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Newly identified CHO ERCC3/XPB mutations and phenotype characterization
Ivana Rybanská1, Ján Gursky, Miriam Fasková
1Laboratory of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava 37, Slovak Republic.
Abstract:
Nucleotide excision repair (NER) is a complex multistage process involving many interacting gene products to repair a wide range of DNA lesions. Genetic defects in NER cause human hereditary diseases including xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy and a combined XP/CS overlapping symptom. One key gene product associated with all these disorders is the excision repair cross-complementing 3/xeroderma pigmentosum B (ERCC3/XPB) DNA helicase, a subunit of the transcription factor IIH complex. ERCC3 is involved in initiation of basal transcription and global genome repair as well as in transcription-coupled repair (TCR). The hamster ERCC3 gene shows high degree of homology with the human ERCC3/XPB gene. We identified new mutations in the Chinese hamster ovary cell ERCC3 gene and characterized the role of hamster ERCC3 protein in DNA repair of ultraviolet (UV)-induced and oxidative DNA damage. All but one newly described mutations are located in the protein C-terminal region around the last intron-exon boundary. Due to protein truncations or frameshifts, they lack amino acid Ser751, phosphorylation of which prevents the 5' incision of the UV-induced lesion during NER. Thus, despite the various locations of the mutations, their phenotypes are similar. All ercc3 mutants are extremely sensitive to UV-C light and lack recovery of RNA synthesis (RRS), confirming a defect in TCR of UV-induced damage. Their limited global genome NER capacity averages approximately 8%. We detected modest sensitivity of ercc3 mutants to the photosensitizer Ro19-8022, which primarily introduces 8-oxoguanine lesions into DNA. Ro19-8022-induced damage interfered with RRS, and some of the ercc3 mutants had delayed kinetics. All ercc3 mutants showed efficient base excision repair (BER). Thus, the positions of the mutations have no effect on the sensitivity to, and repair of, Ro19-8022-induced DNA damage, suggesting that the ERCC3 protein is not involved in BER.
Insights
New mutations in the ERCC3 gene of Chinese hamster cells reveal its crucial role in DNA repair, particularly in repairing UV-induced damage through nucleotide excision repair (NER) and transcription-coupled repair (TCR).
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Genetics
Background:
- Nucleotide excision repair (NER) is vital for correcting diverse DNA lesions.
- Defects in NER genes, like ERCC3/XPB, cause human hereditary diseases (e.g., Xeroderma Pigmentosum).
- ERCC3 is a subunit of transcription factor IIH, essential for transcription initiation and DNA repair.
Purpose of the Study:
- To identify and characterize new mutations in the Chinese hamster ERCC3 gene.
- To elucidate the role of hamster ERCC3 protein in repairing UV-induced and oxidative DNA damage.
- To investigate the impact of mutation location on ERCC3 protein function and DNA repair phenotypes.
Main Methods:
- Identification of novel mutations in the Chinese hamster ovary cell ERCC3 gene.
- Characterization of mutant phenotypes, including sensitivity to UV-C light and photosensitizer Ro19-8022.
- Assessment of DNA repair capacities: global genome NER, transcription-coupled repair (TCR), and base excision repair (BER).
Main Results:
- Newly identified mutations, primarily in the C-terminal region, lead to protein truncations or frameshifts, lacking the critical Ser751 residue.
- All ERCC3 mutants exhibit extreme UV-C sensitivity and defective transcription-coupled repair (TCR), with limited global genome NER capacity (approx. 8%).
- Mutants show modest sensitivity to Ro19-8022 (oxidative damage inducer) but efficient base excision repair (BER), indicating ERCC3 is not involved in BER.
Conclusions:
- ERCC3 mutations, regardless of location, result in similar severe phenotypes related to UV-induced DNA damage repair.
- The ERCC3 protein is essential for transcription-coupled repair (TCR) of UV damage and plays a role in global genome NER.
- ERCC3 is not implicated in the repair of oxidative DNA damage induced by Ro19-8022, suggesting distinct repair pathways.
