title: Newly identified CHO ERCC3/XPB mutations and phenotype characterization.
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.
authors: Laboratory of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava 37, Slovak Republic.
authors: Ivana
authors: I
authors: Rybanská
authors: Ján
authors: J
authors: Gursky
authors: Miriam
authors: M
authors: Fasková
authors: Edmund P
authors: EP
authors: Salazar
authors: Erika
authors: E
authors: Kimlícková-Polakovicová
authors: Karol
authors: K
authors: Kleibl
authors: Larry H
authors: LH
authors: Thompson
authors: Miroslav
authors: M
authors: Pirsel
mesh_terms: D000818:Animals; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D020552:Comet Assay; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage / Q000187:drug effects* / Q000528:radiation effects*; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D043382:DNA-Formamidopyrimidine Glycosylase / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D011759:Pyrrolidines / Q000494:pharmacology; D011807:Quinolizines / Q000494:pharmacology; D014466:Ultraviolet Rays / Q000009:adverse effects
chemical_list: D004268:DNA-Binding Proteins; D011759:Pyrrolidines; D011807:Quinolizines; C076809:Ro 19-8022; C072022:XPBC-ERCC-3 protein; D043382:DNA-Formamidopyrimidine Glycosylase; D004265:DNA Helicases