT. 89,90 Nonhomologous end joining (NHEJ) is the DNA repair pathway involved in the resolution of double-strand breaks in cells that are mainly in the G1 phase of the cell cycle. NHEJ requires the activity of the DNA-PK complex, which recognizes the DNA break and catalyzes the end processing, then ligated by the activity of XRCC4 and DNA ligase IV.…”

“…Interestingly, AS of the endonuclease ERCC1, which catalyzes the excision reaction, correlates with a reduction in the cellular capability to repair PT‐induced DNA adducts 88 . Also XPG (ERCC5) and XPF (ERCC4) endonucleases can be transcribed in several splicing variants, differently expressed in human tissues and displaying different repair abilities following exposure to PT 89,90 …”

Experimentelle Forschung an der Klinik und Poliklinik für Dermatologie und Venerologie

“…Zuletzt konnte in unserer Arbeitsgruppe eine XPF-Knockout-Zelllinie mittels CRISPR/Cas9-Technologie generiert werden [11]. Im Vergleich mit Wildtyp-Zellen wurden anschließend spontane mRNA-Splice-Varianten der beiden Endonukleasen XPF und XPG an dieser XPF-Knockout-Zelllinie und an primären Fibroblasten von einem XPG-Patienten analysiert [12]. Dabei wurden Splice-Varianten ohne Reparaturkapazität, mit einer residualen Reparaturkapazität und Varianten mit einem inhibitorischen Effekt auf die NER identifiziert.…”

“…Darüber hinaus zeigten sich interindividuelle Unterschiede in den Expressionsmustern der Splice-Varianten in gesunden Spendern und in verschiedenen Geweben. Funktionell relevante spontane Splice-Varianten von XPF und XPG stellen interessante potenzielle prognostische Biomarker-Kandidaten für das individuelle Krebsrisiko, den Krankheitsverlauf oder Therapieerfolg dar [12]. Diese müssen jedoch noch in weiteren Untersuchungen und klinischen Studien validiert werden.…”